Duplex
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摘要:
目的 实现猪流行性腹泻病毒（PEDV）和猪传染性胃肠炎病毒（TGEV）的快速鉴别诊断，并调查分析福建省2019–2021年PEDV和TGEV流行情况。 方法 根据PEDV的N 基因和TGEV的S 基因序列分别设计特异性引物和标记FAM、VIC荧光报告基团的探针，建立、优化双重荧光定量PCR反应条件和体系，并检测其敏感性、特异性和重复性。应用该方法对福建省2019–2021年收集的297份疑似腹泻病料进行病原检测。 结果 建立的方法对PEDV和TGEV的最低检测限均为10拷贝·μL−1，比普通PCR检测灵敏度提高100倍；对其他常见猪病原不发生非特异性反应；批内、批间变异系数均小于1%。福建省297份临床样本检测结果显示，单PEDV感染率为88.89%，单TGEV感染率为1.01%，PEDV和TGEV混合感染率为3.37%。 结论 本研究建立的方法具有敏感性高、特异性强、稳定性好等特点。福建省内2019–2021年以PEDV感染为主，TGEV感染率低，存在PEDV、TGEV混合感染的情况。 Abstract:Objective A TaqMan probe-based duplex real-time PCR for rapid detection of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) was developed. A study was conducted using the methodology to analyze the related 2019–2021 epidemic occurred in Fujian. Method Specific primers and probes labeled with FAM and VIC were designed to amplify the N gene of PEDV and the S gene of TGEV, respectively. A reaction system for the assay was established, optimized, and tested for sensitivity, specificity, and repeatability. The assay was used for the viral detection on 297 suspected clinic specimens collected from 2019 to 2021 for an epidemiology study. Result The newly developed duplex qPCR methodology showed a sensitivity of 10 copies·μL−1 on PEDV and TGEV, which was 100 times higher than that of regular PCR. There were no cross reactions with other common viruses. The inter- and intra-assays had variations on Ct values below 1%. On the 297 specimens, the infection rate of PEDV was 88.89%, that of TGEV 1.01%, and that of both PEDV and TGEV 3.37%. Conclusion The established duplex qPCR had high sensitivity, specificity, repeatability, and reproducibility for detecting PEDV and TGEV. The 2019–2021 epidemic involving the viruses appeared to be mostly PEDV with low incidents of mixed TGEV and PEDV/TGEV infection. -
图 1 PEDV和TGEV阳性重组质粒鉴定结果
M为Marker DL2000；1为PEDV；2为TGEV。
Figure 1. Identification of PEDV- and TGEV-positive recombinant plasmids
M: Marker DL2000; 1: PEDV; 2: TGEV.
图 2 PEDV(N)和TGEV(S) real-time PCR标准曲线
Figure 2. qPCR standard curve for PEDV (N) and TGEV (S)
图 3 双重荧光PCR体系的特异性检测
1为PEDV；2为TGEV；3–8分别为CSFV、PRV、PRRSV、PoRV、PDCoV、PCV-2。
Figure 3. Specificity of qPCR assay for PEDV and TGEV detection
1: PEDV; 2: TGEV; 3–8: CSFV, PRV, PRRSV, PoRV, PDCoV, and PCV-2.
图 4 PEDV(A)和TGEV(B)荧光定量PCR体系敏感性检测
Figure 4. Sensitivity of qPCR assay for PEDV (A) and TGEV (B)
图 5 PEDV和TGEV普通PCR体系敏感性检测
M：Marker DL2000；1–6分别为PEDV质粒浓度1×106 ～ 1×101拷贝·μL−1；8–13分别为TGEV质粒浓度1×106 ～ 1×101拷贝·μL−1，7和14为阴性对照。
Figure 5. Sensitivity of regular PCR for PEDV and TGEV
M: Marker DL2000; PEDV (1-6) and TGEV (8-13) with numbers of copies of 1×106, 1×105, 1×104, 1×103, 1×102, and 1×101; 7 and 14: negative.
图 6 2019–2021年福建省各地区不同季节PEDV阳性检出比例
Figure 6. Proportion of PEDV-positive pathogenic cases in different seasons 2019-2021 in Fujian
表 1 PEDV、TGEV双重荧光定量PCR引物及探针序列
Table 1. Primers and probes of PEDV and TGEV for qPCR
病毒
Virus引物名称
Primer序列(5'- 3')
Sequence (5'- 3')片段大小
Amplified fragment/bp猪流行性腹泻病毒
PEDVPEDV-F CTCCCGAGTGTAGTTGAGATTG 175 PEDV-R CTCCACGACCCTGGTTATTT PEDV-P FAM–CAACCCAACACACCTCCTACTTCACG-BHQ1 传染性胃肠炎病毒
TGEVTGEV-F TGAGGGTGCTGGCTTTGAT 149 TGEV-R CAAGAGTGACACCACCCGTT TGEV-P VIC-CACTGTGGCACCCTTAC
CTGATTGT-BHQ1
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表 2 疑似腹泻病料收集的来源信息
Table 2. Information on collection of suspected diseased tissues
地区
Region样本数（份）/来源猪场（个）
Samples/ Farms2019年 2020年 2021年 福州 Fuzhou 7/2 25/6 28/19 宁德 Ningde 5/3 15/6 24/13 泉州 Quanzhou 3/2 12/4 21/18 漳州 Zhangzhou 4/3 13/5 18/17 南平 Nanping 7/5 15/7 26/19 三明 Sanming 6/4 11/8 17/12 龙岩 Longyan 3/2 14/12 23/11 合计 Total 35/21 105/48 157/109
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表 3 PEDV和TGEV双重荧光定量PCR重复性试验
Table 3. Repeatability and stability of duplex qPCR on PEDV and TGEV detections
质粒 Plasmid 浓度Concentration/
（拷贝·μL−1）组内变异试验 Intra-assay variability 组间变异系数 Inter-assay variability 平均数$\overline { X} $±SD 变异系数
CV/%平均数$\overline { X} $±SD 变异系数
CV/%T-PEDV 1.0×102 31.57±0.22 0.69 31.50±0.30 0.93 1.0×103 28.46±0.13 0.45 28.35±0.2 0.71 1.0×104 25.44±0.11 0.45 25.31±0.19 0.75 T-TGEV 1.0×102 32.06±0.28 0.87 31.83±0.33 0.96 1.0×103 28.87±0.11 0.38 28.36±0.19 0.69 1.0×104 25.82±0.16 0.62 25.39±0.23 0.91
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表 4 临床样品检测结果
Table 4. Test results on clinical specimens
检测结果 Test results 猪流行性腹泻病毒PEDV 猪传染性胃肠炎病毒TGEV 猪流行性腹泻病毒+猪传染性胃肠炎病毒PEDV+TGEV 样本阳性率 Rate of positive samples/% 88.89 (264/297) 1.01 (3/297) 3.37 (10/297) 病毒拷贝数 Virus copies 1.8×102～6.5×107 3.3×103～7.5×106 1.5×102～7.5×105 阳性猪场比例 Rate of positive farms/% 61.24 (109/178) 1.12 (2/178) 2.81 (5/178)
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