Establishment and Preliminary Application of a Multiple PCR Assay for NDRV, NGPV, and DTMUV Detections
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摘要:
目的 建立能同时检测新型鸭呼肠孤病毒（New-type duck reovirus，NDRV）、新型鹅细小病毒（Novel goose parvovirus，NGPV）和鸭坦布苏病毒（Duck Tembusu virus，DTMUV）的方法。 方法 根据NDRV、NGPV和DTMUV基因组的保守区域设计3对特异性引物，预期特异性扩增NDRV、NGPV和DTMUV的片段大小分别为594、467、328 bp，建立可同时检测NDRV、NGPV和DTMUV的多重PCR检测方法，对其进行特异性、敏感性及重复性检验，并在临床上进行初步应用。 结果 特异性检测结果表明，该多重PCR方法可同时扩增NDRV、NGPV、DTMUV的目的片段，且未扩增出其他鸭常见病原；敏感性结果显示NDRV、NGPV和DTMUV的检测下限分别为8.80×104、4.03×104、2.15×104 copies·μL−1；重复性试验表明该方法具有良好的重复性。采用建立的多重PCR方法对167份临床样品进行检测结果显示，其检测结果与该3种病毒的常规单一PCR检测结果符合率为100%。 结论 建立的多重PCR检测方法具有特异性强、敏感性高、重复性好等优点，适用于临床样品中存在以上3种病原感染的检测和流行病学调查。 Abstract:Objective A PCR method for simultaneous detection of new-type duck reovirus (NDRV), novel goose parvovirus (NGPV), and duck tembusu virus (DTMUV) was developed and tested for clinic applications. Method Separate sets of specific primers were designed based on the conserved regions of NDRV, NGPV, and DTMUV genomes. The target specific amplifications of NDRV, NGPV, and DTMUV were in the regions of 594 bp, 467 bp, and 328 bp, respectively. The specificity, sensitivity, and repeatability of the assay method were determined prior to a trial determination on clinic specimens. Result The newly developed multiplex PCR methodology delivered highly repeatable results and showed a high specificity on the 3 viruses with negative results on other commonly found duck pathogens. The detection sensitivity on NDRV was 8.80×104 copies·μL−1, 4.03×104 copies·μL−1 on NGPV, and 2.15×104 copies·μL−1 on DTMUV. On 167 clinical samples a perfect 100% total coincidence rate between the new and conventional PCR methods were achieved in detecting the 3 viruses. Conclusion The newly established multiplex PCR assay was specific, sensitive, and repeatable in simultaneously detecting NDRV, NGPV, and DTMUV. It was considered adequate for clinic applications. -
Key words:
- NDRV /
- NGPV /
- DTMUV /
- multiplex PCR
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图 1 NDRV、NGPV、DTMUV单一和多重PCR扩增
注：M为DNA Marker DL2000；1为NDRV、NGPV、DTMUV混合液；2为NDRV ；3为NGPV ；4为DTMUV；5为阴性对照。
Figure 1. Single and multiple PCR amplifications of NDRV, NGPV, and DTMUV
Note: M: DNA marker DL2000; 1: mixture of NDRV, NGPV, and DTMUV; 2: NDRV; 3: NGPV; 4: DTMUV; 5: negative control.
图 2 多重PCR特异性检测
注：M为DNA Marker DL2000；1为NDRV、NGPV、DTMUV混合液；2为NDRV；3为NGPV；4为DTMUV；5为MDRV；6为MPV；7为DuCV；8为DHV；9为H9AIV；10为DPV；11为NDV；12为阴性对照。
Figure 2. Specificity of multiplex PCR assay
Note: M: DNA marker DL2000; 1: mixture of NDRV, NGPV, and DTMUV; 2: NDRV; 3: NGPV; 4: DTMUV; 5: MDRV; 6: MPV; 7: DuCV; 8: DHV; 9: H9AIV; 10: DPV; 11: NDV; 12: negative control.
图 3 多重PCR敏感性试验
注：M为DNA Marker DL2000；1～7分别为107～101 copies·μL−1 NDRV+107～101 copies·μL−1 NGPV+107～101 copies·μL−1 DTMUV；8为阴性对照。
Figure 3. Sensitivity of multiplex PCR assay
Note: M: DNA marker DL2000; 1-7: 107−101 copies·μL−1 NDRV+107−101 copies·μL−1 NGPV+107−101 copies·μL−1 DTMUV; 8: negative control.
图 4 多重PCR的重复性试验
注：A、B、C分别代表第1、3、5周的PCR ；M为DNA Marker DL2000；1为NDRV、NGPV、DTMUV混合液；2为NDRV；3为NGPV；4为DTMUV；5为阴性对照。
Figure 4. Repeatability of multiplex PCR assay
Note: A-C: PCR results at week 1, 3, and 5; M: DNA marker DL2000; 1: mixture of NDRV, NGPV, and DTMUV; 2: NDRV; 3: NGPV; 4: DTMUV; 5: negative control.
表 1 本研究使用的引物序列
Table 1. Primer sequences used in this study
引物名称
Primer name引物序列（5′-3′）
Primer sequence目的片段
Target fragment/bp登录号
Login number参考毒株
Reference strainsNDRV-F TCGTCACTACTGTCAAGCTC 594 JQ664689 TH11 NDRV-R TATGTATGAGAGGAGCCACA NGPV-F GGCTCACTGAGAACCGAGAC 467 MW147180 PL157/2019 NGPV-R AGACCCTCCCAAAATTGCTT DTMUV-F GCAGGGTTTGAAGCTGAAAG 328 MN966680 CHN-YC DTMUV-R CCCACTTCTATGCCACTGGT 
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表 2 常规PCR与多重PCR临床样品检测结果比较
Table 2. Results of clinical samples using the multiplex PCR and conventional PCR
临床样品
Clinical samples单项PCR检测结果
Single PCR results多重PCR检测结果
Multiplex PCR results阳性数
Positive阴性数
Negative检出率
Detection rate/%阳性数
Positive阴性数
Negative检出率
Detection rate/%NDRV 4 167 2.39 4 167 2.39 NGPV 9 167 5.39 9 167 5.39 DTMUV 11 167 6.58 11 167 6.58 NDRV+ NGPV 1 167 0.60 1 167 0.60 NGPV+ DTMUV 1 167 0.60 1 167 0.60
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