Development and evaluation of TaqMan real-time reverse transcription PCR assays for detection of Salmonella Choleraesuis
ZHANG Jing-shan1, LI Xu2, YAN Mei-ying1, KAN Biao1, FAN Fen-xia1
1. State Key Laboratory for Infections Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Beijing 102206, China; 2. School of Light Industry, Beijing Technology and Business University, Beijing 100048, China
Abstract：A TaqMan real-time reverse transcription polymerase chain reaction (TaqMan-rRT-PCR) assay was established for the detection of Salmonella Choleraesuis on the basis of the SC0358 gene. The specific S. Choleraesuis gene SC0358 was identified through alignment of the genome sequences of S. Choleraesuis with those of other Salmonella serotypes in GenBank and the human genome, and the specificity of SC0358 was confirmed by PCR. The specificity and detection limit of the TaqMan-rRT-PCR assay were evaluated on pure culture strains and S. Choleraesuis simulated blood specimens. All S. Choleraesuis strains in our study were amplified and found to be positive with TaqMan-rRT-PCR assays, whereas other isolates were negative. The detection limit of total RNA from the pure cultured isolates was 5 fg/reaction, equivalent to 10 molecular copies per reaction. For RNA from stimulated blood, the sensitivity was 25 cfu/mL. The established assay based on the conserved and specific gene SC0358 for detecting S. Choleraesuis had high sensitivity and specificity in distinguishing S. Choleraesuis from other serotypes, particularly S. Paratyphi C, with the same antigenic formula. This method should be suitable for rapid detection and diagnosis in early clinical treatment.
张景山, 李旭, 闫梅英, 阚飙, 樊粉霞. TaqMan-rRT-PCR检测猪霍乱沙门菌方法的建立和实验室评价[J]. 中国人兽共患病学报, 2021, 37(5): 410-415.
ZHANG Jing-shan, LI Xu, YAN Mei-ying, KAN Biao, FAN Fen-xia. Development and evaluation of TaqMan real-time reverse transcription PCR assays for detection of Salmonella Choleraesuis. Chinese Journal of Zoonoses, 2021, 37(5): 410-415.
[1] Nair S, Farzan A, O’Sullivan TL, et al. Time course of Salmonella shedding and antibody response in naturally infected pigs during grower-finisher stage[J]. Can J Vet Res, 2018, 82(2):139-145. [2] Chiu CH, Su LH, Chu C.Salmonella enterica serotype Choleraesuis: epidemiology, pathogenesis, clinical disease, and treatment[J]. Clin Microbiol Rev, 2004, 17(2):311-322. DOI: 10.1128/cmr.17.2.311-322.2004 [3] Chen YH, Chen TP, Tsai JJ, et al.Epidemiological study of human salmonellosis during 1991-1996 in southern Taiwan[J]. Kaohsiung J Med Sci, 1999, 15(3):127-136. [4] Chiu CH, Chuang CH, Chiu S, et al.Salmonella enterica serotype Choleraesuis infections in pediatric patients[J]. Pediatrics, 2006, 117(6):e1193-1196. DOI: 10.1542/peds.2005-2251 [5] Feasey NA, Dougan G, Kingsley RA, et al.Invasive non-typhoidal Salmonella disease: an emerging and neglected tropical disease in Africa[J]. Lancet, 2012, 379(9835):2489-2499. DOI: 10.1016/S0140-6736(11)61752-2 [6] Chiu CH, Wu TL, Su LH, et al.Fluoroquinolone resistance in Salmonella enterica serotype Choleraesuis, Taiwan, 2000-2003[J]. Emerg Infect Dis, 2004, 10(9):1674-1676. DOI: 10.3201/eid1009.030596 [7] 张洲慧,彭珉娟,陈娟.34例儿童败血症病原菌及耐药性分析[J].四川医学,2011,32(6):838-841. DOI: 10.3969/j.issn.1004-0501.2011.06.018 [8] 余丰,梁飞立,邓梅花,等.艾滋病合并败血症44例临床特点和病原菌分析[J].中国热带医学,2012,12(8):967-969. DOI:10.13604/j.cnki.46-1064/r.2012.08.031 [9] Anderson RC, Nisbet DJ, Buckley SA, et al.Experimental and natural infection of early weaned pigs with Salmonella choleraesuis[J]. Res Vet Sci, 1998, 64(3):261-262. DOI:10.1016/S0034-5288(98)90136-9 [10] Osumi T, Asai T, Namimatsu T, et al.Enrichment for isolating Salmonella Choleraesuis and other Salmonella spp. from pigs[J]. J Vet Med Sci, 2003, 65(8):949-951. DOI: 10.1292/jvms.65.949 [11] Vassiliadis P. The Rappaport-Vassiliadis (RV) enrichment medium for the isolation of Salmonellas: an overview[J]. J Appl Bacteriol, 1983, 54(1):69-76. DOI: 10.1111/j.1365-2672.1983.tb01302.x [12] Janyapoon K, Korbsrisate S, Thamapa H, et al.Rapid detection of Salmonella enterica serovar Choleraesuis in blood cultures by a dot blot enzyme-linked immunosorbent assay[J]. Clin Diagn Lab Immunol, 2000, 7(6):977-979. DOI: 10.1128/cdli.7.6.977-979.2000 [13] Chiu TH, Pang JC, Hwang WZ, et al.Development of PCR primers for the detection of Salmonella enterica serovar Choleraesuis based on the fliC gene[J]. J Food Prot, 2005, 68(8):1575-1580.DOI: 10.4315/0362-028x-68.8.1575 [14] 朱超,许学斌.沙门菌属血清型诊断[M].上海:上海同济大学出版社,2009:302-325. [15] Popoff MY, Bockemuhl J, Gheesling LL.Supplement 2001 (no. 45) to the Kauffmann-White scheme[J]. Res Microbiol, 2003, 154(3):173-174. DOI:10.1016/S0923-2508(03)00025-1 [16] McQuiston JR, Parrenas R, Ortiz-Rivera M, et al. Sequencing and comparative analysis of flagellin genes fliC, fljB, and flpA from Salmonella[J]. J Clin Microbiol, 2004, 42(5):1923-1932. DOI: 10.1128/jcm.42.5.1923-1932.2004 [17] 陈弟诗,郭万柱,徐志文,等.猪霍乱沙门菌的分离与鉴定及PCR检测方法的建立[J].安徽农业科学,2007,35(20):6020-6023. DOI:10.3969/j.issn.0517-6611.2007.20.063 [18] 林一曼,石晓路,朱玉梅,等.实时荧光PCR同时检测丙型副伤寒沙门菌和猪霍乱沙门菌方法的建立和应用[J]. 热带医学杂志,2012,12(2):168-171. [19] 高瑞红,曹阳,闫梅英.基于单基因的实时荧光定量反转录-聚合酶链反应定性检测猪霍乱沙门菌[J].疾病监测,2016,31(6):507-511. DOI:10.3784/j.issn.1003-9961.2016.06.015 [20] Echeita MA, Herrera S, Garaizar J, et al.Multiplex PCR-based detection and identification of the most common Salmonella second-phase flagellar antigens[J]. Res Microbiol, 2002, 153(2):107-113. DOI: 10.1016/s0923-2508(01)01295-5 [21] Echeita MA, Usera MA.Rapid identification of Salmonella spp. phase 2 antigens of the H1 antigenic complex using "multiplex PCR"[J]. Res Microbiol, 1998,149(10):757-761. DOI: 10.1016/s0923-2508(99)80022-9 [22] Oliveira SD, Santos LR, Schuch DM, et al.Detection and identification of Salmonellas from poultry-related samples by PCR[J]. Vet Microbiol, 2002, 87(1):25-35. DOI: 10.1016/s0378-1135(02)00028-7 [23] Woo PC, Fung AM, Wong SS, et al.Isolation and characterization of a Salmonella enterica serotype Typhi variant and its clinical and public health implications[J]. J Clin Microbiol, 2001, 39(3):1190-1194. DOI: 10.1128/JCM.39.3.1190-1194.2001 [24] Fan F, Du P, Kan B, et al.The development and evaluation of a loop-mediated isothermal amplification method for the rapid detection of Salmonella enterica serovar Typhi[J]. PLoS One, 2015,10(4):e0124507. DOI: 10.1371/journal.pone.0124507 [25] Fan F, Yan M, Du P, et al.Rapid and sensitive Salmonella Typhi detection in blood and fecal samples using reverse transcription loop-mediated isothermal amplification[J]. Foodborne Pathog Dis, 2015,12(9):778-786. DOI: 10.1089/fpd.2015.1950
2021, Vol. 37 
