| 摘要: |
| 【目的】利用PfAgo蛋白结合环介导等温扩增技术（LAMP）建立一种新的PCV3检测方法LAMP-PfAgo法，用于PCV3的快速鉴别诊断。【方法】PCR扩增PCV3 Rep基因，构建重组阳性质粒pMD19-T-PCV3-Rep，对其进行PCR和测序鉴定。根据PCV3 Rep基因的保守区域设计PCV3-T1～PCV3-T4等4组引物（每组包括1对内引物和1对外引物），以pMD19-T-PCV3-Rep为模板进行LAMP，筛选最佳引物组，并对LAMP反应的Mg2+浓度、内外引物浓度、反应温度、反应时间进行优化。根据PCV3 Rep基因的保守序列，设计PCV3-gs1～PCV3-gs5等5组gDNAs和一条特异性探针，进行LAMP-PfAgo反应，筛选最佳的gDNAs，并对LAMP-PfAgo反应体系中 PfAgo蛋白浓度、 gDNAs浓度、Mn2+浓度和反应时间进行优化。 对该LAMP-PfAgo方法的特异性、敏感性进行评价；并用该方法和实时荧光定量PCR方法对疑似感染PCV3的46份临床样品进行检测，比较结果的一致性。【结果】重组阳性质粒pMD19-T-PCV3-Rep PCR和测序均获得了891 bp的序列，表明重组阳性质粒构建成功。LAMP最佳引物为PCV3-T1，最佳Mg2+终浓度为6 mmol/L，最佳内外引物终浓度分别为1.6和1.2 μmol/L，最适的反应温度65 ℃，最佳反应时间60 min。使用优化后的LAMP反应条件对PCV3 Rep基因保守序列进行扩增，并将其用于后续试验。LAMP-PfAgo反应最佳的gDNAs为PCV3-gs1；优化后的PfAgo蛋白终浓度为40 U/μL，gDNAs终浓度为1.50 μmol/L，Mn2+终浓度为1.50 mmol/L，反应时间为30 min。LAMP-PfAgo法的灵敏度为10 拷贝/μL，且与猪圆环病毒2型、猪细小病毒、猪繁殖与呼吸综合征病毒、猪传染性胃肠炎病毒、猪瘟病毒及猪伪狂犬病毒等常见猪源病原核酸无交叉反应。临床样本检测结果显示，LAMP-PfAgo法与实时荧光定量PCR方法检测结果的符合率为100%。【结论】基于PfAgo蛋白结合LAMP技术建立了对PCV3的核酸检测方法，该方法灵敏度高和特异性强，具有潜在的临床应用价值。 |
| 关键词: 猪圆环病毒3型 PfAgo 环介导等温扩增 即时检验 猪 |
| DOI：10.13207/j.cnki.jnwafu.2025.02.001 |
| 分类号: |
| 基金项目:国家自然科学基金青年基金项目(32202891)；安徽省重点实验室开放项目(XM2201) |
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| Establishment of LAMP-PfAgo detection method for porcine circovirus 3 |
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QI Zhao, YU Xiangyu, CHEN Weiyuan, SONG Xiangjun
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Anhui Key Laboratory of Veterinary Pathobiology and Disease Prevention and Control,Anhui Key Laboratory of Animal Food Quality and Biosafety Engineering,Anhui Agricultural University,Hefei,Anhui 230036,China
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| Abstract: |
| 【Objective】A new detection method for porcine circovirus 3 (PCV3) by Pyrococcus furiosus Argonaute (PfAgo) protein combined with loop-mediated isothermal amplification assay (LAMP) was established in this study.【Method】PCV3 Rep gene was amplified using PCR,and the recombinant positive plasmid pMD19-T-PCV3 Rep was constructed.PCR and sequencing were conducted to confirm its identity.According to the conserved region of PCV3 Rep gene,four sets of primers (one pair of inner primers and one pair of outer primers),including PCV3-T1-PCV3-T4,were designed.The LAMP reaction was carried out using pMD19-T-PCV3-Rep as a template,the optimal primer set was screened,and Mg2+ concentration,inner and outer primers concentrations,reaction temperature and reaction time of the LAMP reaction were optimized.Based on the conserved sequence of PCV3 Rep gene,a total of 5 sets of gDNAs of PCV3-gs1-PCV3-gs5 and specific probes were designed.Then,the LAMP-PfAgo reaction was performed to screen the best gDNAs and optimize the PfAgo protein concentration,gDNAs concentration,Mn2+ concentration and reaction time in the LAMP-PfAgo reaction system.Subsequently,the specificity and sensitivity of the LAMP-PfAgo method were evaluated.A total of 46 clinical samples suspected to be infected with PCV3 were detected with the established method,and the results were compared with qPCR results.【Result】The PCR and sequencing of the recombinant positive plasmid pMD19-T-PCV3-Rep consistently produced an 891 bp sequence,indicating that the construction of recombinant positive plasmid was successful.The optimal primer set for LAMP was PCV3-T1,with an optimal Mg2+ final concentration of 6 mmol/L,optimal inner primers and outer primers final concentrations of 1.6 and 1.2 μmol/L,an optimal reaction temperature of 65 ℃ and an optimal reaction time of 60 min.The conserved sequence of PCV3 Rep gene was amplified using optimized LAMP reaction conditions and used for subsequent experiments.The optimal gDNAs for the LAMP-PfAgo reaction were PCV3-gs1,the optimized final concentration of PfAgo protein was 40 U/μL,the final concentration of gDNAs was 1.50 μmol/L,the final concentration of Mn2+ was 1.50 mmol/L,and the reaction time was 30 min.The LAMP-PfAgo method had a sensitivity of 10 copies/μL and no crossreactivity with pig-derived pathogens such as porcine circovirus 2 (PCV2),porcine parvovirus (PPV),porcine reproductive and respiratory syndrome virus (PRRSV),transmissible gastroenteritis virus (TGEV),classical swine fever virus (CSFV) and porcine pseudorabies virus (PRV).The established method and qPCR were 100% in compliance for the detection of PCV3 clinical samples.【Conclusion】A nucleic acid detection method based on PfAgo protein combined with LAMP technology was established for PCV3,and it had high sensitivity and strong specificity with potential clinical application. |
| Key words: porcine circovirus 3 (PCV3) PfAgo loop-mediated isothermal amplification (LAMP) point-of-care testing(POCT) pig |
