TECHNICAL FIELD

The present invention relates generally to the detection of specific DNA amplicons generated from a polymerase chain reaction (PCR) or reverse-transcriptase polymerase chain reaction (RT-PCR) sample by using Sybr Green without performing DNA separation using agarose gel electrophoresis.

BACKGROUND ART

Developing a reliable and accurate technique for rapid detection of specific DNA or cDNA remains to be one of the most important challenges in molecular biology. Some application of this platform includes identifying or detecting pathogens (virus or bacteria) and also pests. Polymerase chain reaction is one of the most applied tools that has been applied in the field of diagnostics (Frey & Pfunder 2006). While DNA sequencing has been applied as a tool for species identification due to its specificity and accuracy (Winder et al. 2005; Ball & Armstrong 2006; Pereira, Carneiro, & Amorim 2008), this technique remains laborious, costly and unsuitable for large sample size (Frey & Pfunder 2006).

Another type of known technique described as real-time or quantitative PCR (RT- or qPCR) is based on a real-time monitoring of specific DNA which accumulates in every cycle allowing quantification and detection of specific DNA sequences. Quantitative PCR applies a fluorescent dye which intercalates with double-stranded DNA or a modified DNA oligonucleotide (DNA probe) that fluoresces when hybridised with complementary DNA (VanGuilder, Vrana, & Freeman 2008). This method decreases post-processing steps and reduces potential experimental error (VanGuilder, Vrana, & Freeman 2008). The use of intercalating dyes having the ability to bind to double stranded DNA are inexpensive, simple to use (not sequence specific) and can be used for any reaction or type of molecular test (VanGuilder, Vrana, & Freeman 2008). However, the use of DNA base probes remains expensive and requires further optimization to be used as it is highly dependent on obtaining substantial sequence information to design the custom probe and different probes are requited for each target DNA when used in a multiplex format (Pereira, Carneiro, & Amorim 2008; VanGuilder, Vrana, & Freeman 2008).

Melt peak method and analyses, is based on an observation of the mechanism of these non-specific DNA-binding dyes. These dyes will fluoresce when intercalated with double-stranded DNA, but not in the form of single-stranded DNA (Reed, Kent, & Wittwer 2007). Melt peak method is mostly used in the end of the qPCR cycle to indicate a specific DNA product (target DNA or expected PCR product) exists to differentiate it from possible non-specific amplification (VanGuilder, Vrana, & Freeman 2008).

During PCR, the concentration of double-stranded amplicons increases during the thermocycling cycle which involves DNA duplex to undergo disassociation (or melts). In return, deactivates the fluorescent dye molecules as the characteristic of the dye which only binds to double-stranded DNA. The decrease of fluorescence is then plotted against increasing temperature thus producing a melt curve. In return, a chart representing melt peak(s) is derived by plotting the negative derivative of fluorescence vs. temperature. The observation of the melt profile will define the specific existence of the PCR product (s). Compared to PCR based on amplification and separation of DNA by agarose gel electrophoresis (PCR-AGE), detection of specific DNA or PCR product(s) is solely based on the approximate amplicon size. The practice if melt peak analysis offers rapid identification of target DNA as AGE analysis is not required.

Although the fluorescent dye Sybr Green can inhibit PCR reaction and used in melt peak analysis (Zipper et al. 2004), the dye has to be applied at subsaturating concentration (Reed, Kent, & Wittwer 2007). The lack of dye saturation will cause a risk based on published reports causing ‘dye jumping’ where the dye released from the melting DNA duplex may get re incorporated into regions of doublestranded which have not melted (Reed, Kent, & Witter 2007).

The characteristics of the PCR product and its relationship to the melt temperature profile is primarily based on the nucleotide sequence, ratio of GC/AT and length of the target DNA (Ririe, Rasmussen, & Wittwer 1997; Varga & James 2006). There are also other parameters which may affect the melt temperature such as the dye and its concentration (Gudnason et al. 2007), settings used for the melt run (Varga & James 2006) and also capability of the qPCR machine itself (Herrmann et al. 2006). It was speculated that the fluorescence profile and precision of melt temperature control may be insufficient of produce variability on many standard qPCR instruments thus reducing the robustness for discrimination of PCR amplicons (Herrmann et al. 2006). A better solution to overcome this problem is by introducing another technique called high-resolution melt-curve analysis (HRM) that enables the discrimination of PCR amplicons used for the application of single nucleotide polymorphisms (SNPs) (Norambuena et al. 2009; Vossen et al. 2009).

However, Lipsky et al. (2001) was able to demonstrate by detection of three SNPs and a single-base deletion/insertion in human DNA using melt peak analysis on a standard qPCR equipment. Standard melt peak analysis has also been demonstrated by Berry and Sarre (2007) for discriminating mammalian carnivores. Therefore, a standard melt peak analysis is still reliable to ensure the specificity of the amplicon and thus further verified by the introduction of a positive PCR control as a ‘profile marker’.

SUMMARY OF THE INVENTION

The present invention is defined in the appended independent claims. Some optional features of the present invention are defined in the appended dependent claims. According to one aspect of the present invention, there is provided a method forthe detection of specific DNA in a test sample, wherein the method comprises:

(a) the final concentration of the Sybr Green in the PCR mix is prepared from a 10X Sybr Green stock that does not inhibit Taq DNA polymerase at the minimum concentration of 0.5 Units under 1X Sybr Green in a final 10-20 pl PCR reaction;

(b) the assay is done by testing on a positive control DNA with the ability of the Sybr Green PCR to detect the control DNA from 10 ⁶ copy numbers to at least 1 copy number (or depending on the annealing ability of the primer); wherein the minimum amount of effective MgCl2 is set at a threshold of 2 mM, and the minimum amount of primer is 0.2 pM.

In a preferred embodiment, the method further further comprises the DNA amplification and detection steps that are conducted via a thermocycling proses and detection by melt peak analysis, the amplification and detection steps comprising:

(a) adding the Sybr Green PCR mixture in a 0.2 ml PCR tube;

(b) reacting the mixture in a conventional thermocycler equipment, which comprises a profile of denaturation, annealing and elongation condition conducted between 35-40 cycles, wherein a positive and negative PCR control is included in the process;

(c) after completion of the PCR, the tube containing the Sybr Green PCR reaction mixture is furthertransferred to a qPCR equipment and a melt-curve analysis is conducted by detecting the fluorescence of the labelled DNA by Sybr Green at temperature range from 50-95°C wherein the chosen green channel is: source: 470 nm I detector: 510 nm to detect the change of Sybr Green mixture profile; wherein; the detection of the target DNA is compared by the melt peak with a positive and negative control PCR to allow discrimination of the target DNA within the test sample and the control sample thus allowing proper validation, and the interpretation of a positive sample is based on detecting a similar melt-curve temperature within the sample and a specific physical melt curve when compared to the positive control (with an expected + 0.5°C), wherein the negative control must not have a specific melt curve similar to the positive control as this indicates external DNA contamination.

Advantageously, the Sybr Green used in the PCR process, possess the ability to not inhibit the PCR reaction at a minimum specified unit of activity. This will further prevent the event of ‘dye jumping’ and reduce the risk of uncertainty. As the objective of the Sybr Green PCR is to detect the existence of specific amplified DNA in the sample, no ‘real-time’ monitoring is required.

Advantageously, the time of detecting and reporting of the test sample is approximately 10-15 minutes thus providing a rapid detection when compared to AGE (approximately 45 minutes by DNA separation) or even qPCR (approximately 90 minutes by real-time monitoring of samples). The transfer of liquid in the Sybr Green PCR occurs only during the preparation of the PCR mix, this eliminates further liquid transfer and issue of contamination when compared to AGE by reducing an extra procedure.

BREIF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the Sybr Green PCR method will now be described by way of example with reference to the accompanying figures in which:

Figure 1 (a) is a description of melt curve for sensitivity of Sybr Green PCR under 1X Sybr Green that does not inhibit Taq DNA polymerase (at 0.5 units) to detect the positive control DNA of African Swine Fever (ASF) virus from 10 ⁶ DNA copy numbertol O’ ¹ copy number using the method of King et al. 2003.

Figure 1 (b) is a description of melt data and melt-curve analysis for sensitivity of Sybr Green PCR under 1X Sybr Green that does not inhibit Taq DNA polymerase (at 0.05 units) to detect the positive control DNA of African Swine Fever (ASF) virus from 10 ⁶ DNA copy number to10’ ¹ copy number using the method of King et al. 2003.

Figure 2(a) is a description of melt curve of Sybr Green PCR under 1X Sybr Green that does not inhibit Taq DNA polymerase (at 0.05 units) to detect the positive control DNA of African Swine Fever (ASF) virus from 10 ⁶ DNA copy number to10’ ¹ copy number using the method of Tignon et al. 2011 .

Figure 2(b) is a description of melt data and melt-curve analysis for sensitivity of Sybr Green PCR under 1X Sybr Green that does not inhibit Taq DNA polymerase (at 0.05 units) to detect the positive control DNA of African Swine Fever (ASF) virus from 10 ⁶ DNA copy number to10’ ¹ copy number using the method of Tignon et al. 201 1 .

Figure 3(a) is a description of melt curve for Sybr Green PCR under 1X Sybr Green that does not inhibit Taq DNA polymerase (at 0.05 units) to identify isolated Salmonella spp. using the method of Oliveira et al. 2002.

Figure 3(b) is a description of melt data and melt-curve analysis for using Sybr Green PCR under 1X Sybr Green that does not inhibit Taq DNA polymerase (at 0.05 units) to identify isolated Salmonella spp. using the method of Oliveira et al. 2002. Figure 4(a) is a description of melt curve for reverse-transcriptase Sybr Green PCR under 1X Sybr Green that does not inhibit Taq DNA polymerase (at 0.05 units) to detect Newcastle disease virus RNA using the method of Kianizadeh et al. 1999.

Figure 4(b) is a description of melt data and melt-curve analysis for using Sybr Green PCR under 1X Sybr Green that does not inhibit Taq DNA polymerase (at 0.05 units) to to detect Newcastle disease virus RNA using the method of Kianizadeh et al. 1999.

DETAILED DESCRIPTION OF THE PREFFERED EMBODIMENT

Before the present methods for Sybr green PCR are disclosed and described, it is to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

This invention relates generally to detect specific DNA or cDNA using a Sybr Green polymerase chain reaction (PCR) mixture, the PCR is conducted in a conventional thermocycler instrument followed by analysing via melt peak profile using a quantitative PCR (qPCR) instrument. More specifically, the present invention relates to observing the melt curve profile by comparing it to the profile of the positive control reaction allowing discrimination of the generated amplicon(s) without detection by DNA separation using agarose gel electrophoresis (AGE).

According to the present invention, the Sybr Green used in the PCR process, possess the ability to not inhibit the PCR reaction at a minimum specified unit of activity. This will further prevent the event of ‘dye jumping’ and reduce the risk of uncertainty. As the objective of the Sybr Green PCR is to detect the existence of specific amplified DNA in the sample, no ‘real-time’ monitoring is required. The time of detecting and reporting of the test sample is approximately 10-15 minutes thus providing a rapid detection when compared to AGE (approximately 45 minutes by DNA separation) or even qPCR (approximately 90 minutes by real-time monitoring of samples). The transfer of liquid in the Sybr Green PCR occurs only during the preparation of the PCR mix, this eliminates further liquid transfer and issue of contamination when compared to AGE by reducing an extra procedure.

EXAMPLE 1

Figure 1 (a) and 1 (b), depicts the ability of the primers, Taq DNA polymerase and 1X Sybr Green mixture to detect the positive ASF control DNA from 10 ⁶ to 10' ¹ copy numbers at a final concentration of 2 mM MgCl2. The positive DNA fragment (250 bp) was extracted from an agarose gel and purified, quantified and converted to a unit of ‘copy number’ which depicts the physical amount of the DNA in the solution. To test the sensitivity of Sybr Green PCR, a test was conducted to detect the ASF DNA from 10 ⁶ to 10' ¹ copy numbers under the condition of 1X PCR buffer, 1X Sybr Green, 2 mM MgCl2, 0.2 pM primers (forward and reverse), 0.2 mM dNTP and 0.05 Unit of Taq DNA polymerase in a final 20 pl volume (containing 1 pl of DNA). The Sybr Green PCR also included a non-template control (negative control).

This PCR reaction is carried out in a conventional thermocycler. The PCR condition consisted of denaturation at 95°C for 3 minutes, followed by 40 cycles of pre-denaturation (95°C, 20 seconds), annealing (50°C, 20 seconds) and elongation (72C°, 20 seconds). Next, the tubes containing the Sybr Green PCR reactions are transferred into a qPCR equipment. The pre-installed melt-curve program was chosen with the temperature range was set to 50-95°C and the green channel was selected for detection.

The positive ASF control DNA used by King et al. (2003), represents a DNA encoding the VP72 gene with a size of 250 bp and represented as a melt-curve with a peak at 85° (+0.5°C). The non-template control (negative control) does not show any melt-curve similar to the positive ASF control DNA. This demonstrates the ability of the Sybr Green PCR to differentiate between a positive control DNA reaction and a negative control reaction. The sensitivity of the Sybr Green PCR indicated its ability to detect the mentioned target DNA to a minimum 10' ¹ copy numbers without any evidence of reaction inhibition.

EXAMPLE 2

Figure 2(a) and 2(b), depicts the ability of the primers, Taq DNA polymerase and 1X Sybr Green mixture to detect the positive ASF control DNA from 10 ⁶ to 10' ¹ copy numbers at a final concentration of 2 mM MgCl2. The positive DNA fragment (159 bp) was extracted from an agarose gel and purified, quantified and converted to a unit of ‘copy number’ which depicts the physical amount of the DNA in the solution. To test the sensitivity of Sybr Green PCR, a test was conducted to detect the ASF DNA from 10 ⁶ to 10' ¹ copy numbers under the condition of 1X PCR buffer, 1X Sybr Green, 2 mM MgCl2, 0.2 pM primers (forward and reverse), 0.2 mM dNTP and 0.05 Unit of Taq DNA polymerase in a final 20 pl volume (containing 1 pl of DNA). The Sybr Green PCR also included a non-template control (negative control).

This PCR reaction is carried out in a conventional thermocycler. The PCR condition consisted of denaturation at 95°C for 3 minutes, followed by 40 cycles of pre-denaturation (95°C, 20 seconds), annealing (50°C, 20 seconds) and elongation (72°C, 20 seconds). Next, the tubes containing the Sybr Green PCR reactions are transferred into a qPCR equipment. The pre-installed melt-curve program was chosen with the temperature range was set to 50-95°C and the green channel was selected for detection.

The positive ASF control DNA used by Tignon et al. (2011), represents a DNA encoding the VP72 gene with a size of 159 bp and represented as a melt-curve with a peak at 84.5° (+0.5°C). The non-template control (negative control) does not show any melt-curve similar to the positive ASF control DNA. This demonstrates the ability of the Sybr Green PCR to differentiate between a positive control DNA reaction and a negative control reaction. The sensitivity of the Sybr Green PCR indicated its ability to detect the mentioned target DNA to a minimum 10’ ¹ copy numbers without any evidence of reaction inhibition.

EXAMPLE 3

Figure 3(a) and 3(b), depicts the ability of the primers, Taq DNA polymerase and 1X Sybr Green mixture to identify 14 isolated Salmonella spp. at a final concentration of 2 mM MgCh The suspected colonies were cultured overnight in bacto-peptone water broth (2.0 ml) and incubated at 37°C overnight. The next day, 5 pl of the overnight culture was added to a 0.2 ml PCR tube containing 45 pl of sterile water. The lysis process was carried out by incubating the tubes in a thermocycler. The tubes were heated to 85°C for 15 minutes and cooled down to room temperature. To test the capability of Sybr Green PCR for the identification Salmonella isolates, the reaction condition consisted of; 1X PCR buffer, 1X Sybr Green, 2 mM MgCl2, 0.2 pM primers (forward and reverse), 0.2 mM dNTP and 0.05 Unit of Taq DNA polymerase in a final 20 pl volume (containing 2 pl of DNA). A positive Salmonella DNA control and non-template control (negative control) is added in the test.

This PCR reaction is carried out in a conventional thermocycler. The PCR condition consisted of denaturation at 95°C for 3 minutes, followed by 40 cycles of pre-denaturation (95°C, 20 seconds), annealing (55°C, 20 seconds) and elongation (72°C, 20 seconds). Next, the tubes containing the Sybr Green PCR reactions are transferred into a qPCR equipment. The pre-installed melt-curve program was chosen with the temperature range was set to 50-95°C and the green channel was selected for detection.

The positive Salmonella spp. control DNA used by Oliveira et al. (2002), represents a DNA encoding the InvA gene with a size of 258 bp and represented as a melt-curve with a peak at 87.2° (+0.5°C) when referring to the positive control. The non-template control (negative control) does not show any meltcurve similar to the positive Salmonella spp. control DNA. This demonstrates the ability of the Sybr Green PCR to differentiate between a positive control DNA reaction and a negative control reaction. In the 14 tested samples, 13 samples were detected positive for Salmonella spp and 1 sample was detected as negative Salmonella spp. This ability indicates that the Sybr Green PCR technique can differentiate between Salmonella spp. and non-Salmonella spp.

EXAMPLE 4

Figure 4(a) and 4(b), depicts the ability of the reverse-transcriptase (RT), primers, Taq DNA polymerase and 1X Sybr Green mixture to detect Newcastle disease virus (NDV) in an RT-Sybr Green PCR reaction. The test was conducted on a purified RNA isolated from a suspected NDV positive allantoic fluid. In a 0.2 ml PCR tube, total RNA (2 pl) was reverse-transcribed in a 10 pl reaction containing; 1X RT-Buffer, 0.4 |JM of primers (forward and reverse), 0.4 mM of dNTPs, 10 units of M-MuLV and 4 mM MgCl2. The tube is then incubated in a conventional thermocycler where the reaction takes place at 42°C for 30 minutes. To test the capability of Sybr Green PCR, a second reaction (10 pl) consisted of; 1X PCR buffer, 1X Sybr Green, and 0.05 Unit of Taq DNA polymerase was added to the previous tube in a final 20 pl. A positive NDV RNA control and non-template control (negative control) is added in the test.

This PCR reaction is carried out in a conventional thermocycler. The PCR condition consisted of denaturation at 95°C for 3 minutes, followed by 40 cycles of pre-denaturation (95°C, 20 seconds), annealing (57°C, 20 seconds) and elongation (72°C, 20 seconds). Next, the tubes containing the Sybr Green PCR reactions are transferred into a qPCR equipment. The pre-installed melt-curve program was chosen with the temperature range was set to 50-95°C and the green channel was selected for detection.

The positive NDV control DNA used by Kianizadeh et al. (1999), represents an amplicon with size of 242 bp and represented as a melt-curve with a peak at 88.3° (+0.5°C). The non-template control (negative control) does not show any melt-curve similar to the positive NDV control RNA. This demonstrates the ability of the Sybr Green PCR to differentiate a positive control DNA reaction and a negative control reaction. This method shows that the Sybr Green PCR method can be used to detect cDNA in an RT-PCR condition.