MAGNETIC NANOPARTICLES BASED NUCLEIC ACID EXTRACTION FROM BIOLOGICAL SAMPLES FOR MOLECULAR DIAGNOSTICS

FIELD OF THE INVENTION

[1] The present invention relates to a method of magnetic nanoparticles (MNPs) based nucleic acid extraction from biological fluids. Moreover, the invention relates to the use of MNPs and nucleic acid complex mixture directly for molecular diagnostics of various diseases.

BACKGROUND OF THE INVENTION

[2] The basic properties of nucleic acids have been extensively used in the development of various molecular biology techniques. These techniques are used to classify microorganisms and genetic predispositions, to identify various mutations and to establish their role in the resistance of antibiotics, to study phylogenetic relationships, etc. what all these approaches have in common is collecting a sample of purified nucleic acid.

[3] Since the isolation of nucleic acid is a starting point for a wide variety of downstream applications, the high quality of nucleic acid isolation from the samples is a critical factor in the performance of the subsequent phases of study. Nucleic acid extraction may therefore be characterized as a series of steps to obtain samples of nucleic acid/materials of particular purity that are free of impurities and are appropriate for various downstream application steps. The aim of nucleic acid extraction methods is to disintegrate the cell envelope and to achieve the full removal of lipids and proteins for the processing of pure DNA and/or RNA. This primarily focused on thermal adsorption of silica membranes/beads, anion exchange chromatography, sedimentation/precipitation and the use of magnetic particles. These techniques yield initial nucleic acid samples with differing purity and concentration based on the original sample (bacteria, viruses, tissues).

[4] The choice of approach - taking into account the optimum time/quality balance - depends on the intent of the research, the form of analysis, the type of nucleic acid and the expense. It is necessary to provide suitable conditions for the extraction of nucleic acid in order to prevent the degradation of nucleic acid due to oxidation by reactive oxygen species generated during in vivo respiration or, extracellularly, by mechanisms involving metal ions.

[5] What accounts for the differences between the methods for extraction of DNA and RNA is their different stability. RNA contains ribosome RNA (rRNA) 80%, mitochondrial RNA (mtRNA), messenger RNA (polyadenylate in eukaryotic cells) (mRNA) 1-5%, transfer RNA (tRNA) and microRNA molecules (miRNA). There can be varying concentrations of mRNA in the cells: from massive numbers to just five copies per cell. In fact, mRNA is the preferred RNA for reverse transcription and cDNA synthesis. RNA molecules are vulnerable to degradation by ozone in the air; ozone is extremely reactive regardless of whether the RNA sample is liquid or solid.

[6] Water is another element that plays a role in the degradation of the RNA, as it makes proton transfer possible and acts as a source of hydronium or hydroxyl ions. That's why dehydration has a beneficial role against RNA degradation. Nucleic acids, particularly RNA molecules, are also responsive to nucleases. In RNA extraction procedures, therefore, it is important to ensure an RNase-free fraction and a means of rapidly cooling the sample.

[7] The methods for preparation of nucleic acid samples can be grouped into thermal extraction, solution-based methods (homemade and commercial kits), column -based methods, and ones that use magnetic particles. [8] As a whole, solution-based methods, and especially their homemade versions, have the disadvantage that they use toxic reagents and, therefore, a fume hood is a must; moreover, precision in phase separation is difficult to achieve; the extracted nucleic acid samples are of lower purity (especially in homemade methods) compared to those obtained by column -based methods; the obtained samples have limited use - mainly for conventional PCR. Commercial kits and protocols, albeit validated for downstream applications, do not solve the problem of reagent toxicity and subjective factors related to the person who performs the procedure.

[9] In the present invention MNPs without any covalent modifications have been used for nucleic acid extraction. Conjugation of nucleic acids to be extracted, comprises of coordinate bond (Fe2+ cation is directly coordinated to the N(7) atom of guanine and indirectly, through water molecules, to the 0(6) atom of guanine and phosphate groups, thus forming an octahedral coordination complex of iron) and electrostatic interaction of the PEG coated magnetic nanoparticles with nucleic acid to be extracted. This interaction allows the method to be independent of the type of nucleic acid to be extracted. Moreover, this method requires minimal use of toxic reagents for extraction of nucleic acid. The process is beneficial by cutting down the labor, time and expertise needed for the extraction of nucleic acids.

OBJECTS OF THE INVENTION

[10] Accordingly, it is an object of the present invention is to extract nucleic acid (RNA/DNA (as a model we have used chikungunya virus nucleic acid)) from biological samples using MNPs.

[11] Still another object of the present invention is nucleic acid extraction from biological samples without any covalent modifications on the MNPs for interaction.

[12] Yet another object of the present invention is single step extraction of nucleic acid from various biological fluids. [13] Still another object of the present invention is single step extraction of nucleic acids from prokaryotic and eukaryotic cells.

[14] Yet another object of the present invention is single step extraction of nucleic acids from nanoparticles-nucleic acid complex using physical/mechanical or chemical treatment

[15] Still another object of the present invention is to use these extracted nucleic acids for molecular diagnostic techniques such as Polymerase Chain Reaction (PCR), microarray, etc.

[16] Yet another object of the present invention is to enhance stability of nucleic acids and preventing its degradation from nucleases during shelf storage and transport.

SUMMARY OF THE INVENTION

[17] The present invention describes the methodology for nucleic acid isolation by MNPs from biological fluids as well as from prokaryotic and eukaryotic cells. The method of the present invention requires no covalent modification for the conjugation of nucleic acid with MNPs and hence extraction of nucleic acid is not specific to the type of the nucleic acid to be extracted.

[18] In accordance to the present invention MNPs have been synthesized according to the synthesis protocol mentioned in the filed Patent Application in Indian Patent Office (IPO). Application No. 201911043317 Dated 24th October 2019 (Title of the Invention: Dual Stimuli Responsive Magnetic Nanoparticles for Sustained Drug Release; Applicant: Jawaharlal Nehru University; Inventors: Bhattacharya Jaydeep, Gandhi Harsh A., MondalTitas).

[19] According to the present invention the PEG coated MNPs are incubated with the lysed biological samples for extraction of nucleic acids. Moreover, this extraction method requires minimal use of toxic reagents for extraction of nucleic acid. Later on, this complex can be directly used for molecular diagnostic techniques such as PCR for detection of nucleic acid.

[20] Further, MNPs-Nucleic acid complex has enhanced stability and shelf life compared to nucleic acid preventing its degradation from nucleases. This can be further explored for transport and storage related problems of nucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

[21] The present invention may be better understood and its numerous objects, features and advantages made apparent to those skilled in the art, by referring to the accompanying drawings:

[22] FIGURE 1: PCR: Digital image of PCR products run on 2% (w/v) agarose gel stained with ethidium bromide Lane 1: lOObp Ladder Lane 3: PCR reaction

[23] FIGURE 2: PCR in presence on MNPs:Digital image of PCR products run on 2% (w/v) agarose gel stained with ethidium bromide Lane 1: lOObp Ladder Lane 3: PCR reaction Lane 4: PCR reaction with MNPs

[24] FIGURE 3: PCR in presence of different concentration of MNPs: Digital image of PCR products run on 2% (w/v) agarose gel stained with ethidium bromide Lane 1 : PCR reaction Lane 2: PCR reaction with 25mg/mL MNPs Lane 3: PCR reaction with 12.5mg/mL MNPs Lane 4: PCR reaction with 6.25mg/mL MNPs Lane 5: PCR reaction with 3.75mg/mL MNPs Lane 6: PCR reaction with 1.5mg/mL MNPs Lane 7: PCR reaction with 0.75mg/mL MNPs Lane 8: PCR reaction with 0.375mg/mL MNPs Lane 9: PCR reaction with 0.0375mg/mL MNPs

[25] FIGURE 4: PCR in presence of different concentration of MNPs with additives: Digital image of PCR products run on 2% (w/v) agarose gel stained with ethidium bromide Lane 1: PCR reaction Lane 2: PCR reaction with 25mg/mL MNPs Lane 3: PCR reaction with 12.5mg/mL MNPs Lane 4: PCR reaction with 6.25mg/mL MNPs Lane 5: PCR reaction with 3.75mg/mL MNPs Lane 6: PCR reaction with 1.5mg/mL MNPs Lane 7: PCR reaction with 0.75mg/mL MNPs Lane 8: PCR reaction with 0.375mg/mL MNPs Lane 9: PCR reaction with 0.0375mg/mL MNPs

[26] FIGURE 5: MNPs based DNA isolation and detection from spiked serum: Digital image of PCR products run on 2% (w/v) agarose gel stained with ethidium bromide Lane 1: lOObp Ladder Lane 3: 10 Copy no./mL Lane 4: 10 Copy no./mL Lane 5: 10 Copy no./mL Lane 6: lOCopy no./mL Lane 7: 1 Copy no./mL

[27] FIGURE 6: MNPs based DNA isolation and detection from spiked saliva: Digital image of PCR products run on 2% (w/v) agarose gel stained with ethidium bromide Lane

1: 107 Copy no./mL Lane 2: 105 Copy no./mL Lane 3: 103 Copy no./mL Lane 4: lOCopy no./mL Lane 5 : 1 Copy no./mL

[28] FIGURE 7: MNPs based DNA isolation and detection from spiked urine: Digital image of PCR products run on 2% (w/v) agarose gel stained with ethidium bromide Lane

1: 107 Copy no./mL Lane 2: 105 Copy no./mL Lane 3: 103 Copy no./mL Lane 4: lOCopy no./mL Lane 5 : 1 Copy no./mL

[29] FIGURE 8: MNPs based RNA isolation and detection from spiked serum: Digital image of PCR products run on 2% (w/v) agarose gel stained with ethidium bromide Lane 1: lOObp Ladder Lane 3: Normal Serum Lane 4: Spiked Serum Lane 5: Normal Serum Lane 6: Spiked Serum

[30] FIGURE 9: MNPs based pseudo virus isolation and detection from spiked serum: Digital image of PCR products run on 2% (w/v) agarose gel stained with ethidium bromide Lane 1: lOObp Ladder Lane 2: Spiked Serum Lane 3: Spiked Serum Lane 4: Spiked Serum Lane 5: Spiked Serum Lane 6: Spiked Serum Lane 7: Spiked Serum Lane 8: Spiked Serum Lane 9: Spiked Serum Lane 10: Spiked Serum Lane 11: Spiked Serum

DETAILED DESCRIPTION OF THE INVENTION [31] The The aspects of the present invention are achieved by the protocol mentioned as follows:

1. Synthesis of PEG coated MNPs:

[32] MNPs have been synthesized according to the synthesis protocol mentioned in the filed Patent Application in Indian Patent Office (IPO). Application No. 201911043317 Dated 24th October 2019 (Title of the Invention: Dual Stimuli Responsive Magnetic Nanoparticles for Sustained Drug Release; Applicant: Jawaharlal Nehru University; Inventors: Bhattacharya Jaydeep, Gandhi Harsh A., MondalTitas).

2. Preparation of Biological samples:

[33] Biological samples are pre -heated at 95oC for 5-10 minutes and then rapidly cooling at 4oC for 5-10 minutes to denature most of the protein contents present in the sample. Later on, depending on the sample type some of the mild chemical treatment or mechanical or physical melting can be done to lyse the biological components except nucleic acids of the sample.

3. Incubation of synthesized PEG coated MNPs with Biological Samples:

[34] The complex mixture obtained in step 2 has been incubated with PEG coated MNPs (0.25 mg) for 20-30 minutes and later on centrifuged to obtain a MNPs-Nucleic acid complex.

4. Using MNPs-Nucleic acid complex for molecular diagnostics:

[35] The complex obtained in step 3 is directly used for PCR. For PCR reaction in presence of MNPs, the reaction is carried out in presence of various additives such as 2.5 mM MgC12, 6 m g/m L BSA and 7.5% DMSO along with PCR components.

[36] The present invention is illustrated with examples below, which should in no way be construed as limiting the invention.

[37] EXAMPLE 1 Using CHIKV (chikungunya virus) nsp4 gene cloned in pGME-T easy cloning vector as template, thermal cycle has been performed using Bio-Rad Cl 000 Touch Thermal Cycler as follows: 1 cycle 95 o C for 5 minutes, 30 cycles 95 o C for 30 seconds, 51 o C for 30 seconds 72 o C for 30 seconds, 1 cycle 72 o C for 5 minutes and 4 o C hold. PCR product (243 bp) is analyzed using 2% w/v agarose gel electrophoresis (Bio Rad Gel Electrophoresis Unit) using IX TAE buffer and 0.3 pg/mL of ethidium bromide. The DNA bands were detected under UV (Bio-Rad Gel Doc System EQ) (FIGURE 1).

[38] EXAMPLE 2

PCR in presence of MNPs has been performed, where the reaction is carried in presence of various additives. PCR product is analyzed using 2% w/v agarose gel electrophoresis (Bio Rad Gel Electrophoresis Unit) using IX TAE buffer and 0.3 pg/mL of ethidium bromide. The DNA bands were detected under UV (Bio-Rad Gel Doc System EQ) (FIGURE 2, FIGURE 3, FIGURE 4).

[39] EXAMPLE 3

Isolation and detection of DNA from spiked serum, saliva, urine is carried in presence of MNPs (FIGURE 5, FIGURE 6, FIGURE 7).

[40] EXAMPLE 4

Isolation and detection of RNA from spiked serum is carried in presence of MNPs (FIGURE 8).

[41] EXAMPLE 5

Isolation and detection of pseudo virus from spiked serum is carried in presence of MNPs (FIGURE 9).

[42] EXAMPLE 6

Specificity, Sensitivity, Positive Predictive Value, Negative Predictive Value of the isolation and diagnostic method (TABLE 1). [43] Moreover, MNPs-Nucleic acid complex has enhanced stability and shelf life compared to nucleic acid preventing its degradation from nucleases. This can be further explored for transport and storage related problems of nucleic acids.

[44] While this invention has been described in certain embodiments, the present invention can be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the invention using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this invention pertains and which fall within the limits of the appended claims and their equivalents.

[45] Table 1: Specificity, Sensitivity, Positive Predictive Value, Negative Predictive Valueof the isolation and diagnostic method.

[46] CHIKV nsp4 Sequence:

>gb:JF272476|Organism:Chikungunya virus|Strain Name:IND-06-Guj|Protein Name:nonstructural protein P4|Gene Symbol:nsP4 TATATATTCTCGTCGGACACTGGTCCAGGTCATTTACAACAGAAGTCAGTACG

CCAGTCAGTGCTGCCGGTGAACACCCTGGAGGAAGTCCACGAGGAGAAGTGT

TACCCACCTAAGCTGGATGAAGCAAAGGAGCAACTATTACTTAAGAAACTCC

AGGAGAGTGCATCCATGGCCAACAGAAGCAGGTATCAGTCGCGCAAAGTAG

AAAACATGAAAGCAGCAATCATCCAGAGACTAAAGAGAGGCTGTAGACTAT

ACTTAATGTCAGAGACCCCAAAAGTCCCTACTTACCGGACTACATATCCGGC

GCCTGTGTACTCGCCTCCGATCAACGTCCGATTGTCCAATCCCGAGTCCGCAG

TGGCAGCATGCAATGAGTTCTTAGCTAGAAACTATCCAACTGTCTCATCATAC

CAAATTACCGACGAGTATGATGCATATCTAGACATGGTGGACGGGTCGGAGA

GTTGCCTGGACCGAGCGACATTCAATCCGTCAAAACTCAGGAGCTACCCGAA

ACAGCACGCTTACCACGCGCCCTCCATCAGAAGCGCTGTACCGTCCCCATTCC

AGAACACACTACAGAATGTACTGGCAGCAGCCACGAAAAGAAACTGCAACG

TCACACAGATGAGGGAATTACCCACTTTGGACTCAGCAGTATTCAACGTGGA

GTGTTTCAAAAAATTCGCATGCAACCAAGAATACTGGGAAGAATTTGCTGCC

AGCCCT ATT AGG AT A AC A ACTG AG A ATTT AGC A ACCT ATGTT ACT AA ACT A A

AAGGGCCAAAAGCAGCAGCGCTATTCGCAAAAACCCATAATCTACTGCCACT

ACAGGAAGTACCAATGGATAGGTTCACAGTAGATATGAAAAGGGACGTGAA

GGTGACTCCTGGTACAAAGCATACAGAGGAAAGACCTAAGGTGCAGGTTATA

CAGGCGGCTGAACCCTTGGCGACAGCATACCTATGTGGGATTCACAGAGAGC

TGGTTAGGAGGCTGAACGCCGTCCTCCTACCCAATGTACATACACTATTTGAC

ATGTCTGCCGAGGATTTCGATGCCATCATAGCCGCACACTTTAAGCCAGGAG

ACACTGTTTTGGAAACGGACATAGCCTCCTTTGATAAGAGCCAAGATGATTC

ACTTGCGCTTACTGCTTTGATGCTGTTAGAGGATTTAGGGGTGGATCACTCCC

TGCTGGACTTGATAGAGGCTGCTTTCGGAGAGATTTCCAGCTGTCACCTACCG

ACAGGTACGCGCTTCAAGTTCGGCGCCATGATGAAATCAGGTATGTTCCTAA

CTCTGTTCGTCAACACATTGTTAAACATCACCATCGCCAGCCGAGTGCTGGAA

GATCGTCTGACAAAATCCGCGTGCGCGGCCTTCATCGGCGACGACAACATAA

TACATGGAGTCGTCTCCGATGAATTGATGGCAGCCAGATGTGCCACTTGGAT

GAACATGGAAGTGAAGATCATAGATGCAGTTGTATCCTTGAAAGCCCCTTAC

TTTTGTGGAGGGTTTATACTGCACGATACTGTGACAGGAACAGCTTGCAGAG TGGCAGACCCGCTAAAAAGGCTTTTTAAACTGGGCAAACCGCTAGCGGCAGG

TGACGAACAAGATGAAGATAGAAGACGAGCGCTGGCTGACGAAGTGATCAG

ATGGCAACGAACAGGGCTAATTGATGAGCTGGAGAAAGCGGTATACTCTAGG

TACGAAGTGCAGGGTATATCAGTTGTGGTAATGTCCATGGCCACCTTTGCAA

GCTCCAGATCCAACTTCGAGAAGCTCAGAGGACCCGTCATAACTTTGTACGG

CGGTCCTAAA

[47] pGEM-T Easy Vector Sequence:

>pGEM-T Easy Vector

GGGCGAATTGGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGG

AATTCGATnATCACTAGTGAATTCGCGGCCGCCTGCAGGTCGACCATATGGGA

GAGCTCCCAACGCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAA

TAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGC

TCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGG

TGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTT

TCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGC

GGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACT

CGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGC

GGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTG

AGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGC

_{GTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCA}

AGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCC

CTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC

CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG

TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACG

AACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAG

TCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACA

GGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTG

GCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGA

AGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAAC CACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAA

AAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG

TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGA

TCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGT

ATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCAC

CTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCG

TGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAAT

GATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAG

CCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCA

TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAAT

AGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTC

GTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACAT

GATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTT

GTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGC

ATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAG

TACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTG

CCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTG

CTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCT

GTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCAT

CTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGC

CGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTC

_{CTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATA}

CAT ATTTG A ATGTATTT AG A A A A AT A A AC A A AT AGGGGTTCCGCGC AC ATTT

CCCCGAAAAGTGCCACCTGATGCGGTGTGAAATACCGCACAGATGCGTAAGG

AGAAAATACCGCATCAGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGC

GTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCA

AAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCC

AGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGCTCCAACGTCAAAGGGC

GAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATC

AAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGG

AGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAG GAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCG

GTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGG

GCGCGTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTG

CGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGC

GATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGAC

GGCC AGTG A ATTGT A AT ACG ACTC ACT AT A

[48] Primer Sequence:

CHIKV nsp4 forward: CGGAGAGATTTCCAGCTGTC CHIKV nsp4 reverse: ATGTTCATCCAAGTGGCACA