|
|
|
| Establishment and Application of Liquid Chip Method for Rapid Detection of Seven Porcine (Sus scrofa) Reproductive Diseases |
| WU Xu-Long1, XIAO Lu2,*, YANG Miao3, XIE Li3, AN Wei3, GUO Yang3, ZHANG Qin3, WANG Yin4, LIN Hua3,* |
1 Animal Epidemic Disease Detection Center, Chengdu Agricultural College, Chengdu 611130, China;
2 Key Laboratory of Animal Genetics and Breeding of Sichuan Province, Sichuan Animal Science Academy, Chengdu 610066, China;
3 Key Laboratory of Food Safety Testing of Sichuan Province, Technology Center of Chengdu Customs, Chengdu 610041, China;
4 College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China |
|
|
|
|
Abstract With the development and progress of the pig industry in China, higher expection are required for the differential diagnosis and big data analysis of diseases. Here, a novel method was established, which promoted the development and application of high-throughput detection technology in animal pathogen detection. In detail, specific primers and probes were designed for the Pseudorabies virus (PRV)、Japanese encephalitis virus (JEV)、Classical swine fever virus (CSFV)、Porcine circovirus type 2 (PCV-2)、Porcine reproductive and respiratory syndrome virus (PRRSV)、Porcine parvovirus (PPV)、African classical swine fever virus (ASFV) 7 pathogens of swine (Sus scrofa) reproductive diseases. After optimizing the reaction conditions, coupling the probe with microspheres, and hybridization capture reaction, a liquid chip detection method to detect 7 pig reproductive diseases at the same time was successfully established and applied to clinical practice. The results showed that the specific target gene could be captured and detected in the liquid environment, the detection limit of 7 specific pathogens was 103 copies/μL. The method had good specificity and no cross reaction with other pathogens. The established method was used to detect the 65 samples collected from large-scale pig farms in Sichuan province. A total of 49 positive samples were detected, with a positive rate of 75.38% (49/65). The positive rate of mixed infection of 2 or more pathogens was 23.08% (15/65). The results of single PCR detection were consistent with the established method, which confirmed that the established liquid chip detection method can be applied to clinical detection. This study provides a powerful technical reserve for rapid differential diagnosis of multiple pathogens of swine disease, and provides a reference for the further development of high-throughput detection technology.
|
|
Received: 11 June 2022
|
|
|
|
Corresponding Authors:
*yaanxiaolu@163.com; cdcclh@126.com
|
|
|
|
[1] 陈焕春. 2015. 当前猪病流行动态与防控对策[J]. 国外畜牧学(猪与禽), 35(4): 7-13.
(Chen H C.Current epidemic trend of swine disease and its prevention and control countermeasures[J]. Animal Science Abroad (Pigs and Poultry), 35(4): 7-13.)
[2] 陈铮铮, 吕火烊. 2021. 液相芯片技术进展及在检验医学中应用[J]. 现代实用医学, 33(8): 981-983.
(Chen Z Z, Lv H Y, Progress of liquid chip technology and its application in laboratory medicine[J]. Modern Practical Medicine, 33(8): 981-983.)
[3] 德西措姆, 王印, 杨泽晓, 等. 2019. 2016-2018年四川地区猪流行性腹泻病毒流行病学调查与ORF3、S1部分基因序列分析[J]. 浙江农业学报, 31(9): 1423-1428.
(Dexi C M, Wang Y, Yang Z X, et al.2019. Epidemiological investigation of Porcine epidemic diarrhea virus and sequence analysis of ORF3 and S1 genes in Sichuan from 2016 to 2018[J]. Acta Agriculturae Zhejiangensis, 31(9): 1423-1428.)
[4] 何海健, 刘正奎, 吴瑗, 等. 2020. 猪病毒性腹泻病相关病原的MLPA检测技术的建立及应用[J]. 农业生物技术学报, 28(9): 183-194.
(He H J, Liu Z K, Wu Y, et al.2020. Establishment and application of multiplex ligation-dependent probe amplification assay for identification of pathogens causing porcine viral diarrhea diseases[J]. Journal of Agricultural Biotechnology, 28(9): 183-194.)
[5] 邬旭龙, 肖璐, 姜睿姣, 等. 2016. 靶序列富集多重PCR技术的发展及其应用[J]. 湖南农业大学学报(自科版), 42(2): 172-176.
(Wu X L, Xiao L, Jiang R J, et al.2016. A review on the application of target enriched multiplex PCR[J]. Journal of Hunan Agricultural University (Natural Sciences), 42(2): 172-176.)
[6] 王莹, 刘雨田, 张齐政, 等. 2020. 液相芯片技术在动物疫病诊断中的应用[J]. 动物医学进展, 41(9): 115-118.
(Wang Y, Liu Y T, Zhang Q Z, et al.2020. Application of liquichip technology in diagnose of animal infectious diseases[J]. Progress in Veterinary Medicine, 41(9): 115-118.)
[7] 王子良, 王颖, 彭少杰, 等. 2010. 新型液相芯片系统在食源性致病菌快速检测中的应用研究[J]. 中国卫生检验杂志, 20(3): 462-463.
(Wang Z L, Wang Y, Peng S J, et al.2010. Application of new liquid-chip system in rapid detection of foodborne pathogens[J]. Chinese Journal of Health Laboratory Technology, 20(3): 462-463.)
[8] 肖璐, 邬旭龙, 王印, 等. 2016. 一种新型同时检测6种猪病毒病简化靶序列富集多重PCR的建立和初步应用[J]. 中国兽医科学, 46(6): 695-701.
(Xiao L, Wu X L, Wang Yet al.2016. Development of a novel simplified target enriched multiplex PCR for simultaneously detection of 6 porcine pathogens and its preliminary application[J]. Chinese Veterinary Science, 46(6): 695-701.)
[9] 肖璐. 2017. 7种猪病毒性疫病QIAxcel及Bio-Plex检测方法的建立与初步应用[D]. 硕士学位论文, 四川农业大学, 导师: 王印, pp. 66-70.
(Xiao L.2017. Simultaneous typing of seven porcine virus pathogens by QIAxcel and Bio-Plex and its preliminary application[D]. Thesis for M.S., Sichuan Agricultural University, Supervisor: Wang Y, pp. 66-70.)
[10] 于金辉. 2020. 应用液相芯片技术快速检测常见食源性致病菌的研究[J]. 现代食品, 5(10): 192-194.
(Yu J H.2020. Rapid detection of common foodborne pathogens using liquid chip technology[J]. Modern Food, 5(10): 192-194.)
[11] Chao G Q, Wang Y Y, Zhang S.2016. Colonic mucosal gene expression in irritable bowel syndrome rats by the liquid chip technology[J]. International Journal of Clinical & Experimental Medicine, 9(10): 10751-10755.
[12] Chen R, Yu X L, Gao X B, et al.2015. Bead-based suspension array for simultaneous differential detection of five major swine viruses[J]. Applied Microbiology & Biotechnology, 99(2): 919-28.
[13] Deng J, Luo X, Wang R, et al.2015. A comparison of Luminex xTAG gastrointestinal pathogen panel (xTAG GPP) and routine tests for the detection of enteropathogens circulating in southern China[J]. Diagnostic Microbiology & Infectious Disease, 83(3): 325-330.
[14] Guo X, Wang M, Wang L, et al.2018. Establishment of a molecular serotyping scheme and a multiplexed luminex-based array for Enterobacter aerogenes[J]. Frontiers in Microbiology, 9: 501-510.
[15] Hu L, Lin X Y, Nie F P, et al.2016. Simultaneous typing of seven porcine pathogens by multiplex PCR with a GeXP analyser[J]. Journal of Virological Methods, 232: 21-28.
[16] He M, Wang Y, Wang J, et al.2020, The potential markers involved in newly diagnosed graves' disease and the development of active graves' orbitopathy[J]. Cytokine, 127(23): 154998.
[17] Hiseni P, Wilson R C, Storrø O, et al.2019. Liquid array diagnostics: A novel method for rapid detection of microbial communities in single-tube multiplex reactions[J]. Biotechniques, 66(3): 143-149.
[18] Kiat L C, Kai L H, Siong N, et al.2017. Comparison of luminex NxTAG respiratory pathogen panel and xTAG respiratory viral panel FAST version 2 for the detection of respiratory viruses[J]. Annals of Laboratory Medicine, 37(3): 267-271.
[19] Lombardelli L, Logiodice F, Kullolli O, et al.2021. Evaluation of secreted cytokines by multiplex bead-based assay (X MAP Technology, Luminex)[J]. Methods in Molecular Biology 2285: 121-130.
[20] Liang D W, Lu J H, Wu Q, et al.2016, Comparing the ability of luminex xMAP salmonella serotyping assay and traditional serotyping method for serotyping salmonella isolated from southern Chinese population[J]. Journal of Applied Microbiology, 120(6): 1668-1676.
[21] Lin A, Salvador A, Carter J M.2015. Multiplexed microsphere suspension array-based immunoassays[J]. Methods in Molecular Biology, 1318: 107-118.
[22] Liu Z, Xu Y, Luo J, et al.2016. Target-Enriched multiplex PCR (Tem-PCR) assay for simultaneous detection of Salmonella spp. listeria monocytogenes and Escherichia coli O157:H7 in food[J]. Journal of Food Safety, 36(2): 180-185.
[23] Preeti S, Ningyan W, Adam S C, et al.2015. Comparison of sensitivities of toxin detection in capture ELISA versus a bead-based, singleplex, flow cytometry assay[J]. PLOS ONE, 10(8): e0135986.
[24] Wang N, Song L, Deng T, et al.2020. Microsphere-based suspension array for simultaneous recognition and quantification of multiple cancer-associated miRNA via DNAzyme-mediated signal amplification[J]. Analytica Chimica Acta, 1140: 69-77.
[25] Wu X L, Xiao L, Lin H, et al.2017. A Novel capillary electrophoresis-based high-throughput multiplex polymerase chain reaction system for the simultaneous detection of nine pathogens in swine[J]. BioMed Research International, (1): 1-8. |
|
|
|
