Pig bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR detection primer and application

Abstract

The invention relates to the technical field of molecular biology, and particularly discloses a porcine bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR (polymerase chain reaction) detection primer and application thereof. A synthetic primer is designed according to a highly homologous conserved region of a VP1 gene of a PBoV-G3 gene group, an SYBR Green I fluorescent quantitative PCR method for detecting PBoV-G3 is established, and the SYBR Green I fluorescent quantitative PCR method has the advantages of strong specificity, high sensitivity and good repeatability. Compared with a TaqMan probe method, the method has the advantages that no complex and expensive probe is used, and the detection cost is saved. Compared with common PCR, SYBR Green I fluorescent quantitative PCR can directly quantify the virus load without treatment after amplification, so that the possibility of pollution is reduced, and the method is more suitable for clinical detection of the porcine bocavirus G3 gene group.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and specifically discloses a porcine bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR detection primer and application thereof. Background Art

[0002] Porcine bocavirus (PBoV) was first discovered in Swedish pigs in 2009. Since then, the virus has become prevalent in pigs around the world and has been reported in many countries, including China, South Korea, the United States, Mexico, Canada, Northern Ireland, Romania and Uganda. Various different PBoV strains have been found in pigs suffering from Postweaning Multisystemic Wasting Syndrome (PMWS), respiratory diseases and diarrhea. PBoV, a member of the Parvoviridae family, Parvovirinae subfamily, and Bocavirus genus, is commonly associated with diarrhea and respiratory tract infections in piglets. It is susceptible to co-infection with porcine reproductive and respiratory syndrome virus (PSSRV), classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2), and porcine parvovirus (PPV), among others. PBoV is considered to have potential immunosuppressive properties. ORF1 and ORF2 encode two nonstructural proteins, NS1 and NP1, respectively, while ORF3 encodes two capsid proteins, VP1 and VP2. VP1 may influence its tissue tropism and potentially contribute to pathogenesis. In 2012, some scholars proposed a unified naming method for PBoV based on the VP1 gene, dividing PBoV into PBoV Group 1, PBoV Group 2 and PBoV Group 3, and genotyping the known PBoVs.

[0003] Porcine bocavirus causes disease primarily in young animals, while in adult animals it is usually subclinical, with a high incidence in weaned piglets. In sick pigs with respiratory disease, multisystemic wasting syndrome, and diarrhea, the detection rate of PBoV was significantly higher than in healthy pigs. Another study showed that multiple PBoV genotypes were co-infected in the samples examined, suggesting the complexity of bocavirus. Considering its high co-infection rate with other viruses in clinical practice, porcine bocavirus is believed to have an immunosuppressive function, allowing it to invade host-related tissues together with other viruses. In 2018, PBoV was detected in the nasopharynx of a 3-year-old child in Iran, and the possibility of cross-species transmission of PBoV should be considered.

[0004] Accurate, sensitive, rapid and economical real-time fluorescence quantitative PCR technology has developed into a fast and highly automated nucleic acid quantification technology, which greatly reduces the false positive rate, has high work efficiency, good reproducibility of results, and does not require the use of dyes harmful to the human body. Its application in pathogen detection has become relatively mature.

[0005] The development of a highly accurate and easy-to-operate SYBR GreenⅠ fluorescent quantitative PCR detection method for porcine bocavirus G3 gene group is of great significance. Summary of the Invention

[0006] The purpose of the present invention is to address the problems existing in the prior art and provide a porcine bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR primer and its application.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A SYBR Green I fluorescent quantitative PCR detection primer for porcine bocavirus G3 gene group, the sequence of the primer is as follows:

[0009] PBoV-qPCR-F: 5′-TTATCGACTCKTTGCAGTCTG-3′;

[0010] PBoV-qPCR-R: 5′-CGCAAAGTAYAGGTGACGTT-3′.

[0011] Another object of the present invention is to protect a detection kit.

[0012] A porcine bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR detection kit comprises the above-mentioned detection primers.

[0013] Furthermore, the reaction system includes 2×ChamQ Universal SYBR qPCR Master Mix, upstream and downstream primers, ddH2O, and template. Preferably, each 20 μL reaction system includes 10 μL of 2×ChamQ Universal SYBR qPCR Master Mix, 0.4 μL each of upstream and downstream primers, 8.2 μL of ddH2O, and 1 μL of DNA template.

[0014] Furthermore, positive controls and negative controls are also included.

[0015] Furthermore, the positive control was genomic DNA of porcine bocavirus, and the negative control was sterile water.

[0016] Another object of the present invention is to protect the method of performing detection using the detection kit.

[0017] A detection method using the porcine bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR detection kit,

[0018] The following steps are involved:

[0019] Extracting genomic DNA from the sample to be tested, and performing real-time fluorescence quantitative PCR amplification using the kit according to any one of claims 2 to 6;

[0020] Determine whether the sample contains porcine bocavirus based on the amplification curve.

[0021] Furthermore, the PCR reaction conditions were 95°C pre-denaturation for 30 s, 95°C denaturation for 10 s, 61°C annealing and extension for 15 s, and 40 cycles.

[0022] Another object of the present invention is to provide an application of the above detection kit.

[0023] For example, the use of the porcine bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR detection kit in detecting porcine bocavirus reagents.

[0024] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0025] This study designed synthetic primers based on highly homologous conserved regions within the VP1 gene of the PBoV-G3 genogroup, establishing a SYBR Green I fluorescent quantitative PCR method for detecting PBoV-G3. This method exhibits strong specificity, high sensitivity, and good reproducibility. Compared to the TaqMan probe method, this method eliminates the need for complex and expensive probes, saving detection costs. Compared to conventional PCR, SYBR Green I fluorescent quantitative PCR can directly quantify viral load without requiring post-amplification processing, reducing the possibility of contamination and making it more suitable for clinical detection of the porcine bocavirus G3 genogroup. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The figure shows the PCR identification data of the PBoV-G3 VP1 gene in the example; wherein, M: DNA molecular mass standard (DL2000); 1: PBoV-G3 PCR product; 2: negative control.

[0027] Figure 2 This is the PBoV-G3 fluorescence quantitative PCR standard curve in the example.

[0028] Figure 3 This is the melting curve of PBoV-G3 fluorescent quantitative PCR in the example.

[0029] Figure 4 This is a graph showing the sensitivity test data of real-time fluorescence quantitative PCR in the examples.

[0030] Figure 5 This is a graph showing the data of a conventional PCR sensitivity test in the examples.

[0031] Figure 6 This is a graph showing the specificity test data of fluorescent quantitative PCR in the examples. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] Example 1

[0034] 1 Materials and Methods

[0035] 1.1 Source of the strain

[0036] Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine transmissible gastroenteritis virus (TGEV) are all isolated and stored in our laboratory. Positive samples of the G1, G2, and G3 genogroups of PBoV are identified and stored in our laboratory.

[0037] 1.2 Test equipment

[0038] Fluorescence quantitative PCR instrument: Thermo Fisher Scientific, USA, model Applied Biosystems QuantStudio 6Pro; UV spectrophotometer: Thermo Fisher Scientific, USA, model G10S; electrophoresis apparatus power supply and horizontal electrophoresis tank: BIO-RAD, model Sub-Cell192; conventional PCR instrument: BIO-RAD, model T100; desktop high-speed centrifuge: SIGMA freeze dryer Co., Ltd., Germany; fully automatic gel imager: SYNGENE, model Gbox-F3-E.

[0039] 1.3 Test reagents and consumables

[0040] Viral DNA / RNA extraction kit (RC311-01), Ultra-Universal TOPO Cloning Kit (C603-01), plasmid miniprep kit (DC201-01), gel recovery kit (DC311-01), nucleic acid dye and DNA marker, 2× Rapid Taq Master Mix (P222-03), and ChamQ Universal SYBR qPCR Master Mix (Q711-02) were all purchased from Nanjing Novozymes Biotechnology Co., Ltd.; DH-5α competent cells were prepared in our laboratory.

[0041] 1.4 Primer design

[0042] Based on the PBoV-G3 gene sequence registered in GenBank, gene homology analysis was performed using the MegAlign program built into DNAstar software. Primers were designed using Primer (Version 6.0) software based on the base sequence of the highly conserved region of the VP1 gene of the PBoV-G3 genogroup. A pair of primers specifically amplifying the VP1 gene of the PBoV-G3 genogroup was obtained. The primers were synthesized by Shanghai Sangon Biotechnology Technology Service Co., Ltd. The sequences are shown in Table 1 below.

[0043] Table 1 Primer sequences

[0044]

[0045] 1.5 Conventional PCR amplification

[0046] PEDV, PDCoV, TGEV, PBoV-G1, PBoV-G2, and PBoV-G3 nucleic acids were extracted according to the viral DNA / RNA extraction kit instructions. PEDV, PDCoV, and TGEV nucleic acids were reverse transcribed according to the reverse transcription kit instructions. The resulting nucleic acids and cDNA were stored at -80°C. The VP1 gene was amplified by conventional PCR using the extracted DNA as a template. The reaction procedure was: 95°C for 3 min; 95°C for 15 s, 54°C for 15 s, and 72°C for 30 s, for a total of 30 cycles; 72°C for 5 min; and 12°C for ∞. The reaction volume (10 μL) contained: 5 μL of 2× Rapid Taq Master Mix, 3 μL of ddH2O, 0.5 μL each of the upstream and downstream primers, and 1 μL of template DNA.

[0047] 1.6 Preparation of recombinant plasmid standards

[0048] The PCR product was purified by gel electrophoresis and ligated into the pCE3 Blunt Vector to construct the recombinant plasmid pCE3 Blunt-VP1. This recombinant plasmid was then transformed into competent E. coli DH-5α cells. Plasmids that tested positive for PCR were sent to Sangon Biotechnology Services Co., Ltd. for sequencing. The absorbance of the standard plasmid with the correct sequence was measured to calculate the copy number of the standard plasmid.

[0049] 1.7 Establishing standard curve and melting curve

[0050] The prepared recombinant plasmid standard pCE3 Blunt-VP1 was diluted 10-fold, with a total of 8 dilution gradients (the number of plasmid copies after dilution was 4.51×10 6 —4.51×10 -1 The optimized conditions were used for fluorescence quantitative PCR to measure the reaction data, and a standard curve was drawn with the logarithm of the plasmid copy number as the horizontal axis and the Cq value as the vertical axis.

[0051] 1.8 Optimization of Fluorescence Quantitative PCR Conditions

[0052] The reaction system recommended by ChamQ Universal SYBR qPCR Master Mix was used, and the reaction conditions and reaction system were further optimized using the specificity of the melting curve and the correlation coefficient of the standard curve as indicators.

[0053] 1.9 Specificity test

[0054] The fluorescent quantitative PCR method established in this experiment was used to amplify six viral nucleic acids, including PEDV, PDCoV, TGEV, PBoV-G1, PBoV-G2, and PBoV-G3. ddH2O was selected as a negative control, and a real-time reaction curve was established to test the specificity of the method.

[0055] 1.10 Sensitivity test

[0056] Diluted by 10 times (10 4 ~10 10 ) was used as a template. Fluorescence quantitative PCR and conventional PCR were used for amplification detection, and real-time reaction curves were established. The sensitivities of the two methods were compared by comparing the maximum dilution factor at which a positive signal appeared in fluorescence quantitative PCR with the maximum dilution factor at which a positive band appeared in conventional PCR gel electrophoresis. The sensitivity of the fluorescence quantitative PCR method established in this experiment was determined.

[0057] 1.11 Repeatability test

[0058] The copy number used was 4.51×10 6 —4.51×10 0 Using the recombinant plasmid standard pCE3 Blunt-VP1 at 10 copies / μL as a template, three replicates were performed at each dilution using the optimized reaction system and conditions. This was repeated three times at different times as inter-group replicates, and the data were recorded. The intra- and inter-group coefficients of variation were calculated to assess the stability of the method.

[0059] 1.12 Testing of clinical samples

[0060] A total of 212 fecal samples from diarrheal pigs were collected from pig farms in different regions of Henan Province. Total nucleic acid was extracted according to the instructions of the DNA / RNA extraction kit. The nucleic acid was detected using the established SYBR Green I fluorescent quantitative PCR method. At the same time, the nucleic acid was amplified by conventional PCR and subjected to gel electrophoresis. The detection results of the two methods were statistically analyzed and compared.

[0061] 2 Test results

[0062] 2.1 Preparation of recombinant plasmid standards

[0063] Using DNA extracted from PBoV G3-positive pig feces samples as a template, a partial fragment of PBoV-G3VP1 nucleic acid was amplified by conventional PCR method, such as Figure 1The 167 bp target fragment was amplified and ligated into the pCE3Blunt Vector to construct the recombinant plasmid standard, pCE3 Blunt-VP1. Sequencing showed that the inserted sequence in the recombinant plasmid was completely correct. The calculated copy number of this standard plasmid was 4.51×10 6 / μL.

[0064] 2.2 Optimization of PCR conditions

[0065] The optimized reaction volume was 20 μL, consisting of 10 μL of 2× ChamQ Universal SYBR qPCR Master Mix, 0.4 μL of each upstream and downstream primer, 8.2 μL of ddH2O, and 1 μL of DNA template. The optimal reaction conditions were: 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds and 61°C for 15 seconds.

[0066] 2.3 Establishment of standard curve and analysis of dissolution curve

[0067] like Figure 2 and Figure 3 As shown, the recombinant plasmid standard pCE3 Blunt-VP1 (10 4 ~10 10 That is 4.51×10 6 copies / μL~4.51×10 0 qPCR amplification was performed with the logarithmic value of the copy number of the recombinant plasmid standard as the abscissa and the Cq value as the ordinate to draw a standard curve. The standard curve equation was y = -4.0791x + 37.3, and the correlation coefficient R 2 The Cq value was 0.9934, indicating a good linear relationship between the concentration of the recombinant plasmid standard at each gradient and the Cq value. Melting curve results showed that the melting curve peaks of this method were relatively uniform, with an average melting temperature of approximately 86°C and fluctuations of no more than 1°C.

[0068] 2.4 Sensitivity test

[0069] The fluorescent quantitative PCR method established in this example and the conventional PCR method were used to amplify the recombinant plasmid standard diluted tenfold. The results showed that the fluorescent quantitative PCR method could detect 4.51 copies / μL of the standard plasmid, while the detection limit of the conventional PCR method was 4.51×10 2 Copies / μL. It can be seen that the sensitivity of the fluorescent quantitative PCR established in this example is 100 times that of ordinary PCR. Figure 4 and Figure 5 As shown, the copy numbers of the standard plasmids 1-7 are 4.51×10 6 —4.51×100 copies / μL.

[0070] 2.5 Specificity test

[0071] The established fluorescence quantitative PCR was used to amplify PEDV, PDCoV, TGEV, PBoV-G1, PBoV-G2, and PBoV-G3 viruses, and ddH2O was set as a negative control for detection. Only PBoV-G3 amplified a high level of fluorescence signal, while other viruses showed no specific amplification, indicating that the method has good specificity. Figure 6 As shown, 1-7: PBoV-G3, PEDV, PDCoV, TGEV, PBoV-G1, PBoV-G2, ddH2O, respectively.

[0072] 2.6 Repeatability test

[0073] The selected copy number was 4.51×10 6 —4.51×10 0 The recombinant plasmid standard was used as a template and amplified three times using the established fluorescent quantitative PCR method. The intra-group coefficient of variation was within 0.5%, and the inter-group coefficient of variation was within 2%, as shown in Table 2. The test results showed that this method has good repeatability.

[0074] Table 2 Results of the fluorescence quantitative PCR repeatability test

[0075]

[0076] 2.7 Testing of clinical samples

[0077] Among 212 diarrheal fecal samples from pigs in Henan Province, the positive rate detected by the SYBR Green I fluorescent quantitative PCR method was 41%, while the positive rate of conventional PCR was 33.5%. In addition, all samples that tested positive by conventional PCR were positive in the SYBR Green I fluorescent quantitative PCR test results, indicating that the detection rate of the SYBR Green I fluorescent quantitative PCR method established in this experiment is higher than that of conventional PCR and has good sensitivity.

[0078] This example establishes a SYBR Green I fluorescent quantitative PCR method for detecting the PBoV-G3 genogroup based on synthetic primers designed based on highly homologous conserved regions of the VP1 gene of the PBoV-G3 genogroup. This method has the advantages of strong specificity, high sensitivity, and good reproducibility. Compared to the TaqMan probe method, this method does not require complex and expensive probes, saving detection costs. Compared to conventional PCR, SYBR Green I fluorescent quantitative PCR does not require post-amplification processing to directly quantify viral load, reducing the possibility of contamination and making it more suitable for clinical detection of the porcine bocavirus G3 genogroup.

[0079] In summary, the SYBR Green I fluorescent quantitative PCR method established in this study has good specificity, sensitivity, and reproducibility. It provides an excellent method for the clinical detection of PBoV and lays a foundation for epidemiological investigation and comprehensive prevention and control of PBoV.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A SYBR Green I fluorescent quantitative PCR detection primer for porcine bocavirus G3 gene group, characterized in that: The sequences of the primers are as follows: PBoV-qPCR-F: 5′-TTATCGACTCKTTGCAGTCTG-3′; PBoV-qPCR-R: 5′-CGCAAAGTAYAGGTGACGTT-3′.

2. A SYBR Green I fluorescent quantitative PCR detection kit for porcine bocavirus G3 gene group, characterized in that: Comprising the detection primer as claimed in claim 1.

3. The porcine bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR detection kit according to claim 2, characterized in that: Includes 2×ChamQ Universal SYBR qPCR Master Mix, upstream and downstream primers, ddH2O, and DNA template.

4. The porcine bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR detection kit according to claim 3, characterized in that: Each 20 μL reaction system includes 10 μL of 2×ChamQ Universal SYBR qPCR Master Mix, 0.4 μL of upstream and downstream primers, 8.2 μL of ddH2O, and 1 μL of DNA template.

5. The porcine bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR detection kit according to claim 2, characterized in that: Positive and negative controls were also included.

6. The porcine bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR detection kit according to claim 5, characterized in that: The positive control was porcine bocavirus genomic DNA, and the negative control was sterile water.

7. A detection method using the porcine bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR detection kit according to any one of claims 2 to 6, It is characterized by: The following steps are involved: Extracting genomic DNA from the sample to be tested, and performing real-time fluorescence quantitative PCR amplification using the kit according to any one of claims 2 to 6; Determine whether the sample contains porcine bocavirus based on the amplification curve.

8. The detection method according to claim 7, characterized in that The PCR reaction conditions were 95°C pre-denaturation for 30 s, 95°C denaturation for 10 s, 61°C annealing and extension for 15 s, and 40 cycles.

9. Use of the porcine bocavirus G3 gene group SYBR Green I fluorescent quantitative PCR detection kit according to any one of claims 2 to 6 in detecting porcine bocavirus reagents.

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