猪传染性胃肠炎病毒S基因RAA检测方法的建立与初步应用

doi:10.11843/j.issn.0366-6964.2024.08.030

摘要/Abstract

摘要：

本研究旨在建立一种基于猪传染性胃肠炎病毒(porcine transmissible gastroenteritis virus，TGEV)S基因便捷、高效和特异的由重组酶介导的核酸等温扩增(recombinase-aid amplification，RAA)检测方法。通过基因组序列比对，选择TGEV S基因中特定片段为检测靶标，构建重组质粒pUC57-S，设计并筛选RAA引物和探针；建立RAA检测方法；评价该方法灵敏度、特异性和重复性；运用该方法检测腹泻临床样品，与荧光定量PCR方法比较检测结果的符合率。结果确定最佳引物对F2/R1和探针PF2；该方法在40 ℃恒温条件下，30 min即可完成反应；荧光型RAA法最低检出限可达1.34×10¹ copies·μL^-1；与猪流行性腹泻病毒、猪丁型冠状病毒、猪呼吸道冠状病毒等常见猪源病毒核酸无交叉反应；采用该方法检测107份临床样本，阳性率为5.61%(6/107)，与荧光定量PCR法相比较，检测结果一致。本研究成功建立了一种灵敏、特异、可视化的RAA检测方法，可摆脱对昂贵设备、专业人员和检测技术的依赖，为临床检测TGEV提供良好的技术基础。

关键词: 猪传染性胃肠炎病毒, 基础型RAA, 荧光型RAA, 可视化检测

Abstract:

The aim of this study was to establish a convenient, efficient and specific recombinase-aid amplification (RAA) assay based on the porcine TGEV S gene. Comparing the genome sequence, selecting specific segment of TGEV S gene to construct the recombinant plasmid pUC57-S as templete. RAA primers and probes were designed and screened. RAA assays were established. The sensitivity, specificity and repeatability of the method were evaluated. Verifying the established test method by clinical samples of diarrhea, and compare the consistency with the testing results of the fluorescence quantitative PCR assay. Our results showed that the best primer pair F2/R1 and probe were screened out. The assay can be completed in 30 min at 40 ℃. The lowest detection limit of fluorescent RAA assay was 1.34×10¹ copies·μL^-1. There was no cross reaction with other virus nucleic acid, as porcine epidemic diarrhea virus, porcine delta coronavirus, porcine respiratory coronavirus and other common swine viruses. A total of 107 clinical samples were tested using this method, and positive rate was 5.61% (6/107), which was consistent with the test results of fluorescence quantitative PCR. This study successfully established a sensitive, specific and visual RAA test method, which can get rid of the dependence of expensive equipment, the professional and technical detection technology, and provided a reliable technical basis for the clinical detect of TGEV.

Key words: porcine transmissible gastroenteritis virus, basic RAA, fluorescent RAA, visual detection

中图分类号:

S852.659.6

吕林丹, 牟豪, 胡霞, 刘明妮, 李绍梅, 李星, 宋振辉, 杨柳. 猪传染性胃肠炎病毒S基因RAA检测方法的建立与初步应用[J]. 畜牧兽医学报, 2024, 55(8): 3590-3599.

Lindan LÜ, Hao MU, Xia HU, Mingni LIU, Shaomei LI, Xing LI, Zhenhui SONG, Liu YANG. Establishment and Preliminary Application of RAA Assay for the Detection of Porcine Transmissible Gastroenteritis Virus based on S Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3590-3599.

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编号 Number	引物序列 Primer sequences	引物组合 Primer pairing	产物大小/bp Product size/bp
TGEV-F1	5′-GGTGTTAGGTGATTATTTTCCTACTGTACA-3′	TGEV-F1/TGEV-R1	308
TGEV-F2	5′-ACGGTTAAACGTAGTCGTTAATGGATACCC-3′	TGEV-F2/TGEV-R1	164
TGEV-F3	5′-CGGTTAAACGTAGTCGTTAATGGATACCCA-3′	TGEV-F3/TGEV-R1	163
TGEV-R1	5′-ACCTGCACTCACTACCCCAATTGCAAGTCA-3′	TGEV-F1/TGEV-R2	309
TGEV-R2	5′-AACCTGCACTCACTACCCCAATTGCAAGTC-3′	TGEV-F2/TGEV-R2	165
		TGEV-F3/ TGEV-R2	164

编号 Number	探针和标记引物序列 Probe and labeled primer sequences
TGEV-PF1	5′-AACAACCCGCAATTTTAATTCTGCTGAAGGTGCT//THF//TTATATGCATTTGCAA-3′ (5′-端FAM修饰，3′-端C3 Spacer修饰，中间/THF/间隔及BHQ修饰)
TGEV-PF2	5′-AACAACCCGCAATTTTAATTCTGCTGAAGGTGCT//THF//TTATATGCATTTGCAA-3′ (5′-端FITC修饰，3′-端C3 Spacer修饰，中间/THF/间隔及BHQ修饰)
TGEV-PR1	5′-ACCTGCACTCACTACCCCAATTGCAAGTCA-3′(5′-端Biotin修饰)

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