Establishment and preliminary application of fluorescent RT-RAA diagnostic method for porcine epidemic diarrhea virus
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摘要:
目的 建立一种快捷、灵敏、简便检测猪流行性腹泻病毒（porcine epidemic diarrhea virus, PEDV）的RT-RAA检测方法，以提高猪流行性腹泻病毒临床检测效率。 方法 针对PEDV S基因片段保守区设计引物和探针，构建标准质粒PEDV-S，通过特异性、敏感性、重复性测定及条件优化，建立检测PEDV重组酶介导链置换核酸扩增荧光法（RT-RAA）。 结果 在42 ℃恒温作用20 min的条件下，建立的检测方法对检测猪传染性胃肠炎病毒（porcine transmissible gastroenteritis virus, TGEV）、猪瘟病毒（classical swine fever virus, CSFV）、伪狂犬病病毒（porcine pseudorabies virus, PRV）、猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus, PRRSV）和猪轮状病毒（porcine rotavirus, PoRV）等猪源病毒均为阴性，猪流行性腹泻病毒（PEDV）为阳性；最低检出限为4.43×102拷贝·μL−1的标准质粒；重复性结果显示，相同浓度标准质粒的差异性很小；利用建立的RT-RAA方法检测40份猪病毒样本，阳性率均为7.5%（3/40），与实时荧光定量PCR检测结果相同。 结论 本研究建立的PEDV RT-RAA检测方法具有快速简便、耗时短、特异性高、灵敏度强和重复性好等特点，适用于对PEDV的快速诊断。 Abstract:Objective To establish a quick, simple, sensitive detection of porcine epidemic diarrhea virus (PEDV) the RT- RAA detection method, to improve the efficiency of porcine epidemic diarrhea virus clinical detection. Method Primers and probes were designed for the conserved region of PEDVS gene fragment, and a standard plasmid PEDV-S was constructed. Through specificity, sensitivity, repeatability and condition optimization, a recombinant enzyme-mediated chain replacement nucleic acid amplification fluorescence assay (RT-RAA) for detection of PEDV was established. Result Under the condition of constant temperature at 42 ℃ for 20 min,The established assay is effective for the detection of porcine transmissible gastroenteritis virus (TGEV), classical swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV) and other porcine viruses were negative, porcine epidemic diarrhea virus (PEDV) was positive.The minimum detection limit was 4.43× 102 copies·μL−1 standard plasmid.The reproducibility results showed that there was little difference between the standard plasmids with the same concentration.The positive rate of 40 swine virus samples was 7.5% (3/40) by RT-RAA method, which was the same as that of real-time fluorescence quantitative PCR. Conclusion The RT-RAA detection method for PEDV established in this study is suitable for the rapid diagnosis of PEDV due to its rapid and simple detection, short time-consuming, high specificity, strong sensitivity and good reproducibility. -
Key words:
- porcine epidemic diarrhea virus /
- S protein /
- RT-RAA
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图 1 引物及探针所在S基因保守区域
Figure 1. The conserved region of the S gene where the primers and probes are located
图 2 PEDV S 基因PCR扩增
M：DL2000 Marker；1～4：PEDV S 基因扩增；5：阴性对照。
Figure 2. PCR amplification of the PEDV S gene
M：DL2000 Marker；1–4: PEDV S gene amplification; 5: negative control.
图 3 荧光型RT-RAA引物筛选
1：F3/R3；2：F3/R2；3：F1/R1；4：F2/R3；5：F3/R1；6：F1/R2；7：F2R2；8：F1/R3；9：F2/R1；10：阴性对照。
Figure 3. Fluorescent special-pull primer screening
1: F3/R3; 2: F3/R2; 3: F1/R1; 4: F2/R3; 5: F3/R1; 6: F1/R2; 7: F2/R2; 8: F1/R3; 9: F2/R1; 10: negative control.
图 4 荧光型RT-RAA反应温度的筛选
1～3分别为42、39和37 ℃。
Figure 4. Screening of fluorescent RT-RAA reaction temperatures
1–3was 42, 39 and 37 °C, respectively.
图 5 荧光型RT-RAA反应时间的筛选
1～3分别为反应20、25、17min。
Figure 5. Screening of fluorescent RT-RAA reaction time
1–3 was 20, 25 and 17 min for reaction.
图 6 荧光型RT-RAA特异性试验
1：PEDV-S；2～8：TGEV、CSFV、PRV、PCV、PRRSV、PoRV、阴性对照。
Figure 6. Fluorescent RT-RAA specificity test
1: PEDV-S; 2–8: TGEV, CSFV, PRV, PCV, PRRSV, PoRV, negative control.
图 7 荧光型RT-RAA的重复性检测
1～3：1×105拷贝·μL−1；4～6：1×103拷贝·μL−1；7～9：1×102拷贝·μL−1。
Figure 7. Repeatability detection of fluorescent RT-RAA
1–3: 1×105 copies·μL−1; 4–6: 1×103 copies·μL−1; 7–9: 1×102 copies·μL−1.
图 8 荧光型RT-RAA敏感性试验
1～7：标准质粒依次为106～100 拷贝·μL−1；8：阴性对照。
Figure 8. Fluorescence RT-RAA sensitivity test
1–7: the standard plasmid was 106–100 copies·μL−1; 8: negative control.
表 1 疑似猪流行性腹泻病料收集的来源信息
Table 1. Source information of sample collection for suspected porcine epidemic diarrhea
地区
Region样本数（份）/来源猪场（个）
Samples/Farms宁德 Ningde 5/2 三明 Sanming 6/2 龙岩 Longyan 10/6 漳州 Zhangzhou 8/3 莆田 Putian 11/6 合计 total 40/19 
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表 2 引物和探针
Table 2. Primers and probes
名称
Name序列（5′-3′）
SequencePEDV S-DF AAATCTGGCAGTATTGGCTAC PEDV S-DR ATCGGCTGAAAGAATGTCC PEDV S-F1 TATTCCCACCAACTTTAGTATGAGTATTAG PEDV S-F2 GTATTCCCACCAACTTTAGTATGAGTATTA PEDV S-F3 AGTATTCCCACCAACTTTAGTATGAGTATT PEDV S-R1 TAATGCTGACTCTATGGTCTTACATGCTGC PEDV S-R2 GTTGTAATGCTGACTCTATGGTCTTACATG PEDV S-R3 TGTAATGCTGACTCTATGGTCTTACATGCT PEDV S-P GACAGAATATTTACAGCTTTACAACACGCC(i6FAMdT)(THF)(iBHQ1dT)TAGTGTTGATTGTGC-C3spacer
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表 3 临床样本的检测
Table 3. Detection of clinical samples
方法
Method阳性样品
Number of
positives/份阴性样品
Number of
negatives/份总数
Total/份阳性率
Positivity
rate/%RT-RAA 3 37 40 7.5 RT-qPCR 3 37 40 7.5
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