Establishment and application of multiplex RT-PCR methods for akabane virus, bovine herpesvirus 1 and bovine viral diarrhea virus
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摘要:
目的 旨在建立一种可同时检测赤羽病病毒（Akabane virus, AKAV）、牛疱疹病毒1型（Bovine herpesvirus 1, BoHV-1）和牛病毒性腹泻病毒（Bovine viral diarrhea virus, BVDV）的多重RT-PCR检测方法，为牛繁殖障碍类疫病鉴别诊断及防控奠定基础。 方法 针对AKAV S基因、BoHV-1 gH基因和BVDV 3'UTR基因保守区域设计3对引物，通过反应条件参数的优化，建立AKAV、BoHV-1和BVDV多重RT-PCR检测方法。 结果 特异性结果显示，该方法仅对AKAV、BoHV-1和BVDV阳性样品检测为阳性，对牛细小病毒（BPV）、牛呼吸道合胞体病毒（BRSV）、牛副流感病毒3型（BPIV3）、口蹄疫病毒（FMDV）和牛流行热病毒（BEFV）等阳性样品检测为阴性。敏感性结果显示，该方法对AKAV、BoHV-1和BVDV重组质粒的下限阈值均为1.0×103 拷贝·μL−1。重复性结果显示，批内、批间重复性均较好。利用建立的方法对143份患繁殖障碍的牛全血/组织样品进行测定，并与现有的地方标准进行对比，验证本检测方法的实际临床应用效能。结果显示，AKAV、BoHV-1和BVDV的阳性率分别为2.80%（4/143）、21.68%（31/143）、38.46%（55/143），存在混合感染现象，与AKAV、BoHV-1和BVDV地方标准的符合率为99.3%、98.6%和97.2%，Kappa系数分别为0.885、0.960和0.942，表明本研究建立的方法与地方标准两者一致性良好。 结论 本研究开创性地建立了一种特异性强、灵敏度高和成本低的多重RT-PCR检测方法，适用于AKAV、BoHV-1和BVDV的鉴别检测。 Abstract:Objective To establish a multiplex RT-PCR method for the rapid and simultaneous detection of akabane virus （AKAV）, Bovine herpesvirus 1 （BoHV-1） and bovine viral diarrhea virus （BVDV）, so as to lay a foundation for the differential diagnosis, prevention and control of bovine reproductive disorder epidemics. Method Three pairs of primers were designed for the conservative regions of AKAV S gene, BoHV-1 gH gene and BVDV 3'UTR gene, and a multiplex RT-PCR assay for AKAV, BoHV-1 and BVDV was established through the optimal of reaction condition parameters. Result The specificity results showed that the method was positive only for AKAV, BoHV-1 and BVDV, and negative for the nucleic acids of bovine parvovirus （BPV）, bovine respiratory syncytial virus （BRSV）, bovine parainfluenza virus 3 （BPIV3）, foot-and-mouth disease virus （FMDV）, and bovine ephemeral fever virus （BEFV）. The sensitivity results showed that the lower threshold of this method for AKAV, BoHV-1 and BVDV recombinant plasmids were all 1.0×103 copies/μL. The results of the reproducibility test showed good intra- and inter-batch reproducibility. The established method was used to determine 143 whole blood/tissue samples of cattle with reproductive disorders and compared with the existing local standards to verify the efficacy of this test for practical clinical application. The results showed that the positivity rates of AKAV, BoHV-1 and BVDV were 2.80% （4/143）, 21.68% （31/143） and 38.46% （55/143）, respectively, with the presence of several mixed infections; and the conformity rates of this method with the local standards for AKAV, BoHV-1 and BVDV were 99.3%, 98.6% and 97.2%, respectively. The Kappa coefficients of the two were 0.885, 0.960 and 0.942 respectively, indicating that the method established in this study was in good agreement with the local standards. Conclusion In this study, we pioneered a multiplex RT-PCR assay with high specificity, sensitivity and low cost for the differential detection of AKAV, BoHV-1 and BVDV. -
图 1 AKAV、BoHV-1和BVDV单重和多重PCR扩增
M：DL2 000 DNA Marker；1：AKAV、BoHV-1和BVDV；2：AKAV和BoHV-1；3：AKAV和BVDV；4：BoHV-1和BVDV；5：AKAV；6：BoHV-1；7：BVDV；8：阴性对照。
Figure 1. Single and multiplex PCR products of AKAV, BoHV-1 and BVDV
1: AKAV, BoHV-1 and BVDV; 2: AKAV and BoHV-1; 3: AKAV and BVDV; 4: BoHV-1 and BVDV; 5: AKAV; Lane 6: BoHV-1; Lane 7: BVDV; 8: negative control; M: DL2 000 DNA Marker.
图 2 AKAV、BoHV-1和BVDV多重PCR特异性试验
M：DL2 000 DNA Marker；1：AKAV、BoHV-1和BVDV多重PCR扩增产物； 2～6分别为BPV、BRSV、BPIV3、FMDV和BEFV；7：阴性对照。
Figure 2. Validation of the specificity of the multiplex PCR for BoHV-1, AKAV and BVDV detection
M: DL2 000 DNA Marker; 1: BoHV-1, AKAV and BVDV; 2–6: BPV, BRSV, BPIV3, FMDV and BEFV, respectively; 7: negative control.
图 3 多重PCR敏感性试验
M：DL2 000 DNA Marker；1～9依次为1.0×108～1.0×100拷贝·μL−1；10：阴性对照。
Figure 3. Validation of the sensitivity of the multiplex PCR
M: DL2 000 DNA Marker; 1～9: 1.0×108～1.0×100 copies·μL−1; 10: negative control.
图 4 建立方法的重复性试验
M：DL2 000 DNA Marker 1–3：同一批次的3个重复。
Figure 4. Repeatability of the established multiplex PCR
M: DL2 000 DNA Marker; 1–3: three groups of plasmids from the same batch.
表 1 引物序列Fig.1 The information of primer sequences
病毒
Virus引物序列（5'-3'）
Sequences of the primers for PCR（5'-3'）靶基因
Target gene产物大小（bp）
Products size/bpAKAV AKAV-F：ACATAAGACGCCACAACCAAGT S 623 AKAV-R：CGAGCAGCTGAACAAAGGTG BoHV-1 BoHV-1-F：TCCTGCCTCTGGAGTTTATGG gH 433 BoHV-1-R：TTGAGGTGGTAGTCAAGGTCG BVDV BVDV-F：CAGCGAAGGCCGAAAAGAGG 3'UTR 308 BVDV-R：CCATGTGCCATGTACAGCAGAG 
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表 2 临床样本检测结果对比
Table 2. Comparison results of positive rates of two methods
病毒
Virus本研究方法
This study地方标准检测方法
Local standardsKappa值
Kappa value阳性样本数
No. of positive samples阳性率
Positivity rate阳性样本数
No. of positive samples阳性率
Positivity rate赤羽病病毒 AKAV 4 2.80% 5 3.50% 0.8852 牛疱疹病毒1型 BoHV-1 31 21.68% 33 23.08% 0.9597 牛病毒性腹泻病毒 BVDV 55 38.46% 59 41.26% 0.9417
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