A Rapid Real Time Fluorescence Recombinase-aided Amplification Method for Detecting Porcine Pseudorabies Virus
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摘要:目的 基于荧光重组酶介导核酸扩增（Recombinase-aided amplification, RAA）技术，建立一种猪伪狂犬病毒（Porcine pseudorabies virus, PRV）快速检测方法。方法 根据PRV gE基因序列，设计特异性引物及探针，优化扩增体系，建立PRV荧光重组酶介导核酸扩增检测方法，检验其特异性、敏感性和重复性，应用该方法对临床样品进行检测。结果 该方法在43 ℃恒温反应23 min即可完成PRV核酸扩增，最低检出限为111 copies·μL−1；与猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus, PRRSV）、猪流行性腹泻病毒（Porcine epidemic diarrhea virus, PEDV）、猪轮状病毒（Porcine rotavirus, PoRV）、猪传染性胃肠炎病毒（Transmissible gastroenteritis virus, TGEV）、猪圆环病毒2型（Porcine circovirus 2, PCV2）、猪圆环病毒3型（Porcine circovirus 3, PCV3）均无交叉反应。重复性试验显示，组内和组间变异系数均小于5%；40份临床样品检测结果显示PRV阳性率为15%（6/40），检测结果与常规聚合酶链式反应（PCR）一致。结论 成功建立了简便快速、高效准确的PRV实时荧光RAA检测方法，为PRV的快速检测和流行病学调查提供了新的检测手段。
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关键词:
- 猪伪狂犬病毒 /
- 荧光重组酶介导核酸扩增 /
- 检测方法
Abstract:Objective A rapid method applying the recombinase-aided amplification (RAA) method for detecting porcine pseudorabies virus (PRV) was developed.Methods Based on the sequence of gE gene in PRV, specific primers and probes were designed. Amplification conditions were optimized, and assay specificity, sensitivity, and reproducibility scrutinized by a verification trial on clinical samples.Results The newly developed assay successfully amplified the PRV nucleic acids in merely 23m under the constant temperature of 43 ℃ with a detection limit of 111 copies·μL−1. There were no cross reactions with viruses that produced reproductive and respiratory syndromes, epidemic diarrhea, rotavirus, transmissible gastroenteritis, circovirus 2, or circovirus 3 on pigs. The coefficients of variation within a group and between groups on the repeatability test were less than 5%. And, on 40 clinical samples, the positive detection on PRV of the assay was 15% (6/40), which was comparable to that of the conventional PCR.Conclusion A simple, rapid, efficient, and accurate method of fluorescence RAA detection on PRV was established for laboratory testing and epidemiological investigation of the disease. -
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图 7 临床样品PRV的PCR和RAA检测
A：临床样品RAA结果。B：临床样品PCR结果；M：DL 2000 Marker；1：阴性对照；2：阳性对照；3~42：临床样品。
Figure 7. PRV detections by conventional PCR and fluorescent RAA on clinical samples
A: results on clinical samples by fluorescent RAA; B: results on clinical samples by conventional PCR; M: DL 2000 marker; 1: negative control ; 2: positive control ; 3–42: clinical samples.
表 1 疑似伪狂犬病料收集的来源信息
Table 1 Tissue collection of suspected diseased pigs
地区
Region样本数（份）/来源猪场（个）
Samples/Farms宁德 Ningde 4/2 泉州 Quanzhou 5/3 漳州 Zhangzhou 10/4 南平 Nanping 12/5 龙岩 Longyan 9/4 合计 Total 40/18 
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表 2 PRV荧光RAA引物与探针
Table 2 Primers and probes of PRV for fluorescent RAA
引物/探针
Primer/Probe引物序列（5'-3'）
Sequence（5'-3'）用途
UsagePRV-F1 CGATCTACGTGGACGGCATCACGACGCCG 识别并结合到PRV-gE DNA片段的特定区域，启动扩增过程。 PRV-R1 TAGTAGTCCTCGTGCGTGGGCAGGCTGGTGTA PRV-F2 CGAGTACGTCACGGTCATCAAGGAGCTGAC PRV-R2 GCTGGTGTACACCGGAGAGAGCATGTGCGT PRV-Probe GCTGTTTGTGCTGGCGCTGGGCTCCTTCG[FAM-dT]
[THF]A[BHQ1-dT]GACGTGCGTCGTC-C3用于检测和量化扩增过程中产生的特定核酸序列
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表 3 荧光重复性试验结果
Table 3 Repeatability of fluorescent RAA
质粒浓度
Plasmid
concentration/
（copies·μL−1）组内变异试验
Intra-assay variability组间变异试验
Inter-assay variability循环数
¯X+SD变异系数CV/% 循环数
¯X+SD变异系数CV/% 1.11×104 16.9±0.33 1.98 17.27±0.77 4.52 1.11×105 15.09±0.34 2.31 12.09±0.49 4.09 1.11×106 9.38±0.25 2.76 8.74±0.30 3.45
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