Direct Detection of Enterovirus 71 (EV71) in Clinical Specimens from a Hand, Foot, and Mouth Disease Outbreak in Singapore by Reverse Transcription-PCR with Universal Enterovirus and EV71-Specific Primers

SUMMARYOver the past 8 years, human enteroviruses (HEVs) have caused 27 227 cases of hand, foot and mouth disease (HFMD) in Xiamen, including 99 severe cases and six deaths. We aimed to explore the molecular epidemiology of HFMD in Xiamen to inform the development of diagnostic assays, vaccines and other interventions. From January 2009 to September 2015, 5866 samples from sentinel hospitals were tested using nested reverse transcription PCR that targeted the HEV 5′ untranslated region and viral protein 1 region. Of these samples, 4290 were tested positive for HEV and the amplicons were sequenced and genotyped. Twenty-two genotypes were identified. Enterovirus 71 (EV71) and coxsackieviruses A16, A6 and A10 (CA16, CA6 and CA10) were the most common genotypes, and there were no changes in the predominant lineages of these genotypes. EV71 became the most predominant genotype every 2 years. From 2013, CA6 replaced CA16 as one of the two most common genotypes. The results demonstrate the vast diversity of HFMD pathogens, and that minor genotypes are able to replace major genotypes. We recommend carrying-out long-term monitoring of the full spectrum of HFMD pathogens, which could facilitate epidemic prediction and the development of diagnostic assays and vaccines.

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Reverse transcription-PCR using a Primer Set Targeting the 3D Region Detects Foot-and-Mouth Disease Virus with High Sensitivity

10.1101/305086 ◽

2018 ◽

Author(s):

Tatsuya Nishi ◽

Toru Kanno ◽

Nobuaki Shimada ◽

Kazuki Morioka ◽

Makoto Yamakawa ◽

...

Keyword(s):

Reverse Transcription ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Clinical Samples ◽

Rt Pcr ◽

Pcr Assay ◽

Reverse Transcription Pcr ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Pcr Assays

AbstractBecause foot-and-mouth disease (FMD) has the potential to spread extensively, methods used for its diagnosis must be rapid and accurate. Therefore, reverse transcription-PCR (RT-PCR) plays an important diagnostic role. Here we designed the primer set FM8/9 to amplify 644 bases of the conserved 3D region of all seven serotypes of FMD virus (FMDV). We compared the performance of RT-PCR assays using FM8/9 with that using the primer set 1F/R targeting the 5’-UTR described in the manual of the World Organization for Animal Health. The detection limits of the RT-PCR assays were determined for 14 strains representing all serotypes. Compared with the sensitivities of the RT-PCR assay using 1F/R, those using FM8/9 were 101-to 104-fold higher for eight strains. To assess the validity of the methods for analyzing clinical samples, sera and saliva samples from pigs and cows infected with FMDV were collected daily and analyzed using the two PCR assays. The FM8/9 assay detected FMDV from all infected pigs and cows for longer times compared with the 1F/R assay, therefore revealing higher sensitivity for the clinical samples. Our results suggest that the FM8/9 RT-PCR assay is highly sensitive and is therefore suitable for the diagnosis of FMD.

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Robust Real-Time Reverse Transcription-PCR for Detection of Foot-and-Mouth Disease Virus Neutralizing Carryover Contamination

Journal of Clinical Microbiology ◽

10.1128/jcm.01944-15 ◽

2015 ◽

Vol 54 (1) ◽

pp. 216-219 ◽

Cited By ~ 1

Author(s):

Ji-Hyeon Hwang ◽

Yong-Keol Shin ◽

So-Yeon Park ◽

Jeesoo Kim ◽

Su-Mi Kim ◽

...

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Diagnostic Method ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Reverse Transcription Pcr ◽

Outbreak Period ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Specific Restriction

During an outbreak of foot-and-mouth disease (FMD), real-time reverse transcription-PCR (rRT-PCR) is the most commonly used diagnostic method to detect viral RNA. However, while this assay is often conducted during the outbreak period, there is an inevitable risk of carryover contamination. This study shows that the carryover contamination can be prevented by the use of target-specific restriction endonuclease in that assay.

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Reverse transcription‐PCR using a primer set targeting the 3D region detects foot‐and‐mouth disease virus with high sensitivity

Transboundary and Emerging Diseases ◽

10.1111/tbed.13202 ◽

2019 ◽

Cited By ~ 1

Author(s):

Tatsuya Nishi ◽

Toru Kanno ◽

Nobuaki Shimada ◽

Kazuki Morioka ◽

Makoto Yamakawa ◽

...

Keyword(s):

Disease Virus ◽

Reverse Transcription ◽

Foot And Mouth Disease ◽

High Sensitivity ◽

Mouth Disease ◽

Reverse Transcription Pcr ◽

Mouth Disease Virus ◽

Foot And Mouth

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Development of Reverse Transcription-PCR (Oligonucleotide Probing) Enzyme-Linked Immunosorbent Assays for Diagnosis and Preliminary Typing of Foot-and-Mouth Disease: a New System Using Simple and Aqueous-Phase Hybridization

Journal of Clinical Microbiology ◽

10.1128/jcm.38.12.4604-4613.2000 ◽

2000 ◽

Vol 38 (12) ◽

pp. 4604-4613 ◽

Cited By ~ 21

Author(s):

Soren Alexandersen ◽

Morag A. Forsyth ◽

Scott M. Reid ◽

Graham J. Belsham

Keyword(s):

Reverse Transcription ◽

Pcr Amplification ◽

Foot And Mouth Disease ◽

Variable Region ◽

Mouth Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Reverse Transcription Pcr ◽

Mouth Disease Virus ◽

Foot And Mouth

A reverse transcription-PCR (RT-PCR)–enzyme-linked immunosorbent assay system that detects a relatively conserved region within the RNA genome of all seven serotypes of foot-and-mouth disease virus (FMDV) has been developed. The high specificity of the assay is achieved by including a rapid hybridization step with a biotin-labeled internal oligonucleotide. The assay is highly sensitive, fast, and easy to perform. A similar assay, based on a highly variable region of the FMDV genome and employing a single asymmetric RT-PCR and multiple hybridization oligonucleotides, was developed to demonstrate the method's ability to type FMDV. Based on our theoretical and practical knowledge of the methodology, we predict that similar assays are applicable to diagnosis and strain differentiation in any system amenable to PCR amplification.

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Molecular strategy for the direct detection and identification of human enteroviruses in clinical specimens associated with hand, foot and mouth disease

PLoS ONE ◽

10.1371/journal.pone.0241614 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0241614

Author(s):

Yonghong Zhou ◽

Qi Qiu ◽

Kaiwei Luo ◽

Qiaohong Liao ◽

Yu Li ◽

...

Keyword(s):

Real Time ◽

Direct Detection ◽

Foot And Mouth Disease ◽

Public Health Problem ◽

Mouth Disease ◽

Coxsackie Virus ◽

Global Public Health ◽

Rt Pcr ◽

Clinical Specimens ◽

Foot And Mouth

Background Diseases caused by human enteroviruses (EVs) are a major global public health problem. Thus, the effective diagnosis of all human EVs infections and the monitoring of epidemiological and ecological dynamic changes are urgently needed. Methods Based on two comprehensive virological surveillance systems of hand, foot and mouth disease (HFMD), real-time PCR and nested RT-PCR (RT-snPCR) methods based on the enteroviral VP1, VP4-VP2 and VP4 regions were designed to directly detect all human EVs serotypes in clinical specimens. Results The results showed that the proposed serotyping strategy exhibit very high diagnostic efficiency (Study 1: 99.9%; Study 2: 89.5%), and the variance between the study was due to inclusion of the specific Coxsackie virus A6 (CVA6) real-time RT-PCR and VP4 RT-snPCR in Study 1 but not Study 2. Furthermore, only throat swabs were collected and analyzed in Study 2, whereas in Study 1, if a specific EV serotype was not identified in the primary stool sample, other sample types (rectal swab and throat swab) were further tested where available. During the study period from 2013 to 2018, CVA6 became one of the main HFMD causative agents, whereas the level of enterovirus A71 (EV-A71) declined in 2017. Conclusion The findings of this study demonstrate the appropriate application of PCR methods and the combination of biological sample types that are useful for etiological studies and propose a molecular strategy for the direct detection of human EVs in clinical specimens associated with HFMD.

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