A specific detection of 16s rII group of areca yellowing plant pathogen qPCR primer probe combination, kit and method

Abstract

This invention discloses a qPCR primer-probe combination, kit, and method for specifically detecting 16SrII group areca etiolated phytoplasma, belonging to the interdisciplinary field of molecular biology and plant pathology. The primer-probe combination includes a forward primer qTuf-F4-2 (SEQ ID NO:1), a reverse primer qTuf-R4 (SEQ ID NO:2), and a fluorescent probe qTuf-P4-1 (SEQ ID NO:3), targeting 16SrII group phytoplasma. tuf This gene conserved region demonstrates high specificity for 16SrII group areca yellowing phytoplasma in qPCR reactions, exhibiting no cross-reactivity with 16SrI group, 16SrXXXII group phytoplasma, areca genome, common pathogens, or endophytes. The limit of detection is 14.35 copies / μL, and the sensitivity meets the requirements for detecting low-load samples. The coefficient of variation for intra- and inter-group repeatability is less than 1%, indicating good stability. The kit and detection method developed based on this combination are simple to operate, highly sensitive, and provide stable results, making them suitable for the accurate diagnosis, pathogen typing, and field epidemic monitoring of areca yellowing disease, providing key technical support for the scientific control of 16SrII group phytoplasma.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of molecular biology and plant pathology. Specifically, it relates to a qPCR primer and probe combination, kit, and method for the specific detection of 16SrII group areca etiolated phytoplasma. Background Technology

[0002] Areca nut, as an important tropical economic crop in my country, holds an irreplaceable core position in the agricultural economic structure of Hainan Province. However, areca palm yellow leaf disease (AYLD), caused by phytoplasma infection, has spread rapidly in recent years, becoming a devastating disease that seriously restricts the sustainable development of the areca nut industry. Molecular biological studies have clearly shown that areca palm yellow leaf disease in my country is mainly caused by phytoplasmas from multiple phylogenetic groups, including the 16SrI group, the 16SrII group, and the 16SrXXXII group. These different groups of phytoplasmas differ in terms of host range, genetic variation, and pathogenicity. For example, Che et al. (2024) found that in Hainan Island, the 16SrII group of phytoplasmas has a wide host range, richer genetic variation, and more diverse pathogenic types, making it the absolute dominant group of phytoplasmas in Hainan Province and posing a greater threat to tropical agricultural security; while the 16SrI group of phytoplasmas has a limited host range, concentrated distribution, and more specific symptoms. Therefore, accurately distinguishing different phytoplasm groups, especially the specific identification of the 16SrII group, is a key prerequisite for achieving early warning, dynamic monitoring, risk assessment, and scientific prevention and control by region and category.

[0003] Currently, domestic detection techniques for areca nut yellowing disease are mainly used for specific detection of the 16SrI group, or for broad-spectrum detection of the 16SrI, 16SrII, and 16SrXXXII groups simultaneously. Meng Xiuli et al. (2022) established a nested PCR detection method, but due to sensitivity limitations, this method has not been widely used in YLD detection. Che Haiyan (2010) established a TaqMan probe-based real-time fluorescent quantitative PCR (qPCR) primer-probe combination detection method, but this technique has low sensitivity (1.16 × 10⁻⁶). 4 The probe has a specificity of copies / μL for 16SrI group phytoplasmas and can be distinguished from phytoplasmas of other groups. Yu et al. (2020) established a loop-mediated isothermal amplification (LAMP) detection method based on 16SrI group phytoplasmas. This LAMP primer set has good specificity and stability, with a detection limit of 5.3 × 10⁻⁶. 1copies / μL. Yu et al. (2022) based on the 16SrI-B subgroup of *Onion xanthophytoplasma* strain OY-M. tuf A digital droplet PCR (ddPCR) detection method for areca nut yellowing phytoplasma was established using gene sequence-designed primers Atf / Atr and probe AtProbe. This detection method has a minimum detection limit of 0.07 copies / μL for phytoplasma, but specificity assessment was not observed. Although this technique has high sensitivity, its high cost and expensive equipment limit its widespread use. Lin Zhaowei et al. established qPCR detection methods for areca nut yellowing phytoplasma using the TaqMan probe in 2023 and 2024, respectively, but these methods primarily target broad-spectrum detection of areca nut yellowing phytoplasma (16SrI, 16SrII, and 16SrXXXII groups) and specific detection of the 16SrI group. Zhu Xiaoqiong et al. (2024) invented a method for detecting areca yellowing phytoplasma based on the CRISPR-Cas12a method. This method can detect 16SrI group phytoplasma, but not 16SrXXXII group phytoplasma, and the detection limit is 2.29 × 10⁻⁶. 2 Copies / μL. Ge et al. (2025) established a nested PCR detection method that can detect 16SrI and 16SrII phytoplasmas. This method is a broad-spectrum detection technique, but its sensitivity is relatively low, with a detection rate of 7.5 × 10⁻⁶ for 16SrI and 16SrII phytoplasmas. −7 ng / μL, 4×10 −7 ng / μL. The invention of Tang Qinghua et al. (2025) shows that the RPA-CRISPRCas12a-based detection method is only specific to 16SrI group areca xanthophytoplasma.

[0004] Crucially, to date, no publicly reported qPCR primer-probe combination simultaneously meets the following core requirements: (i) high specificity for 16SrII group areca nut etiolated phytoplasma, without cross-reactivity with 16SrI, 16SrXXXII, and other common plant pathogens; and (ii) high sensitivity suitable for detection in latent or low-load samples. This technological gap directly hinders the accurate assessment of the actual occurrence frequency, distribution range, and dynamic changes of 16SrII group phytoplasma in the field, thus severely impeding the scientific formulation and effective implementation of targeted control measures (such as precise control of vector insects, regional distribution of disease-resistant varieties, and strategies for clearing diseased plants).

[0005] Therefore, there is an urgent need to develop a real-time quantitative PCR (qPCR) detection system based on the specific gene sequence of 16SrII phytoplasma, which is highly specific, sensitive, reproducible, and suitable for field application, to provide reliable technical support for the accurate diagnosis and regional management of areca nut yellowing disease. Summary of the Invention

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides a real-time quantitative PCR primer and probe combination for the specific detection of 16SrII group areca xanthophylloides. The real-time quantitative PCR primer and probe combination consists of a forward primer qTuf-F4-2 with nucleotide sequence as shown in SEQ ID NO:1, a reverse primer qTuf-R4 with nucleotide sequence as shown in SEQ ID NO:2, and a fluorescent probe qTuf-P4-1 with nucleotide sequence as shown in SEQ ID NO:3.

[0007] Furthermore, the fluorescent probe qTuf-P4-1 is labeled with a FAM fluorescent reporter group at its 5' end and with MBG at its 3' end.

[0008] Furthermore, the primer-probe combination is based on the 16SrII group of areca yellow phytoplasma. tuf The conserved regions of the gene were designed and synthesized. tuf The conserved regions of the gene are shown in SEQ ID NO:4.

[0009] The present invention also provides applications of the primer-probe combination, wherein the application is any of the following: (1) Application of specific detection in 16SrII group of areca nut yellowing phytoplasma; (2) Application in the preparation of products that specifically detect 16SrII group areca xanthophyta.

[0010] The present invention also provides a kit for the specific detection of 16SrII group areca xanthophyta, the kit comprising: 2×SuperReal PreMix, the primer and probe combination, DNA template, 50×ROX reference dye, and ddH2O.

[0011] The present invention also provides the application of the kit in the specific detection of 16SrII group areca catechu yellow phytoplasma.

[0012] The present invention also provides a method for specifically detecting 16SrII group areca xanthophytum, using the aforementioned kit.

[0013] Furthermore, the method includes the following steps: S1. Sample processing: Total DNA was extracted from the sample to be tested using a nucleic acid extraction kit; S2. Real-time quantitative PCR: Using the DNA as a template, perform real-time quantitative PCR reaction using the kit. S3. Result Detection: The result is determined based on the Ct value corresponding to the amplification curve plotted by the software of the real-time fluorescence quantitative PCR instrument. When the Ct value of the fluorescent group FAM detection channel is ≤38 and a specific amplification curve appears, the sample is determined to be positive for Areca catechu yellow phytoplasma in group 16SrII; when the Ct value of the fluorescent group FAM detection channel is >38 or no specific amplification curve appears, the sample is determined to be negative for Areca catechu yellow phytoplasma in group 16SrII.

[0014] Furthermore, the reaction system for the real-time quantitative PCR reaction is as follows: 10 μL of 2×SuperReal PreMix, 0.6 μL each of forward and reverse primers (10 μM), 0.4 μL of fluorescent probe (10 μM), 2 μL of DNA template, 0.2 μL of 50×ROX reference dye, and ddH2O added to make up to 20 μL.

[0015] Furthermore, the amplification program for the real-time quantitative PCR reaction is as follows: pre-denaturation at 95°C for 15 minutes, followed by 40 cycles, each cycle consisting of denaturation at 95°C for 3 seconds, annealing at 59°C, and extension for 30 seconds.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention is based on 16SrII group areca xanthophytoplasma. tuf Based on the specific sequences of the gene, a set of highly specific, highly sensitive, and highly reproducible real-time quantitative PCR primer and probe combinations and corresponding detection methods were developed. This method achieves a detection limit as low as 14.35 copies / μL for 16SrII group phytoplasmas, with an amplification efficiency of 90.01% and a correlation coefficient... R 2 The effective value was 0.9925, and there was no cross-reactivity with 16SrI and 16SrXXXII phytoplasmas, as well as other common areca nut pathogens and endophytes, overcoming the technical bottleneck of existing qPCR methods' inability to specifically identify the 16SrII group. Furthermore, this method is simple to operate, time-efficient, and has high throughput, making it suitable for quantitative monitoring of large batches of field samples. It provides reliable technical support for early warning, accurate diagnosis, regional control, and the development of disease-resistant varieties for areca nut yellowing disease, significantly improving the scientific rigor and effectiveness of disease control in the areca nut industry. Attached Figure Description

[0017] Figure 1This is an electrophoresis diagram verifying the accuracy of conventional PCR amplification using the primer combination of this invention; where M is DL2000 DNA Marker; 1-6 are samples T52, E81, S97, T48, H66, and E90, respectively; 7 is the blank control.

[0018] Figure 2 This is an amplification curve used to verify the accuracy of qPCR amplification in this invention; where 1 to 6 are samples S97, T48, E81, H66, T52, and E90, respectively; and 7 is the blank control.

[0019] Figure 3 This is a sensitivity test amplification curve of the primer-probe combination of the present invention; where 1 to 6 are 4.81 × 10⁻⁶ respectively. 6 copies / μL ~4.81×10 1 copies / μL; 7 and 8 were 3.18 × 10⁻⁶ respectively. 0 copies / μL and blank control.

[0020] Figure 4 This is the standard curve for qPCR detection in this invention.

[0021] Figure 5 This is a specificity test amplification curve of the primer-probe combination of the present invention; where 1 is a positive strain of Areca catechu T48 from group 16SrII; 2-17 are, respectively, a negative control of healthy areca, sterile and enzyme-free ddH2O, strains H70, S31 and S40 of Areca catechu from group 16SrI, Areca catechu arbuscular mycorrhizal phytoplasma from group 16SrXXXII, Areca palmvelarivirus 1 (APV1), Areca palm necrotic ringspot virus (ANRSV), and Areca anthrax. Colletotrichum sp.), Rhizoctonia solani (sp.), Leuconostoc mirabilis ( Thielaviopsis paradoxa ‌ Burkholderia spp. ( Burkholderia andropogonis ), Pineapple Pantothecin ( Pantoea ananatis ), Sphingosine mononucleosis ( Sphingomonas yantingensis ), white short bacillus ( Curtobacterium albidum ), Bacillus cereus ( Bacillus cereus ) and leaf-dwelling fungi ( Frondihabitans australicus ).

[0022] Figure 6 This is a schematic diagram illustrating the application and detection results of this invention in field samples. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0024] Example 1: Primer and Probe Design and Synthesis According to Areca yellow phytoplasma tuf Gene sequences (GenBank accession numbers: OQ586072, OQ586085, OQ586086, OQ586087) were compared with other phytoprotoplasmic genomes (GenBank accession numbers: AJ271309, AJ271323, KY130430, AB095674, AY685053, AF086617, Y18215). Real-time quantitative PCR specific primers and probes were designed using Primer Express 3.0 software in highly conserved regions with intergroup differences. The sequence information of the forward primer qTuf-F4-2, the reverse primer qTuf-R4, and the probe qTUF-P4-1 is as follows: (1) The forward primer qTuf-F4-2 has the following nucleotide sequence as shown in SEQ ID NO:1: 5'-CGGTGCTATCCTGGTAGTTGCTG-3'; (2) The reverse primer qTuf-R4 has the following nucleotide sequence as shown in SEQ ID NO:2: 5'-CGTGGAGGGATTGGAATGTAGTTAT-3'; (3) Fluorescent probe qTuf-P4-1, whose nucleotide sequence is shown in SEQ ID NO:3: 5'-CATCCTGTTAGCTCGTCA-3', and its 5' end is modified with FAM fluorescent group and its 3' end is modified with MBG quenching group.

[0025] Primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and purified by HPLC.

[0026] Example 2: Validation of the accuracy of primer / probe combinations for real-time quantitative PCR Place 0.1 g of fresh or liquid nitrogen-frozen areca leaf tissue into a 2 ml centrifuge tube, add two 5 mm diameter stainless steel grinding beads, place the tube in a high-speed tissue homogenizer, set the homogenization frequency to 60 times per minute for 3 minutes, extract total DNA according to the instructions of the plant total DNA extraction kit, and store at -20℃.

[0027] Six positive samples were used as positive controls, and enzyme-free and sterile ddH2O was used as a blank control. Conventional PCR and qPCR were performed respectively.

[0028] Standard PCR amplification reaction system: 2× TaqThe following PCR reaction mixtures were prepared: 12.5 μL PCR Mix, 2.0 μL DNA template, 8.5 μL ddH2O, and 1.0 μL each of forward and reverse primers (10 μmol / L). The standard PCR reaction program was: 95 °C pre-denaturation for 5 min, 95 °C denaturation for 45 s, 59 °C annealing for 30 s, and 72 °C extension for 30 s, for a total of 35 cycles. Amplification results were analyzed by 1% agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0029] qPCR reaction system: 10 μL of 2×SuperReal PreMix (Probe), 0.6 μL each of forward and reverse primers (10 μM), 0.4 μL of fluorescent probe (10 μM), 2 μL of DNA template, 0.2 μL of 50×ROX Reference Dye*3, and ddH2O to 20 μL; qPCR reaction program: 95℃ pre-denaturation for 15 min; 95℃ denaturation for 3 s, 59℃ annealing / extension for 32 sec, 40 cycles.

[0030] The results are as follows Figure 1 As shown, the six positive samples amplified target bands of the same size, while the blank control did not show any lines; Figure 2 As shown, all six positive samples were detected, while no amplification curve was observed in the blank control. In summary, this primer set has good accuracy against 16SrII group Areca xanthophytum, therefore, further characterization tests were conducted on the primer / probe combination.

[0031] Example 3: Sensitivity Test The PCR fragments obtained above were excised from gels containing the target bands, and the target bands were purified using an agarose gel extraction kit. The purified product was then ligated into pMD. TM The 18-T vector was transformed into DH5α competent cells, and plasmids of positive clones were screened and extracted, and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0032] After assembly, the sequencing sequences were aligned online using NCBI's BLAST database and uploaded to the NCBI database (GenBank accession numbers: PX257781, PX257782, PX257783, PX257784, PX257785, PX257786). The concentration of correctly sequenced plasmids was determined using an ultra-micro spectrophotometer and stored at -20°C for later use.

[0033] The copy number in the recombinant plasmid was calculated using Shirima's method (Shirima RR et al, 2017). The recombinant plasmid copy number (copies / μL) = (Avogadro's constant × C) / (10^6)^2.9 × number of recombinant plasmid base pairs × 660 dalton / bp). The Avogadro constant is 6.02 × 10⁻⁶. 23 C represents the concentration of the recombinant plasmid (ng / μL). A recombinant plasmid containing the target fragment from *Areca catechu* strain T48 (group 16SrII) was selected as the standard, with a copy number of 3.18 × 10⁻⁶. 9 copies / μL.

[0034] Using the positive plasmid standard as the initial template, the plasmid was serially diluted to a concentration of 3.18 × 10⁻⁶. 6 3.18×10 5 3.18×10 4 3.18×10 3 3.18×10 2 3.18×10 1 3.18×10 0 The above 7 gradient concentrations of positive plasmid standards were used as templates, and sterile, enzyme-free ddH2O was used as a blank control for real-time quantitative PCR. The reaction system consisted of 10 μL of 2×SuperReal PreMix (Probe), 0.6 μL each of forward and reverse primers (10 μM), 0.4 μL of fluorescent probe (10 μM), 2 μL of DNA template, 0.2 μL of 50×ROX Reference Dye*3, and ddH2O to a final volume of 20 μL. The reaction program was as follows: 95℃ pre-denaturation for 15 min; 95℃ denaturation for 3 s, 59℃ annealing / extension for 30 sec, for 45 cycles.

[0035] The kinetic amplification curve and PCR amplification cycle threshold (Ct value) were obtained. Using Excel 2020 software, a standard curve was plotted with Ct value on the ordinate and the logarithm of the base plasmid copy number (10) on the abscissa. The regression line equation was obtained, and the amplification efficiency of this system was calculated. Amplification efficiency (%) = 10 （-1 / 斜率） -1.

[0036] The results showed that the combination of primers qTuf-F4-2 / qTuf-R4 and probe qTuf-P4-1 could detect 3.18 × 10⁻⁶. 1 copies / μL level ( Figure 3 This sensitivity meets the detection requirements for areca catechu phytoplasma. Its standard curve shows a linear relationship between the Ct value and the logarithm of the copy number; the equation of the standard curve is as follows. y = -3.5871 x +43.976, amplification efficiency of 90.01%, correlation coefficient R 2It is 0.9925 ( Figure 4 ).

[0037] Example 4: Specificity Test Using strain T48 of areca nut etiolated phytoplasma from group 16SrII as a positive control, healthy areca nut DNA as a negative control, and sterile, enzyme-free ddH2O as a blank control, strains H70, S31, and S40 of areca nut etiolated phytoplasma from group 16SrI, and strains APV1, ANRSV, and APV1 from group 16SrXXXII were tested. Colletotrichum sp.、 T. paradoxa , B. andropogonis , P. ananatis , S. yantingensis , C. albidum , B. cereus and F. australicus The specificity of real-time quantitative PCR was tested, and the reaction system and procedure were as described in "Example 3: Sensitivity Test", for a total of 40 cycles.

[0038] The results are as follows Figure 5 As shown, only strain T48 of Areca catechu etiolated phytoplasma from group 16SrII showed an amplification curve. Healthy areca nuts were used as a negative control, and sterile, enzyme-free ddH2O was used. Strains H70, S31, and S40 of Areca catechu etiolated phytoplasma from group 16SrI, and strains APV1 and ANRSV from group 16SrXXXII of Hemp japonica etiolated phytoplasma were also observed. Colletotrichum sp. , T. paradoxa, B. andropogonis, P. ananatis, S. yantingensis, C. albidum, B. cereus, F. australicus No amplification curves were observed, indicating that the real-time quantitative PCR method has good specificity for the detection of areca etiolated phytoplasma in group 16SrII. Phytoplasma in groups 16SrI and 16SrXXXII, areca genome, other pathogens and endophytes did not cause non-specific interference to this detection method.

[0039] Example 5: Repeatability Test Select 3.18×10 5 3.18×10 4 3.18×10 3 Using plasmid standards at three concentrations (copies / μL) as templates, each concentration was replicated three times for a total of three tests, performing intra-group repeatable qPCR. The plasmid standards were stored at -20℃ for 3, 6, and 9 days, respectively, and inter-group experiments were repeated under the same reaction conditions. Ct values ​​and coefficients of variation were calculated to verify the repeatability of the invention. The reaction system and procedure were as described in "Example 3: Sensitivity Test," with a total of 40 cycles.

[0040] The results are shown in Table 1. The coefficient of variation within groups was less than 1%, and the coefficient of variation between groups was less than 1%, indicating that the real-time quantitative PCR method has good repeatability for the detection of 16SrII group areca xanthophyta.

[0041] Table 1. Repeatability test of qPCR

[0042] Example 6: Field Sample Detection Forty areca nut samples were collected from Qionghai City and Ding'an County, Hainan Province. Genomic DNA was extracted from these samples, and the qPCR method established in this invention was used for detection. Enzyme-free and sterile ddH2O was used as a blank control. The reaction system and procedure were as described in "Example 3: Sensitivity Test", with a total of 45 cycles.

[0043] The results are shown in Table 2. In terms of application effectiveness, 17 out of the 40 samples showed amplification curves, including 11 from Qionghai and 6 from Ding'an County (Table 2). Figure 6 The highest concentration detected was 2478.03 copies / μL, and the lowest was 14.35 copies / μL. These concentrations are consistent with the low concentrations of *Areca catechu* phytoparasite within the areca nut plant. Therefore, this preliminary finding indicates that *Areca catechu* phytoparasite group 16SrII has already caused damage in some major areca nut cultivation areas.

[0044] Table 2. Detection results of field samples

[0045] Note: "-" indicates that there is no matching amplification curve.

Claims

1. A real-time fluorescence quantitative PCR primer and probe combination for specific detection of 16SrII group areca xanthophytum, characterized in that, The real-time quantitative PCR primer-probe combination consists of a forward primer qTuf-F4-2 with a nucleotide sequence as shown in SEQ ID NO:1, a reverse primer qTuf-R4 with a nucleotide sequence as shown in SEQ ID NO:2, and a fluorescent probe qTuf-P4-1 with a nucleotide sequence as shown in SEQ ID NO:

3.

2. The primer-probe combination according to claim 1, characterized in that, The fluorescent probe qTuf-P4-1 is labeled with the fluorescent reporter group FAM at its 5' end and with MBG at its 3' end.

3. The primer-probe combination according to claim 1, characterized in that, The primer-probe combination described above is based on the 16SrII group of areca xanthophylloma. tuf The conserved regions of the gene were designed and synthesized. tuf The conserved regions of the gene are shown in SEQ ID NO:

4.

4. The application of the primer-probe combination according to any one of claims 1-3, characterized in that, The application is any one of the following: (1) Application of specific detection in 16SrII group of areca nut yellowing phytoplasma; (2) Application in the preparation of products that specifically detect 16SrII group areca xanthophyta.

5. A kit for specifically detecting 16SrII group areca xanthophylloides, characterized in that, The kit includes: 2×SuperReal PreMix, the primer-probe combination of claim 1, DNA template, 50×ROX reference dye, and ddH2O.

6. The use of the kit according to claim 5 in the specific detection of 16SrII group areca catechu yellow phytoplasma.

7. A method for specifically detecting 16SrII group areca catechu phytoplasma, characterized in that, The kit described in claim 5 was used for detection.

8. The method according to claim 7, characterized in that, Includes the following steps: S1. Sample processing: Total DNA was extracted from the sample to be tested using a nucleic acid extraction kit; S2. Real-time quantitative PCR: Using the DNA as a template, a real-time quantitative PCR reaction is performed using the kit described in claim 5. S3. Result Detection: The result is determined based on the Ct value corresponding to the amplification curve plotted by the software of the real-time fluorescence quantitative PCR instrument. When the Ct value of the fluorescent group FAM detection channel is ≤38 and a specific amplification curve appears, the sample is determined to be positive for Areca catechu yellow phytoplasma in group 16SrII; when the Ct value of the fluorescent group FAM detection channel is >38 or no specific amplification curve appears, the sample is determined to be negative for Areca catechu yellow phytoplasma in group 16SrII.

9. The detection method according to claim 8, characterized in that, The reaction system for the real-time quantitative PCR reaction is as follows: 10 μL of 2×SuperReal PreMix, 0.6 μL each of forward and reverse primers, 0.4 μL of fluorescent probe, 2 μL of DNA template, 0.2 μL of 50×ROX reference dye, and ddH2O added to make up to 20 μL.

10. The detection method according to claim 8, characterized in that, The amplification program for the real-time quantitative PCR reaction is as follows: pre-denaturation at 95°C for 15 minutes, followed by 40 cycles, each cycle consisting of denaturation at 95°C for 3 seconds, annealing at 59°C, and extension for 30 seconds.

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