PRIORITY INFORMATION

[0001] This application claims the benefit under 35 U.S.C. §119(e) of the U.S. Provisional Application No. 63/378,475, filed on October 5, 2022. The contents of the aforementioned application are herein incorporated by reference in their entirety.

INCORPORATION BY REFERENCE

[0002] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

FIELD OF THE INVENTION

[0003] Example embodiments relate to nucleic acid-based kits, compositions, and methods for determining the presence or absence in a sample of diarrhea causing pathogens including the following: Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC).

BACKGROUND OF THE INVENTION

[0004] Bacterial gastrointestinal (GI) pathogens (such as Campylobacter, Salmonella and Shigella/Enteroinvasive Escherichia coli (EIEC)) are the leading causes of acute gastroenteritis, hemorrhagic colitis and fatal typhoid fever. The presence of GI pathogens is correlated to (a) plethora of highly impactful diseases and (b) an increase in antibiotic resistance. Accordingly, detection of the etiological agents of acute bacterial diarrhea is important for the management of the patient and for public health interventions, while key clinical decisions are driven by bacterial gastrointestinal identification.

[0005] Salmonella infection can cause a variable clinical disease starting from a mild, subclinical infection, or lead to severe systemic infection such as typhoid fever. Salmonella sp. invades the host through the colonic epithelial cells, especially M cells using a type III secretion system. They are also able to survive within phagosomes of macrophages, and evade the host immune system by several ways. Campylobacter jejuni and coli are among the large Campylobacter family predominant human stool pathogens causing watery diarrhea, fever and typically hard abdominal pain; Campylobacter upsaliensis is the most important Campylobacter species after C. jejuni and C. coli. Shigella and EIEC are genetically closely related. Both of these organisms invade the colonic epithelium mediated by the genes located in virulence plasmid pINV coding e.g., Ipa proteins and their transcription regulator invE.

[0006] Accordingly, there is a need to detect the above mentioned pathogens in a single assay that would be sensitive, specific for the targets and not cross-reactive. The need is solved by the presently disclosed method that is directed to provide the detection of Campylobacter, Salmonella and Shigella/Enteroinvasive Escherichia coli (EIEC) in a multiplex qPCR assay.

SUMMARY OF THE INVENTION

[0007] In an exemplary embodiment, a method for determining the presence or absence of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample, comprises the steps of producing at least one amplicon by subjecting a reaction mixture including the sample and at least five primer pairs to reaction conditions suitable to amplify targeted nucleic acids wherein the at least five primer pairs include at least one primer pair A that specifically hybridizes with a portion of Campylobacter coli genome, at least one primer pair B that specifically hybridizes with a portion of Campylobacter jejuni genome, at least one primer pair C that specifically hybridizes with a portion of Campylobacter upsaliensis genome, at least one primer pair D that specifically hybridizes with a portion of Salmonella sp. genome, and at least one primer pair E that specifically hybridizes with a portion of Shigella sp./EIEC genome; and determining the presence or absence of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample based on the at least one amplicon.

[0008] In an example embodiment of the method, the producing of the at least one amplicon includes performing PCR.

[0009] In an example embodiment of the method, the at least one amplicon is one selected from an amplicon produced using the primer pair A and comprising a sequence selected from the sequences specific for Campylobacter coli in “Probe” column of Figure 1 , an amplicon produced using the primer pair B and comprising a sequence selected from the sequences specific for Campylobacter jejuni in “Probe” column of Figure 1, an amplicon produced using the primer pair C and comprising a sequence selected from the sequences specific for Campylobacter upsaliensis in “Probe” column of Figure 1, an amplicon produced using the primer pair D and comprising a sequence selected from the sequences specific for Salmonella sp. in “Probe” column of Figure 1, and/or an amplicon produced using the primer pair E and comprising a sequence selected from the sequences specific for Shigella sp./EIEC in “Probe” column of Figure 1.

[0010] In an example embodiment of the method, the reaction mixture includes probes specific for the at least one amplicon.

[0011] In an example embodiment of the method, the reaction mixture further comprises probes suitable for use with a specific forward and reverse primer pair, and the probes are selected from the probe sequences provided in Figure 1.

[0012] In an example embodiment of the method, the at least one primer pair A comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter coli in Figure 1, the at least one primer pair B comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter jejuni in Figure 1, the at least one primer pair C comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter upsaliensis in Figure 1, the at least one primer pair D comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Salmonella sp. in Figure 1, and the at least one primer pair E comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Shigella sp./EIEC in Figure 1.

[0013] In an example embodiment of the method, the reaction mixture further comprises probes suitable for use with said primer pairs in Figure 1.

[0014] In an example embodiment of the method, the at least one primer pair A comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 1 ; the at least one primer pair B comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 2; the at least one primer pair C comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 3; the at least one primer pair D comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 4; and/or the at least one primer pair E comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 5.

[0015] In an example embodiment of the method, the reaction mixture further contains a control sample and a primer pair F that specifically hybridizes with a portion of the control sample genome, and, optionally, a probe specific for target nucleic acid of the control sample.

[0016] In an example embodiment of the method, the primer pair F comprises forward and reverse primer pair selected from Table 6, and the probe sequence is selected from Table 6.

[0017] In an example embodiment of the method, the probes each include a fluorescent reporter.

[0018] In an example embodiment of the method, the probes each includes a quencher.

[0019] In an example embodiment of the method, each of the probes is labeled at or near the 5’ end with a dye selected from Alexa Fluor, ABY, VIC, JUN, FAM and 6FAM.

[0020] In an example embodiment of the method, each of the probes is labeled at the 3’ end with a quencher selected from QSY, MGBNFQ, BHQ, and DFQ.

[0021] In an example embodiment of the method, the reaction mixture comprises probes specific for amplicons produced using one or more of the at least one primer pair A, the at least one primer pair B and the at least one primer pair C, the probes being labeled with 6FAM, at least one probe specific for amplicons produced using the at least one primer pair D, the probes being labeled with VIC, and at least one probe specific for amplicons produced using the at least one primer pair E, the probe being labeled with ABY.

[0022] In an example embodiment of the method, the probe specific for the target nucleic acid of the control sample is specific for amplicons produced using the primer pair F, the probe being labeled with JUN. [0023] In an exemplary embodiment, a composition for determining the presence or absence of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample, comprises: at least five primer pairs, wherein the at least five primer pairs includes at least one primer pair A that specifically hybridizes with a portion of Campylobacter coli genome, at least one primer pair B that specifically hybridizes with a portion of Campylobacter jejuni genome, at least one primer pair C that specifically hybridizes with a portion of Campylobacter upsaliensis genome, at least one primer pair D that specifically hybridizes with a portion of Salmonella sp. genome, and at least one primer pair E that specifically hybridizes with a portion of Shigella sp./EIEC genome.

[0024] In an example embodiment of the composition, the at least one primer pair A comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter coli in Figure 1, the at least one primer pair B comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter jejuni in Figure 1, the at least one primer pair C comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter upsaliensis in Figure 1, the at least one primer pair D comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Salmonella sp. in Figure 1, and the at least one primer pair E comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Shigella sp./EIEC in Figure 1.

[0025] In an example embodiment of the composition, the composition further comprises probes specific for amplicons produced using one or more of the at least one primer pair A, the at least one primer pair B, the at least one primer pair C, the at least one primer pair D, and the at least one primer pair E.

[0026] In an example embodiment of the composition, the probes are suitable for use with a specific forward and reverse primer pair in Figure 1, and optionally, a probe in Figure 1.

[0027] In an example embodiment of the composition, the composition further comprises a polymerase, a buffer, and nucleotides. [0028] In an example embodiment of the composition, the composition is suitable for producing at least one amplicon when included in a reaction mixture that is subjected to reaction conditions suitable to amplify target nucleic acid of a sample in the reaction mixture.

[0029] In an example embodiment of the composition, the composition further comprises a primer pair F that includes a forward and reverse primer pair selected from Table 6; and optionally, at least one probe selected from Table 6.

[0030] In an example embodiment of the composition, the at least one probe includes a fluorescent reporter.

[0031] In an example embodiment of the composition, the at least one probe includes a quencher.

[0032] In an example embodiment of the composition, the at least one probe is labeled at or near the 5’ end with a dye selected from Alexa Fluor, ABY, VIC, JUN, FAM and 6FAM.

[0033] In an example embodiment of the composition, the at least one probe is labeled at the 3’ end with a quencher selected from QSY, MGBNFQ, BHQ, and DFQ.

[0034] In an example embodiment of the composition, the probes specific for amplicons produced using one or more of the primer pairs A, B and C are labeled with 6FAM, the probe specific for amplicons produced using the primer pair D is labeled with VIC, and the probe specific for amplicons produced using the primer pair E is labeled with ABY.

[0035] In an example embodiment of the composition, the probe specific for amplicons produced using the primer pair F is labeled with JUN.

[0036] In an exemplary embodiment, a kit for determining the presence or absence of Campylobacter sp., Salmonella sp., and Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample, comprising the composition described in the above embodiments.

[0037] In an example embodiment of the kit, the kit further comprises a control sample; a primer pair F that specifically hybridizes with a portion of control sample genome; and optionally, a probe that specifically hybridizes with target nucleic acid of a control sample.

[0038] In an example embodiment of the kit, the primer pair F comprises a forward and reverse primer pair selected from Table 6 and the probe sequence is selected from Table 6. [0039] In an example embodiment of the kit, the at least five primer pairs and probe set comprise at least one set of the primer pairs and probe as provided in a Table 7.

[0040] In an example embodiment of the kit, the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 8.

[0041] In an example embodiment of the kit, the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 9.

[0042] In an example embodiment of the kit, the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 10.

[0043] In an example embodiment of the kit, the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 11.

[0044] In an example embodiment of the kit, the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 12.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG. 1 provides exemplary forward and reverse primer pairs, together with a probe that is used with that primer pair (that is, a primer pair/probe set), as well as, the pathogen (or control organism, in case of process control) that is detected with the primer pair/probe set (as can be determined by the column “pathogen or gene”). “Primer pair No.” denotes different primer pair/probe sets that can be used for detection of a particular pathogen.

DETAILED DESCRIPTION

[0046] All publications and patent applications cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent application were specifically and individually indicated to be so incorporated by reference. Further, although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the spirit and substance of this disclosure and of the appended claims. [0047] This invention relates to the field of detection of diarrhea causing pathogens from patient, food or environmental samples. Particularly, the present invention provides a multiplex polymerase chain reaction (PCR) based assay method for detection of diarrhea causing pathogens, such as Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC). The present invention further provides materials such as primers, primer pairs and probes for use in the method of the invention.

[0048] The present application discloses primers and probes designed for target sequences and compatible for use in a multiplex qPCR determining the presence of multiple diarrhea causing pathogens. Multiplex PCR presents a challenge for quantitation of the pathogen DNA: the different amplicons compete for the same PCR reaction components (such as e.g. DNA polymerase and MgC12) and this can compromise the quantitative comparison between samples. It is commonly known in the art that there is bias in the amplification efficiencies between different template amounts or lengths so that e.g. short amplicons are favored in the expense of longer ones. At the same time, undesired cross-reactions of multiplex set oligo combinations must be avoided. Finding suitable primer and probe sequences for the detection of a diverse group of pathogenic microbes is far from trivial especially when designing multiplex set ups. In addition to primers and probes for detecting Salmonella sp. and Shigella sp./EIEC, the present application discloses primers and probes for detecting each of the Campylobacter jejuni, Campylobacter coli and Campylobacter upsaliensis. The selected primers and probes of the assay do not cross-react with other closely-related pathogens. The disclosed detection method, as well as detection assay may contain internal process control, such as Bacillus atrophaeus (BA). In addition, the assay may have a real time PCR positive control. The probes that target genomic regions of different pathogens utilize three distinct fluorophores, while a fourth fluorophore is designated for the process control detection.

Therefore, also a 4-plex qPCR assay is provided herein, wherein the detection takes place in a 1-well reaction, to meet the speed and throughput needs in gastrointestinal pathogen testing.

[0049] When an example “embodiment” or a particular “assay” is described herein, it will be understood that the features of the embodiment may be applicable to a composition (e.g., the particular physical components of an assay such as primers and/or probes), a kit (e.g., primers and/or probes and additional buffers, reagents, etc.), or a method (e.g., a process for detecting target nucleic acids) as appropriate. For simplicity, many embodiments are presented by describing “assays”, but it will be understood that the associated methods of using the assays are also intended to form part of this disclosure.

[0050] A method for determining the presence or absence of Campylobacter sp., Salmonella sp., and Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample is provided, said method comprising the steps of: (a) providing a reaction mixture containing the sample and at least 5 primer pair set; wherein the primer pair set comprises: at least one primer pair A that specifically hybridizes with a portion of Campylobacter coli genome; at least one primer pair B that specifically hybridizes with a portion of Campylobacter jejuni genome; at least one primer pair C that specifically hybridizes with a portion of Campylobacter upsaliensis genome; at least one primer pair D that specifically hybridizes with a portion of Salmonella sp. genome; and at least one primer pair E that specifically hybridizes with a portion of Shigella sp./EIEC genome; (b) subjecting the reaction mixture to reaction conditions suitable to amplify targeted nucleic acids, thereby producing one or more amplicons; wherein the presence or absence of at least one amplicon in the sample indicates the presence or absence of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample.

[0051] In some embodiments, the primer pair set comprises at least one primers pair A, at least one primer pair B, at least one primer pair C, at least one primer pair D and at least one primer pair E. In some embodiments, the primer pair set further comprises at least one primer pair F. In some embodiments, the primer pairs are used with corresponding probes as set out in Figure 1. In some embodiments, the primer pair set comprises at least two primers pairs A, at least two primer pairs B, at least two primer pairs C, at least two primer pairs D and at least two primer pairs E.

[0052] In some embodiments, the primer pair A comprises at least one of the primer pairs, in a combination with a corresponding probe sequence:

Table 1

[0053] In some embodiments, the primer pair B comprises at least one of the primer pairs, in a combination with a corresponding probe sequence:

Table 2

[0054] In some embodiments, the primer pair C comprises at least one of the primer pairs, in a combination with a corresponding probe sequence: Table 3 [0055] In some embodiments, the primer pair D comprises at least one of the primer pairs, in a combination with a corresponding probe sequence:

Table 4

[0056] In some embodiments, the primer pair E comprises at least one of the primer pairs, in a combination with a corresponding probe sequence:

Table 5 [0057] In some embodiments, the method further comprises at least one primer pair F and a probe for detection of control sample:

Table 6

[0058] In some embodiments, the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:

Table 7

[0059] In some embodiments, the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:

Table 8

[0060] In some embodiments, the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:

Table 9

[0061] In some embodiments, the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:

Table 10

[0062] In some embodiments, the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:

Table 11

[0063] In some embodiments, the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:

Table 12

[0064] In some embodiments, the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:

Table 13 [0065] In some embodiments, the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:

Table 14 [0066] In some embodiments, the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:

Table 15

[0067] In some embodiments, the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:

Table 16

[0068] In some embodiments, the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:

Table 17

[0069] In some embodiments, the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:

Table 18

[0070] Sample or Specimen

[0071] The invention provides compositions, kits and method for the detection (i.e., the presence or absence) of diarrhea-causing pathogens including Salmonella sp., Campylobacter sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a specimen or a sample.

DNA is extracted from one or more specimen/sample, multiplied using real-time amplification, and detected using specific primers and a fluorescent reporter dye probe for Salmonella sp., Campylobacter sp. and/or Shigella sp./EIEC . As will be appreciated by those in the art, the specimen/sample may comprise any number of things, including, but not limited to, include stool samples, rectal swabs or tissue samples (for example, taken during a biopsy) obtained from human patients; research samples; purified samples, such as purified genomic DNA, RNA, proteins, etc.; and raw samples (bacteria, virus, genomic DNA, etc.). As will be appreciated by those in the art, any experimental manipulation can have been performed on the sample before analysis. In some embodiments, the specimen/sample type for diagnosis of diarrhea-causing pathogens is a stool sample or a rectal swab.

[0072] If required, nucleic acid from the sample/specimen is isolated using known techniques. For example, the sample/specimen may be treated to lyse the cells, using known lysis buffers, sonication, electroporation, etc., with purification occurring as needed, as will be appreciated by those in the art. In addition, the reactions outlined herein may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents that may be included in the assays. These include reagents like salts, buffers, neutral proteins, e.g., albumin, detergents, etc., which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Also, reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used, depending on the sample/specimen preparation methods and purity of the target diarrhea-causing pathogens.

[0073] In some embodiments, the total nucleic acid extraction from a stool sample or a rectal swab is performed. The specimen/sample may be collected and transported in the Remel™ Cary-Blair Transport Medium by ThermoFisher Scientific according to appropriate laboratory procedures. Remel™ Cary-Blair Transport Medium is a semisolid medium recommended for use in the collection, transportation and preservation of sample/specimens, especially stool samples and rectal swabs. Nucleic acids may be isolated and purified from the specimen/sample using a nucleic acid isolation, such as, e.g., the MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit with Bead Plate by Applied Biosystems™. Nucleic acid extraction may be performed via an automated process using, e.g., the KingFisher™ Flex Purification System. For RNA viruses, the RNA is reverse transcribed into cDNA. The cDNA and genomic DNA (from DNA viruses) are then subjected for amplification using the currently disclosed composition, method and kit.

[0074] Process control or Control organism or Control sample [0075] In addition, the disclosed kit and method of detection may further include a process control (“process control” is herein referred to, interchangeably, as “control organism” or “control sample”). This is an exogenous control that has the added advantage of being a bacterial lysis control in addition to being a nucleic acid extraction and recovery control. Controls are treated and tested in parallel with target pathogen and are used to generate a predetermined expected result. When the expected result is reported, one or more aspects of the diagnostic test are confirmed to be working as intended, enabling the user of to verify the diagnostic test as valid. The process control may be Bacillus atrophaeus, which is a grampositive, endospore forming bacterium.

[0076] Preferably, the process control can function as a positive control for lysis, purification and amplification within the cartridges described herein.

[0077] One exemplified process control is lyophilized Bacillus atrophaeus, such as, e.g., the TaqMan™ Universal Extraction Control Organism by ThermoFisher Scientific. The process control may be supplied lyophilized in a quantity of 1 * IO copies/vial, and reconstituted in 200 pL of IX PBS, pH 7.4 to a final concentration 5 x I ⁶ copies/pL. During nucleic acid isolation, 10 pL of the process control is processed as a stand-alone sample in a background of universal transport media. The process control can be added to a negative extraction control. The process control may be added to one or more samples/ specimens at the start of the extraction process. The process control is carried through the remainder of the workflow with the samples/specimens. It is recommended that at least one stand-alone control sample is run per extraction plate.

[0078] Another exemplified process control is a bacterial spore, such as a spore of & Bacillus species. Suitable spores can be comprised of any species of Bacillus, including, e.g., Bacillus globgii, Bacillus atrophaeus, Bacillus subtilis, and Bacillus stearothermophilus . The process control may be a Bacillus atrophaeus bacterial spore.

[0079] Vicinal oxygen chelate (VOC) genes (accession number cl 1463) of Bacillus atrophaeus are detected in an exemplified process control.

[0080] Positive control [0081] In addition, the disclosed method of detection may further include a positive control to determine the validity of the assay. The positive control is a mixture of plasmids of the target pathogens. For instance, an exemplified positive control is a mixture of plasmids of Salmonella sp., Campylobacter sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC). Yet another exemplified positive control is a mixture of plasmids of Salmonella sp., Shigella sp./Enteroinvasive Escherichia coli (EIEC), Campylobacter coli (, Campylobacter jejuni and/or Campylobacter upsaliensis.

[0082] Reaction Mixture

[0083] The terms “reaction mixture,” “amplification mixture,” or “PCR mixture” as used herein refer to a mixture of components necessary to amplify at least one amplicon from nucleic acid templates. The mixture may comprise nucleotides (dNTPs), a thermostable polymerase, primers, and a plurality of nucleic acid templates. The mixture may further comprise a Tris buffer, a monovalent salt, and/or Mg2+. The working concentration range of each component is well known in the art and can be further optimized as needed by an ordinary skilled artisan.

[0084] Amplification

[0085] “Amplification” as used herein denotes the use of any amplification procedures to increase the concentration of a particular nucleic acid sequence within a mixture of nucleic acid sequences.

[0086] In one embodiment and as describe more fully herein, a sequence from a sample is amplified to produce a secondary target (e.g., an amplicon) that is detected, as outlined herein.

[0087] Amplification involves the amplification (replication) of the sequence to be detected, such that the number of copies of the sequence is increased. Suitable amplification techniques include, but are not limited to, the polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA) and nucleic acid sequence-based amplification (NASBA). [0088] In one embodiment, the amplification technique is PCR. The polymerase chain reaction (PCR) is widely used and described, and involves the use of primer extension combined with thermal cycling to amplify a target sequence. As used herein, “PCR”, unless specifically defined, refers to either singleplex or multiplex PCR assays, and can be real time or quantitative PCR (wherein detection occurs during amplification), end-point PCR (when detection occurs at the end amplification), or reverse transcription PCR, including but not limited to, “real-time PCR” or “quantitative PCR” or “qPCR”, “digital PCR” or “dPCR”, “reverse transcriptase PCR” or “RT-PCR”, “multiplex PCR”, “nested PCR”, “hot start PCR”, “long-range PCR”, “assembly PCR”, “asymmetric PCR”, “in situ PCR,” “single-cell PCR,” or “fast-cycling PCR,” among others.

[0089] Example embodiments of amplification are not limited to PCR. For instance, signal amplification, single base extension (SBE) or minisequencing, oligonucleotide ligation amplification (OLA) and/or rolling-circle amplification can be used for amplification. In an embodiment, amplification can include OLA followed by RCA.

[0090] A skilled person would be well aware of the real-time PCR systems that can be used for a multiplex detection using several dyes, or nucleic acid amplification assays as described herein. Exemplary systems include a real-time quantitative PCR (qPCR) instrument, including for example a QuantStudio™ Real-Time PCR system, such as the QuantStudio™ 5 Real-Time PCR System (QS5), QuantStudio™ 7 Real-Time PCR System (QS7), QuantStudio™ 12K Flex System (QS12K), QuantStudio™ DX Real-Time PCR System (QS Dx or QS5 Dx), or a 7500 Real-Time PCR system, such as the 7500 Fast Dx system, all from Applied Biosystems™ - a ThermoFisher Scientific brand.

[0091] Amplicon

[0092] The terms “amplified product” or “amplicon” refer to a fragment of DNA amplified by a polymerase using a pair of primers in an amplification method such as PCR.

[0093] Probe [0094] “Probe” as used herein, is a non-extendable oli onucleotide attached to a fluorescent reporter dye and a quencher moiety.

[0095] Primer

[0096] “Primer” as used herein can refer to more than one primer and refers to an oligonucleotide, whether occurring naturally or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase, at a suitable temperature for a sufficient amount of time and in the presence of a buffering agent. Such conditions can include, for example, the presence of at least four different deoxyribonucleoside triphosphates (such as G, C, A, and T) and a polymerization-inducing agent such as DNA polymerase or reverse transcriptase, in a suitable buffer (“buffer” includes substituents which are cofactors, or which affect pH, ionic strength, etc.), and at a suitable temperature. In some embodiments, the primer may be single-stranded for maximum efficiency in amplification. The primers herein are selected to be substantially complementary to the different strands of each specific sequence to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands. A non-complementary nucleotide fragment may be attached to the 5 '-end of the primer, with the remainder of the primer sequence being complementary, or partially complementary, to the target region of the target nucleic acid. Commonly, the primers are complementary, except when non-complementary nucleotides may be present at a predetermined sequence location, such as a primer terminus as described.

[0097] The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in “antiparallel association.” Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. [0098] Dye or Detectable Label or Fluorescent Label

[0099] The primers and/or probes described herein may further comprise a fluorescent or other detectable label. It should be appreciated that when using multiple fluorescent or detectable labels, particularly in a multiplex format, each fluorescent or detectable label preferably differs in its spectral properties from the other detectable labels used therewith such that the labels may be distinguished from each other, or such that together the fluorescent or detectable labels emit a signal that is not emitted by either fluorescent or detectable label alone. Exemplary fluorescent or detectable labels include, for instance, a fluorescent dye or fluorophore (e.g., a chemical group that can be excited by light to emit fluorescence or phosphorescence), “acceptor dyes” capable of quenching a fluorescent signal from a fluorescent donor dye, and the like, as described above. Suitable fluorescent or detectable labels may include, for example, fluoresceins (e.g., 5-carboxy-2,7-dichlorofluorescein; 5- Carboxyfluorescein (5-FAM); 5-Hydroxy Tryptamine (5-HAT); 6-JOE; 6-carboxyfluorescein (6-FAM); Mustang Purple, VIC, ABY, JUN; FITC; 6-carboxy-4’,5’-dichloro-2’,7’- dimethoxy-'fluorescein (JOE)); 6-carboxy-l,4-dichloro-2’,7’-dichloro ^_, fluorescein (TET); 6- carboxy-l,4-dichloro-2’,4’,5’,7’-tetra-chlorofluores cein (HEX); Alexa Fluor fluorophores (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY fluorophores (e.g., 492/515, 493/503, 500/510, 505/515, 530/550, 542/563, 558/568, 564/570, 576/589, 581/591, 630/650-X, 650/665-X, 665/676, FL, FL ATP, FL Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-X SE), Cascade Blue, Cascade Yellow; Cy™ dyes (e.g., 3, 3.18, 3.5, 5, 5.18, 5.5, 7), cyan GFP, cyclic AMP Fluorosensor (FiCRhR), fluorescent proteins (e.g., green fluorescent protein (e g., GFP. EGFP), blue fluorescent protein (e g., BFP, EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow fluorescent protein (e.g., YFP, Citrine, Venus, YPet), FRET donor/acceptor pairs (e.g., fluorescein/fluorescein, fluorescein/tetramethylrhodamine, lAEDANS/fluorescein, EDANS/dabcyl, BODIPY FL/BODIPY FL, Fluorescein/QSY7 and QSY9), LysoTracker and LysoSensor (e.g., LysoTracker Blue DND-22, LysoTracker Blue-White DPX, LysoTracker Yellow HCK-123, LysoTracker Green DND-26, LysoTracker Red DND-99, LysoSensor Blue DND-167, LysoSensor Green DND-189, LysoSensor Green DND-153, LysoSensor Yellow/Blue DND- 160, LysoSensor Yellow/Blue 10,000 MW dextran), Oregon Green (e.g., 488, 488-X, 500, 514); rhodamines (e.g., 1 10, 123, B, B 200, BB, BG, B extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Phallicidine, Phalloidine, Red, Rhod-2, ROX (6-carboxy-X-rhodamine), 5-ROX (carboxy-X- rhodamine), Sulphorhodamine B can C, Sulphorhodamine G Extra, TAMRA (6- carboxytetramethyl-rhodamine), Tetramethylrhodamine (TRITC), WT), Texas Red, Texas Red-X, among others as would be known to those of skill in the art.

[00100] Exemplary fluorescent labels include but are not limited to 6FAM, ABY, VIC, JUN, and FAM. In one exemplary multiplex dye scheme, the fluorescent labels include, for instance, 6FAM, VIC, ABY and JUN, or FAM, VIC, ABY and JUN. In another exemplary multiplex dye scheme, the fluorescent labels include, for instance, FAM, HEX (JOE/VIC), Texas Red, and Cy5 dyes. Exemplary dye schemes are shown in Table 19.

Table 19

[00101] Referring to Table 19, the probes specific for amplicons produced using one or more primer pair A (target - C. coli) are labeled with FAM; the probes specific for amplicons produced using one or more primer pair B (target - C. upsaliensis) are labeled with FAM; the probes specific for amplicons produced using one or more primer pair C (target - C. jejuni) are labeled with FAM; the probes specific for amplicons produced using one or more primer pair D (target - Salmonella sp.) are labeled with VIC; the probes specific for amplicons produced using one or more primer pair E (target - Shigella sp./EIEC) are labeled with ABY; and the probes specific for amplicons produced using one or more primer pair E (target - Bacillus atrophaeus) are labeled with JUN.

[00102] Other detectable labels may be used in addition to or as an alternative to labelled probes. For example, primers can be labeled and used to both produce amplicons and to detect the presence (or concentration) of amplicons produced in the reaction, and such may be used in addition to or as an alternative to labeled probes described herein. As a further example, primers may be labeled and utilized as described in Nazarenko et al. (Nucleic Acids Res. 2002 May 1 ; 30(9): e37), Hayashi et al. (Nucleic Acids Res. 1989 May 11 ; 17(9): 3605), and/or Neilan et al. (Nucleic Acids Res. Vol. 25, Issue 14, 1 July 1997, pp. 2938-39). Those of skill in the art will also understand and be capable of utilizing the PCR processes (and associated probe and primer design techniques) described in Zhu et al. (Biotechniques. 2020 Jul: 10.2144/btn-2020-0057).

[00103] In some embodiments, the primers and/or probes may further comprise a quencher. Suitable quenchers include but are not limited to QSY (e.g., QSY7 and QSY21), BHQ (Black Hole Quencher) and DFQ (Dark Fluorescent Quencher). In an exemplary embodiment, the quencher is QSY7.

[00104] Detector probes may also include two probes, wherein, for example, a fluorophore is associated with one probe and a quencher is associated with a complementary probe such that hybridization of the two probes on a target quenches the fluorescent signal or hybridization on the target alters the signal signature via a change in fluorescence. Detector probes may also include sulfonate derivatives of fluorescein dyes with SO3 instead of the carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of Cy5.

[00105] Any of these systems and detectable labels, as well as many others, may be used to detect amplified target nucleic acids. In some embodiments, intercalating labels can be used such as ethidium bromide, SYBR Green I, SYBR GreenER, and PicoGreen (all products of Applied Biosystems - a brand of ThermoFisher Scientific), thereby allowing visualization in real-time, or end point, of an amplification product in the absence of a detector probe. In some embodiments, real-time visualization may include both an intercalating detector probe and a sequence-based detector probe. In some embodiments, the detector probe is at least partially quenched when not hybridized to a complementary sequence in the amplification reaction and is at least partially unquenched when hybridized to a complementary sequence in the amplification reaction. In some embodiments, probes may further comprise various modifications such as a minor groove binder (MGB) to further provide desirable thermodynamic characteristics.

[00106] In some embodiments, the amplicon is labeled by incorporation of, or hybridization to labeled primer. In some embodiments, the amplicon is labeled by hybridization to a labeled probe. In some embodiments, the amplicon is labeled by binding of a DNA-binding dye. In some embodiments, the dye may be a single-strand DNA binding dye. In other embodiments, the dye may be a double-stranded DNA binding dye. In other embodiments, the amplicon is labeled via polymerization or incorporation of labeled nucleotides in a template-dependent (or template-independent) polymerization reaction. This can be part of the amplifying step or alternatively the labeled nucleotide can be added after amplifying is completed. The labeled amplicon (or labeled derivative thereof) can be detected using any suitable method such as, for example, electrophoresis, hybridization-based detection (e.g., microarray, molecular beacons, and the like), chromatography, NMR, and the like.

[00107] In one exemplary embodiment, the labeled amplicon is detected using qPCR. In some embodiments, a plurality of different amplicons is formed, and optionally labeled, within a single reaction volume via a single amplification reaction. For example, a multiplex reaction (e.g., 4-plex) carried out in a single tube or reaction vessel (e.g., “single-tube” or “1-tube” or “single-vessel” reaction) can produce a plurality of amplicons that are labeled. In some embodiments, the plurality of amplicons can be differentially labeled. In some embodiments, each of the plurality of amplicons produced during amplification is labeled with a different label.

[00108] Assay Mixture

The terms “assay mixture” or “assay mix” or “assay composition,” as used herein, include mixture containing the primer-probe pairs described above that are used in PCR. [00109] Another aspect provided herein is a method of detecting or quantifying a target nucleic acid molecule in a sample by polymerase chain reaction (PCR), such as by quantitative real-time polymerase chain reaction (qPCR). In one embodiment, the method includes: (i) contacting a sample comprising one or more target nucleic acid molecules with a) at least one probe, such as those described herein, being sequence specific for the target nucleic acid molecule, where the at least one probe undergoes a detectable change in fluorescence upon amplification of the one or more target nucleic acid molecules; and with b) at least one oligonucleotide primer pair; (ii) incubating the mixture of step (i) with a DNA polymerase under conditions sufficient to amplify one or more target nucleic acid molecules; and (iii) detecting the presence or absence or quantifying the amount of the amplified target nucleic acid molecules by measuring fluorescence of the probe. In some embodiments, the DNA polymerase comprises 5’ exonuclease activity. In some other embodiments, the DNA polymerase is a Thermus aquaticus (Taq) DNA polymerase. In some embodiments, the probe is a hydrolysis probe, such as a TaqMan probe.

[00110] Another aspect provided herein is a kit for PCR, such as quantitative real-time polymerase chain reaction (qPCR) and reverse transcription polymerase chain reaction (RT- PCR). In an exemplary embodiment, the kit includes the assay mixture, the process control, and the positive control each described above, as well as a multiplex master mix.

[00111] In an embodiment, the multiplex master mix is a RT-qPCR mix that provides for sensitive, reproducible detection of at least four different target pathogens in a single multiplex reaction. In an embodiment, the multiplex master mix may include an enzyme (for instance, DNA polymerase), a thermostable enzyme, enzyme cofactors, deoxynucleotide triphosphates (dNTPs) including dUTP, an enzyme inhibitor (for instance, RNase inhibitor), a dye and/or a buffer agent. In an exemplary embodiment, the multiplex master mix can be, for instance, TaqPath™ 1-step Multiplex Master Mix by Applied Biosystems - a brand of ThermoFisher Scientific.

[00112] In an embodiment, the master mix may be concentrated. For instance, the master mix may be provided at a 4X concentration. In some embodiments, the master mix is prepared such that it requires less than a 3X dilution prior to use in PCR, e.g., 2X dilution, 1.5X dilution, 1.2X dilution, etc. [00113] In some embodiments, the kit also includes instructions for conducting the PCR, and one or more of the following: a buffering agent, deoxynucleotide triphosphates (dNTPs), an organic solvent, an enzyme, enzyme cofactors, and an enzyme inhibitor. In another embodiment, the kit for PCR comprises the described dye and/or quencher moiety, instructions for conjugating or labeling the dye and/or quencher moiety to a biomolecule, such as an oligonucleotide, instructions for conducting the PCR, and one or more of the following: a buffering agent, deoxynucleotide triphosphates (dNTPs), an organic solvent, an enzyme, enzyme cofactors, and an enzyme inhibitor.

[00114] In some embodiments, the systems, compositions, methods, and devices used for nucleic acid amplification comprise a “point-of-service” (POS) system. In some embodiments, samples may be collected and/or analyzed at a “point-of-care” (POC) location. In some embodiments, analysis at a POC location typically does not require specialized equipment and has rapid and easy-to-read visual results. In some embodiments, analysis can be performed in the field, in a home setting, and/or by a lay person not having specialized skills. In certain embodiments, for example, the analysis of a small-volume clinical sample may be completed using a POS system in a short period of time (e.g., within hours or minutes).

[00115] Optionally, a POS system is utilized at a location that is capable of providing a service (e.g., testing, monitoring, treatment, diagnosis, guidance, sample collection, verification of identity (ID verification), and other services) at or near the site or location of the subject. A service may be a medical service, or it may be a non-medical service. In some situations, a POS system provides a service at a predetermined location, such as a subject's home, school, or work, or at a grocery store, a drug store, a community center, a clinic, a doctor's office, a hospital, an outdoor triage tent, a makeshift hospital, a border check point, etc. A POS system can include one or more point of service devices, such as a portable virus/pathogen detector. In some embodiments, a POS system is a point of care system. In some embodiments, the POS system is suitable for use by non-specialized workers or personnel, such as nurses, police officers, civilian volunteers, or the patient.

[00116] In certain embodiments, a POC system is utilized at a location at which medical-related care (e.g., treatment, testing, monitoring, diagnosis, counseling, etc.) is provided. A POC may be, e.g., at a subject's home, work, or school, or at a grocery store, a community center, a drug store, a doctor's office, a clinic, a hospital, an outdoor triage tent, a makeshift hospital, a border check point, etc. A POC system is a system which may aid in, or may be used in, providing such medical-related care, and may be located at or near the site or location of the subject or the subject's health care provider (e.g., subject's home, work, or school, or at a grocery store, a community center, a drug store, a doctor's office, a clinic, a hospital, etc.).

[00117] In embodiments, a POS system is configured to accept a clinical sample obtained from a subject at the associated POS location. In embodiments, a POS system is further configured to analyze the clinical sample at the POS location. In embodiments, the clinical sample is a small volume clinical sample. In embodiments, the clinical sample is analyzed in a short period of time. In embodiments, the short period of time is determined with respect to the time at which sample analysis began. In embodiments, the short period of time is determined with respect to the time at which the sample was inserted into a device for the analysis of the sample. In embodiments, the short period of time is determined with respect to the time at which the sample was obtained from the subject.

[00118] In some embodiments, a POS system or a POC system can include the amplification-based methods, compositions and kits disclosed herein, including any of the described assays and/or assay panels. Such assays are contemplated for use with both thermal cycling amplification workflows and protocols, such as in PCR, as well as isothermal amplification workflows and protocols, such as in LAMP.

[00119] In some embodiments, a POS or a POC system comprises self-collection of a biological sample, such as a stool sample or rectal swab. In some embodiments, the selfcollection may comprise the use of a self-collection kit and/or device, such as a swab or a tube (e g., a stool collection tube or similar sample collection device). In some embodiments, the self-collection kit comprises instructions for use, including collection instructions, sample preparation or storage instructions, and/or shipping instructions. For example, the selfcollection kit and/or device may be used by an individual, such as lay person, not having specialized skills or medical expertise. In some embodiments, self-collection may be performed by the patient themselves or by any other individual in proximity to the patient, such as but not limited to a parent, a care giver, a teacher, a friend, or other family member. [00120] Notably, in some embodiments, the nucleic acid amplification protocol can be configured for rapid processing (e.g., in less than about 45 minutes) and high throughput, allowing for a minimally invasive method to quickly screen large numbers of individuals in a scalable way. This can be particularly useful to perform asymptomatic testing (e.g., high frequency/widespread testing at schools, workplaces, conventions, sporting events, large social gatherings, etc.) or for epidemiological purposes. The disclosed embodiments can also beneficially provide a lower cost sample collection system and method that enables selfcollection (reducing health care professional staffing needs) using a low-cost collection device. This eliminates the requirements for swabs, buffers, virus transmission media (or other specialized transport medium), and the like. The disclosed embodiments also allow for a reduction in Personal Protective Equipment (PPE) requirements and costs. There is also a beneficial reduced dependence on supply-constrained items, and the compatibility of these methods and kit components with existing equipment improves the flexibility and simplicity of their implementation to the masses. Overall, such embodiments allow for a less expensive assay that can be accomplished more quickly from sample collection through result generation.

[00121] In an exemplary embodiment, a method for determining the presence or absence of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample, comprises the steps of producing at least one amplicon by subjecting a reaction mixture including the sample and at least five primer pairs to reaction conditions suitable to amplify targeted nucleic acids wherein the at least five primer pairs include at least one primer pair A that specifically hybridizes with a portion of Campylobacter coli genome, at least one primer pair B that specifically hybridizes with a portion of Campylobacter jejuni genome, at least one primer pair C that specifically hybridizes with a portion of Campylobacter upsaliensis genome, at least one primer pair D that specifically hybridizes with a portion of Salmonella sp. genome, and at least one primer pair E that specifically hybridizes with a portion of Shigella sp./ElEC genome; and determining the presence or absence of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample based on the at least one amplicon.

[00122] In an example embodiment of the method, the producing of the at least one amplicon includes performing PCR. [00123] In an example embodiment of the method, the at least one amplicon is one selected from an amplicon produced using the primer pair A and comprising a sequence selected from the sequences specific for Campylobacter coli in “Probe” column of Figure 1, an amplicon produced using the primer pair B and comprising a sequence selected from the sequences specific for Campylobacter jejuni in “Probe” column of Figure 1, an amplicon produced using the primer pair C and comprising a sequence selected from the sequences specific for Campylobacter upsaliensis in “Probe” column of Figure 1, an amplicon produced using the primer pair D and comprising a sequence selected from the sequences specific for Salmonella sp. in “Probe” column of Figure 1, and/or an amplicon produced using the primer pair E and comprising a sequence selected from the sequences specific for Shigella sp./EIEC in “Probe” column of Figure 1.

[00124] In an example embodiment of the method, the reaction mixture includes probes specific for the at least one amplicon.

[00125] In an example embodiment of the method, the reaction mixture further comprises probes suitable for use with a specific forward and reverse primer pair, and the probes are selected from the probe sequences provided in Figure 1.

[00126] In an example embodiment of the method, the at least one primer pair A comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter coli in Figure 1, the at least one primer pair B comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter jejuni in Figure 1, the at least one primer pair C comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter upsaliensis in Figure 1, the at least one primer pair D comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Salmonella sp. in Figure 1, and the at least one primer pair E comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Shigella sp./EIEC in Figure 1.

[00127] In an example embodiment of the method, the reaction mixture further comprises probes suitable for use with said primer pairs in Figure 1. [00128] In an example embodiment of the method, the at least one primer pair A comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 1; the at least one primer pair B comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 2; the at least one primer pair C comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 3; the at least one primer pair D comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 4; and/or the at least one primer pair E comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 5.

[00129] In an example embodiment of the method, the reaction mixture further contains a control sample and a primer pair F that specifically hybridizes with a portion of control sample genome, and, optionally, a probe specific for target nucleic acid of the control sample.

[00130] In an example embodiment of the method, the primer pair F comprises a forward and reverse primer pair selected from Table 6, and the probe specific for the target nucleic acid of the control sample is selected from Table 6.

[00131] In an example embodiment of the method, the probes each include a fluorescent reporter.

[00132] In an example embodiment of the method, the probes each includes a quencher.

[00133] In an example embodiment of the method, each of the probes is labeled at or near the 5’ end with a dye selected from Alexa Fluor, ABY, VIC, JUN, FAM and 6FAM.

[00134] In an example embodiment of the method, each of the probes is labeled at the 3’ end with a quencher selected from QSY, MGBNFQ, BHQ, and DFQ.

[00135] In an example embodiment of the method, the reaction mixture comprises probes specific for amplicons produced using one or more of the at least one primer pair A, the at least one primer pair B and the at least one primer pair C, the probes being labeled with 6FAM, at least one probe specific for amplicons produced using the at least one primer pair D, the probes being labeled with VIC, and at least one probe specific for amplicons produced using the at least one primer pair E, the probe being labeled with ABY. [00136] In an example embodiment of the method, the probe specific for the target nucleic acid of the control sample is specific for amplicons produced using the primer pair F, the probe being labeled with JUN.

[00137] In an exemplary embodiment, a composition for determining the presence or absence of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample, comprises: at least five primer pairs, wherein the at least five primer pairs include at least one primer pair A that specifically hybridizes with a portion of Campylobacter coli genome, at least one primer pair B that specifically hybridizes with a portion of Campylobacter jejuni genome, at least one primer pair C that specifically hybridizes with a portion of Campylobacter upsaliensis genome, at least one primer pair D that specifically hybridizes with a portion of Salmonella sp. genome, and at least one primer pair E that specifically hybridizes with a portion of Shigella sp./EIEC genome.

[00138] In an example embodiment of the composition, the at least one primer pair A comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter coli in Figure 1, the at least one primer pair B comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter jejuni in Figure 1, the at least one primer pair C comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter upsaliensis in Figure 1, the at least one primer pair D comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Salmonella sp. in Figure 1, and the at least one primer pair E comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Shigella sp./EIEC in Figure 1.

[00139] In an example embodiment of the composition, the composition further comprises probes specific for amplicons produced using one or more of the at least one primer pair A, the at least one primer pair B, the at least one primer pair C, the at least one primer pair D, and the at least one primer pair E.

[00140] In an example embodiment of the composition, the probes are suitable for use with a specific forward and reverse primer pair in Figure 1, and optionally, a probe in Figure 1. [00141] In an example embodiment of the composition, the composition further comprises a polymerase, a buffer, and nucleotides.

[00142] In an example embodiment of the composition, the composition is suitable for producing at least one amplicon when included in a reaction mixture that is subjected to reaction conditions suitable to amplify target nucleic acid of a sample in the reaction mixture.

[00143] In an example embodiment of the composition, the composition further comprises a primer pair F that includes a forward and reverse primer pair selected from Table 6; and optionally, at least one probe selected from Table 6.

[00144] In an example embodiment of the composition, the at least one probe includes a fluorescent reporter.

[00145] In an example embodiment of the composition, the at least one probe includes a quencher.

[00146] In an example embodiment of the composition, the at least one probe is labeled at or near the 5’ end with a dye selected from Alexa Fluor, ABY, VIC, JUN, FAM and 6FAM.

[00147] In an example embodiment of the composition, the at least one probe is labeled at the 3’ end with a quencher selected from QSY, MGBNFQ, BHQ, and DFQ.

[00148] In an example embodiment of the composition, the probes specific for amplicons produced using one or more of the primer pairs A, B and C are labeled with 6FAM, the probe specific for amplicons produced using the primer pair D is labeled with VIC, and the probe specific for amplicons produced using the primer pair E is labeled with ABY.

[00149] In an example embodiment of the composition, the probe specific for amplicons produced using the primer pair F is labeled with JUN.

[00150] In an exemplary embodiment, a kit for determining the presence or absence of Campylobacter sp., Salmonella sp., and Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample, comprising the composition described in the above embodiments.

[00151] In an example embodiment of the kit, the kit further comprises a control sample; a primer pair F that specifically hybridizes with a portion of control sample genome; and optionally, a probe that specifically hybridizes with target nucleic acid of a control sample. [00152] In an example embodiment of the kit, the primer pair F comprises a forward and reverse primer pair selected from Table 6 and the probe sequence is selected from Table 6.

[00153] In an example embodiment of the kit, the at least five primer pairs and probe set comprise at least one set of the primer pairs and probe as provided in a Table 7.

[00154] In an example embodiment of the kit, the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 8.

[00155] In an example embodiment of the kit, the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 9.

[00156] In an example embodiment of the kit, the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 10.

[00157] In an example embodiment of the kit, the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 11.

[00158] In an example embodiment of the kit, the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 12.

[00159] The foregoing is illustrative of example or exemplary embodiments and is not to be construed as limiting thereof. Although a few example embodiments have been described, those skilled in the art will readily appreciate that many modifications are possible in example embodiments without materially departing from the novel teachings. Accordingly, all such modifications are intended to be included within the scope of the disclosure as defined in the claims. Therefore, it is to be understood that the foregoing is illustrative of various example embodiments and is not to be construed as limited to the specific embodiments disclosed, and that modifications to the disclosed embodiments, as well as other embodiments, are intended to be included within the scope of the appended claims.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.