|
|
|
| A rapid GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of 5 subtypes of diarrheogenic Escherichia coli |
| CHEN Jiancai, CHEN Honghu, ZHANG Yunyi, ZHANG Junyan, ZHANG Zheng, PAN Junhang, ZHAN Li
|
| Department of Microbiology,Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, Zhejiang 310051, China |
|
|
|
|
Abstract Objective To establish a rapid GeXP-based multiplex reverse transcription-PCR assay (GeXP assay) for simultaneous detection of 5 subtypes of diarrheogenic Escherichia coli. Methods Specific primers were designed according to reserved sequences of 12 virulence genes in enterotoxigenic E. coli (ETEC), enterinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (ETEC), and PCR amplification was performed with a single pair of primers to validate the specificity of PCR assay with a single template and a single pair of primers. The specificity of the GeXP assay was evaluated with the genomic DNA of 5 subtypes of diarrheogenic E. coli as the template in a mixture of 12 pairs of primers, and the sensitivity of the GeXP assay was evaluated with the mixed suspensions of 5 subtypes of diarrheogenic E. coli at concentrations of 106, 105, 104 and 103 CFU/mL as the template. Foods purchased from supermarkets and agricultural retail markets were prepared into 34 spiked samples, and 5 subtypes of diarrheogenic E. coli were detected using the GeXP assay and compared with the fluorescent real-time multiple PCR assay. Results The sizes of sth, pic, bfpB, astA, lt, escV, aggR, stx1, uidA, invE, stx2 and stp genes amplification products were consistent with expected sizes using a single template and a single pair of primers, with a fluorescent signal intensity of more than 25 000 A.U. The sizes of the GeXP assay amplification products of 12 virulence genes in 5 subtypes of diarrheogenic E. coli were consistent with expected sizes, with a high specificity. If the concentration of the mixed suspensions of 5 subtypes of diarrheogenic E. coli was 103 CFU/mL, the GeXP assay was effective for simultaneous detection of 12 virulence genes, with a high fluorescent signal intensity, consistent repeated detection results and a less than 10% coefficient of variation. The GeXP assay detected 3 ETEC isolates, 12 EAEC isolates, one EIEC isolate, one EPEC isolate and one EHEC isolate among the 34 spiked samples, which was in agreement with the detection of 5 subtypes of diarrheogenic E. coli with commercial fluorescent real-time multiple PCR assay kits. Conclusions A GeXP assay has been successfully established for simultaneous detection of 12 virulence genes in diarrheogenic E. coli, which is effective for clinical differential diagnosis and epidemiological surveys of diarrheogenic E. coli.
|
|
Received: 27 June 2022
Revised: 05 September 2022
Published: 30 September 2022
|
|
|
|
|
|
[1] PHILIPSBORN R,AHMED S M,BROSI B J,et al.Climatic drivers of diarrheagenic Escherichia coli incidence:a systematic review and meta-analysis[J].J Infect Dis,2016,214(1):6-15.
[2] 郑书发,余斐,陈晓,等.2009—2014年浙江省哨点医院急性腹泻患者病原监测研究[J].中华预防医学杂志,2016,50(12):34-39.
ZHENG S F,YU F,CHEN X,et al.Monitoring and research on pathogen spectrum in patients with acute diarrhea from sentinel hospital of Zhejiang Province during 2009 to 2014[J].Chin J Prev Med,2016,50(12):34-39.
[3] 洪程基,李毅,孙宝昌,等.婴幼儿感染性腹泻病原微生物及耐药性分析[J].浙江预防医学,2016,28(5):453-457.
HONG C J,LI Y,SUN B C,et al.An analysis on the distribution and drug resistance of pathogenic bacteria in infectious diarrhea among infants[J].Zhejiang J Prev Med,2016,28(5):453-457.
[4] 陈云,袁永娟,朱建民,等.浙江嘉善2014年—2015年感染性腹泻监测结果分析及菌株耐药性研究[J].中国卫生检验杂志,2017,14(3):400-403.
CHEN Y,YUAN Y J,ZHU J M,et al.Analysis of surveillance results of infectious diarrhea in Jiashan,Zhejiang from 2014 to 2015 and research on the antibiotic resistance of isolates[J].Chin J Health Lab Technol,2017,14(3):400-403.
[5] 张睿,赵雪琴,商晓春,等.杭州地区婴幼儿腹泻病原菌构成与耐药性分析[J].中国卫生检验杂志,2015,12(6):901-903.
ZHANG R,ZHAO X Q,SHANG X C,et al.Analysis of pathogens isolated constitution and drug resistance of nurseling diarrhea in Hangzhou area[J].Chin J Health Lab Technol,2015,12(6):901-903.
[6] 李平,戎江瑞,陆璐,等.鄞州区食源性疾病监测结果分析[J].预防医学,2020,32(1):77-79.
LI P,RONG J R,LU L,et al.Surveillance data analysis of foodborne diseases in Yinzhou District[J].Prev Med,2020,32(1):77-79.
[7] DENAMUR E,CLERMONT O,BONACORSI S,et al.The population genetics of pathogenic Escherichia coli[J].Nat Rev Microbiol,2021,19(1):37-54.
[8] 杨梦婕,任佳,李洋,等.GeXP多重聚合酶链式反应法检测5种常见食源性致病菌[J].中国食品卫生杂志,2020,32(4):386-390.
YANG M J,REN J,LI Y,et al.Detection of 5 kinds of common foodborne pathogens by GeXP multiplex polymerase chain reaction[J].Chin J Food Hyg,2020,32(4):386-390.
[9] MU X,CHEN W,SHI J,et al.Development of a GeXP-based multiplex RT-PCR assay for detection of long noncoding RNA in hepatocellular carcinoma[J].Lab Med,2019(2):180-188.
[10] 杨梦婕,徐玲丽,马学军,等.肉及肉制品中6种致病菌的GeXP多重PCR检测[J].食品研究与开发,2021,42(13):168-173.
YANG M J,XU L L,MA X J,et al.GeXP multiplex PCR assays for detection of six pathogenic bacteria in meat and meat products[J].Food Res Dev,2021,42(13):168-173.
[11] LUO S,XIE Z,HUANG J,et al.Simultaneous differentiation of the N1 to N9 neuraminidase subtypes of avian influenza virus by a GeXP analyzer-based multiplex reverse transcription PCR assay[J/OL].Front Microbiol,2019(10)[2022-09-05].http://doi.org/10.3389/fmicb.2019.01271.
[12] WANG H,GU J,LI X,et al.Broad range detection of viral and bacterial pathogens in bronchoalveolar lavage fluid of children to identify the cause of lower respiratory tract infections[J/OL].BMC Infect Dis,2021,21 (1)[2022-09-05].http://doi.org/10.1186/s12879-021-05834-0.
[13] MARQUATDT P,WERTHMANN B,RÄTZEL V,et al.Quantifying 35 transcripts in a single tube:model-based calibration of the GeXP multiplex RT-PCR assay[J/OL].BMC Biotechnol,2021 ,21(1)[2022-09-05].http://doi.org/10.1186/s12896-021-00689-4.
[14] LI M,XIE Z,XIE Z,et al.Simultaneous detection of eight avian influenza A virus subtypes by multiplex reverse transcription-PCR using a GeXP analyser[J/OL].Sci Rep,2018,8(1)[2022-09-05].http://doi.org/10.1038/s41598-018-24620-8.
[15] HUANG H,CHEN S,ZHANG X,et al.Detection and clinical characteristics analysis of respiratory viruses in hospitalized children with acute respiratory tract infections by a GeXP-based multiplex-PCR assay[J/OL].J Clin Lab Anal,2020,34(4)[2022-09-05].http://doi.org/10.1002/jcla.23127. |
|
|
|
