A TaqMan RT-qPCR Assay for Detecting Porcine Rotavirus Group A
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摘要:目的
建立一种猪A群轮状病毒（porcine rotavirus group A, PoRV A）快速检测方法，用于PoRV检测和流行病学调查。
方法参考GenBank中猪A群轮状病毒（PoRV A）VP6基因序列（登录号MT025937.1、OP978242.1、PP566178.1）设计特异性引物和探针，优化反应体系中引物和探针的浓度，建立TaqMan RT-qPCR检测方法，并通过特异性、灵敏性和重复性的结果以及临床应用对该方法进行评价。
结果该方法可特异性扩增PoRV核酸，最低检出限度为27.0 copies·μL−1，灵敏度高于普通RT-PCR 100倍；与猪流行性腹泻病毒（porcine epidemic diarrhea virus, PEDV）、猪德尔塔冠状病毒（porcine deltacoronavirus, PDCoV）、猪传染性胃肠炎病毒（transmissible gastroenteritis of swine, TGEV）核酸均无交叉反应；组内和组间变异系数均小于1.10%，重复性好。151份疑似PoRV的临床样品使用RT-qPCR进行检测，结果显示检出率为42.38%（64/151），优于常规RT-PCR的检出率（33.11%，50/151）。
结论本研究基于猪A群轮状病毒VP6基因，建立了适用于PoRV A检测及其流行病学调查的TaqMan实时荧光定量PCR检测方法，具有灵敏度高、特异性强、重复性好等优势，为猪轮状病毒检测和流行病学调查提供了技术手段。
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关键词:
- 猪A群轮状病毒 /
- TaqMan探针 /
- 荧光定量RT-PCR
Abstract:ObjectiveA rapid method for detecting porcine rotavirus group A (PoRV A) in disease monitoring and epidemiological investigation was developed.
MethodSpecific primers and probes were selected based on the VP6 gene sequences of PoRV A in GenBank (accession numbers MT025937.1, OP978242.1 and PP566178.1). Reaction conditions of the TaqMan RT-qPCR method were optimized followed by evaluations on assay specificity, sensitivity, repeatability, and clinical application.
ResultsThe newly developed method could specifically amplify PoRV nucleic acid with a minimum detection limit of 27.0 copies·μL−1 and a sensitivity 100 times higher than RTPCR. There was no cross reactivity with the nucleic acids of PEDV, TGEV or PDCoV. The method showed high repeatability with the variation coefficients on intra- and inter-group below 1.10%. On 151 clinical specimens suspected of PoRV, a detection rate of 42.38% (64/151), which was better than that of conventional RT-PCR at 33.11% (50/151), was obtained by the assay.
ConclusionThe new TaqMan RT qPCR method for detecting VP6 gene of PoRV A was high in assay sensitivity, specificity, and repeatability. It was considered suitable for PoRV detection and epidemiological investigations.
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Keywords:
- porcine rotavirus Group A /
- TaqMan probe /
- RT-qPCR
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图 1 质粒标准品PCR扩增
M：DL2000 DNA Maker；1：阴性对照；2：PoRV A阳性质粒PCR产物。
Figure 1. PCR amplification of plasmid standard
M: DL2000 DNA marker; 1: negative control; 2: PoRV positive plasmid PCR product.
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图 4 不同模板拷贝数的RT-qPCR扩增结果
1～8分别是拷贝数为2.7×108～ 2.7×101 copies·μL−1的质粒；9：阴性对照。
Figure 4. RT-qPCR amplifications with different template copy numbers
1–8: 2.7×108–2.7×101 copies·μL−1 plasmid; 9: negative control.
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图 5 不同模板拷贝数的RT-PCR扩增结果
M：DL2500 DNA Maker；1～8：分别是拷贝数为2.7×108～2.7×101copies·μL−1质粒；9：阴性对照；
Figure 5. RT-PCR amplifications of different template copy numbers
M: DL2500 DNA Marker; 1–8: 2.7×108–2.7×101 copies·μL−1 plasmid; 9: negative control.
表 1 PoRV引物和探针
Table 1 PoRV primers and probes
引物/探针
Primer/Probe引物序列（5′-3′）
Sequence（5′-3′）产物大小
Product size/bp用途
UsagePoRV-F1 GGCTTTTAAACGAAGTCTTC 1209 RT-PCR PoRV-R1 GGTCACATCCTCTCACT PoRV-F2 GGCTTTTAAACGAAGTCTTC 598 RT-PCR PoRV-R2 CCAGCTACYTGAATTTCTGA PoRV-F3 ATTAAGTGAGGACTAGGCTAA 114 RT-qPCR PoRV-R3 ACTCTACGTAGCGAGTATGA PoRV-Probe FAM-ATGTAGCTATGTCAAGTCAATCAGA-TAMRA RT-qPCR 
下载: 导出CSV
表 2 PoRV TaqMan RT-qPCR重复性试验结果
Table 2 Results of PoRV TaqMan RT-qPCR assay
质粒浓度
Concentration/
（copies·μL−1）组内变异试验
Intra-assay variability组间变异试验
Inter-assay variability平均数
¯X±SD变异系数
CV/%平均数
¯X±SD变异系数
CV/%2.7×105 21.553±0.201 0.932 22.507±0.231 1.026 2.7×104 24.413±0.098 0.401 24.853±0.127 0.511 2.7×103 27.177±0.248 0.912 27.540±0.099 0.359
下载: 导出CSV
表 3 临床样品检测结果比较
Table 3 Comparison of testing results on clinical specimens
指标
Index阳性
Positive/份可疑
Suspicious/份阴性
Negative/份总计
Total/份RT-PCR 50 0 101 151 荧光定量 RT-PCR 64 9 78 151
下载: 导出CSV
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