Simultaneous detection of major enteric viruses using a combimatrix microarray

Various enteric viruses including norovirus, rotavirus, adenovirus, and astrovirus are the major etiological agents of food-borne and water-borne disease outbreaks and frequently cause non-bacterial gastroenteritis worldwide. Sensitive and high-throughput detection methods for these viral pathogens are compulsory for diagnosing viral pathogens and subsequently improving public health. Hence, we developed a sensitive, specific, and high-throughput analytical assay to detect most major enteric viral pathogens using “Combimatrix” platform oligonucleotide probes. In order to detect four different enteric viral pathogens in a sensitive and simultaneous manner, we first developed a multiplex RT-PCR assay targeting partial gene sequences of these viruses with fluorescent labeling for the subsequent microarray. Then, five olignonucleotides specific to each of the four major enteric viruses were selected for the microarray from the oligonulceotide pools targeting the specific genes obtained by multiplex PCR of these viruses. The oligonucleotide microarray was evaluated against stool specimens containing single or mixed viral species. As a result, we demonstrated that the multiplex RT-PCR assay specifically amplified partial sequences of four enteric viruses and the subsequent microarray assay was capable of sensitive and simultaneous detection of those viruses. The developed method could be useful for diagnosing enteric viruses in both clinical and environmental specimens.     

A method for detecting rash and fever illness‐associated viruses using multiplex reverse transcription polymerase chain reaction

In this study, a new multiplex RT‐PCR method for detecting various viral genes in patients with rash and fever illnesses (RFIs ) was constructed. New primer sets were designed for detection of herpes simplex viruses 1 and 2 (HSV1 and 2), and Epstein–Barr virus (EBV). The newly designed and previously reported primer sets were used to detect 13 types of RFI‐associated viruses by multiplex RT‐PCR assay systems. Moreover, to eliminate non‐specific PCR products, a double‐stranded specific DNase was used to digest double‐stranded DNA derived from the templates in clinical specimens. RFI‐associated viruses were detected in 77.0% of the patients (97/126 cases) by the presented method, multiple viruses being identified in 27.8% of the described cases (35/126 cases). Detected viruses and clinical diagnoses were compatible in 32.5% of the patients (41/126 cases). Sensitivity limits for these viruses were estimated to be 10¹–10³ copies/assay. Furthermore, non‐specific PCR products were eliminated by a double‐stranded specific DNase with no influence on sensitivity. These results suggest that this method can detect various RFI‐associated viruses in clinical specimens with high sensitivity and specificity.

     

Real-time multiplex PCR for rapid detection of enteroviruses, adenoviruses and hepatitis A virus in clinical specimens

Real-time multiplex polymerase chain reaction (PCR) with internal positive control (IPC) was developed for simultaneous detection of adenoviruses (AV), enteroviruses (EV) and hepatitis A virus (HAV). Primes and probes labeled with different fluorophores (FAM, R6G, ROX, and Cy5) and able to detect up to four viral RNAs (DNAs) with high specificity in a single tube in real-time PCR were designed. Sensitivity and specificity of the method was estimated using cultural strains of 8 serotypes of EV, 5 serotypes of AV and 2 clinical specimens containing HAV. Sensitivity of the method for detection of polioviruses types 1, 2, and 3 (Sabin vaccine strains) was 0.5--1 TCID50 per reaction mixture. Thirty clinical specimens were analyzed by the multiplex PCR with and without IPC, and by mono-specific PCR. Comparison of these methods with cultural one revealed results agreement in 86.7% in case of multiplex PCR with IPC and in 100% in case of multiplex PCR without IPC and mono-specific PCR. This method can be used for rapid diagnostics of enteric viral infections as well as for determination of viral contamination level of water. As intermediate results of the study the methods for quantitative assessment of HAV, AV, and EV nucleic acids were developed which are convenient tools for the control of antiviral therapy effectiveness.     

2007年北京地区儿童手足口病病原的初步筛查

2007年4～6月儿童手足口病流行期间,对北京地区51例皮损症状典型、伴/不伴发热、无重症合并症的手足口病患儿采样,建立RT-PCR方法,以5'非编码区(5'UTR)肠道病毒通用引物、CA16和EV71 VP1区特异性引物直接对82份临床标本进行了初步筛查,肠道病毒阳性率达70.6%。检测病例中CA16阳性25例(25/51)、EV71阳性4例(4/51)、非CA16和EV71的肠道病毒阳性病例7例(7/51),三者比例约为6:1:2。2007年北京地区儿童轻症手足口病主要病原包括CA16和EV71,同时还存在一定比例其它肠道病毒。部分EV71毒株经测序验证及系统进化分析显示为C4基因亚型。     

Respiratory virus multiplex RT-PCR assay sensitivities and influence factors in hospitalized children with lower respiratory tract infections

Multiplex RT-PCR assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. In this study, we evaluated the Qiagen ResPlex II V2.0 kit and explored factors influencing its sensitivity. Nasopharyngeal swab (NPS) specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the Shenzhen Children’s Hospital from May 2009 to April 2010. Total nucleic acids were extracted using the EZ1 system (Qiagen, Germany) and 17 respiratory viruses and genotypes including influenza A virus (FluA), FluB, parainfluenza virus 1 (PIV1), PIV2, PIV3, PIV4, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), rhinoviruses (RhV), enteroviruses (EnV), human bocaviruses (hBoV), adenoviruses (AdV), four coronaviruses (229E, OC43, NL63 and HKU1), and FluA 2009 pandemic H1N1(H1N1-p) were detected and identified by the ResPlex II kit. In parallel, 16 real-time TaqMan quantitative RT-PCR assays were used to quantitatively detect each virus except for RhV. Influenza and parainfluenza viral cultures were also performed. Among the total 438 NPS specimens collected during the study period, one or more viral pathogens were detected in 274 (62.6%) and 201(45.9%) specimens by monoplex TaqMan RT-PCR and multiplex ResPlex, respectively. When results from monoplex PCR or cell culture were used as the reference standard, the multiplex PCR possessed specificities of 92.9–100.0%. The sensitivity of multiplex PCR for PIV3, hMPV, PIV1 and BoV were 73.1%, 70%, 66.7% and 55.6%, respectively, while low sensitivities (11.1%–40.0%) were observed for FluA, EnV, OC43, RSV and H1N1. Among the seven viruses/genotypes detected with higher frequencies, multiplex PCR sensitivities were correlated significantly with viral loads determined by the TaqMan RT-PCR in FluA, H1N1-p and RSV (p=0.011?0.000). The Qiagen ResPlex II multiplex RT-PCR kit possesses excellent specificity for simultaneous detection of 17 viral pathogens in NPS specimens in pediatric inpatients at the time of admission. The sensitivity of multiplex RT-PCR was influenced by viral loads, specimen process methods, primer and probe design and amplification condition.     

GeXP多重PCR技术同时检测12种常见呼吸道病毒

本研究建立了一种基于GeXP多重基因表达遗传分析系统的多重RT-PCR检测方法,该方法可以同时检测12种呼吸道病毒,包括流感病毒A型和B型、季节性H1N1、副流感病毒1～3型、人鼻病毒、人偏肺病毒、腺病毒、呼吸道合胞病毒A型和B型、人博卡病毒。针对病原体保守区序列设计12种病毒的特异性引物,分别用已验证的阳性标本为模板检验多重体系的特异性。多重检测体系在10~3拷贝/μL水平可同时检测到12种病毒。另检测24份临床标本,以real-time RT-PCR为参考标准,进一步验证检测体系。结果表明,这种基于GeXP系统的新方法灵敏度高、特异性强,可以快速同时检测12种常见呼吸道病毒。     

建立新型的常见腹泻相关病毒的多重检测方法

利用GenomeLab(tm)GeXP遗传分析系统建立一种同时检测A组轮状病毒、诺如病毒GI、GII型、札如病毒、肠道腺病毒、星状病毒、人博卡病毒II型7种常见腹泻相关病毒的方法。对反应条件进行优化后,非同日三次重复实验表明至少在104拷贝/μL水平可同时特异地检测出7种病毒,对Enterovirus71、Human Parechovirus、Picobir-navirusII阳性标本无交叉反应。本研究初步建立了一种高通量、快速的常见腹泻相关病毒的检测方法,为腹泻病原的分子诊断提供了新的方法。     

2014-2016年南充市儿童手足口病重症流行病学调查及危险因素分析

摘要 目的：了解南充市儿童重症手足口病流行病学特征及其相关危险因素,为降低儿童重症手足口病发病率提供依据。方法：对中国"疾病监测信息报告管理系统"中确诊的南充市（顺庆区、高坪区、嘉陵区、阆中市）2014-2016年儿童手足口病的病例信息进行研究,分析该市儿童手足口病疫情、时间分布、地区分布和人群分布特征,并应用单因素和多因素Logistic回归分析儿童重症手足口病危险因素。结果：2014-2016年顺庆区、高坪区、嘉陵区、阆中市共报告儿童手足口病8068例,其中重症病例426例,占5.28%。全年均有手足口病发生,4~7月为手足口病发病高峰期,2014年峰值明显高于2015年、2016年,重症手足口病时间分布和发病高峰期与以上相同。顺庆区、高坪区、嘉陵区、阆中市均有手足口病发生,阆中市重症病例比例均高于其他辖区,差异有统计学意义（P<0.05）。男性患儿重症病例构成比较高,不同性别患儿重症病例构成比比较差异有统计学意义（P<0.05）;重症病例主要集中在1~3岁儿童,不同年龄段重症病例构成比比较差异有统计学意义（P<0.05）,重症病例主要分布在散居儿童和农村儿童,不同生活方式、不同家庭住址重症病例构成比比较差异有统计学意义（P<0.05）;重症病例主要分布在3~6天时间间隔的就诊患儿,不同就诊时间间隔重症病例构成比比较差异有统计学意义（P<0.05）。多因素Logistic分析显示：年龄为1-3岁、散居、家庭住址为农村是重症手足口病的危险因素（P<0.05）。结论：年龄为1-3岁、散居、家庭住址为农村是重症手足口病的危险因素,应在流动人口集中、生活条件较差的地区开展手足口病的宣传教育,提高人们对手足口病防治的认知,对于1-3岁儿童应作为疾病重点防控对象,提高家长疾病防控意识,以降低重症手足口病的发病率。     

Hand foot and mouth disease due to enterovirus 71 in Malaysia

Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affecting the mouth.The illness is caused by a number of enteroviruses with coxsackievirus A16 and enterovirus 71 as the main causative agents.Human enterovirus 71 (EV71) belongs to the species Human enterovirus A under the genus Enterovirus within the family Picornaviridae.EV71 has been associated with an array of clinical diseases including hand foot and mouth disease (HFMD),aseptic meningitis,encephalitis and poliomyelitis-like acute flaccid paralysis.A large outbreak of HFMD due to highly neurovirulent EV71 emerged in Malaysia in 1997,and caused 41deaths amongst young children.In late 2000,a recurrence of an outbreak of HFMD occurred in Malaysia with S fatalities in peninsular Malaysia.Outbreak of HFMD due to EV71 recurred in 2003 with an unknown number of cases and mortalities.A similar outbreak of HFMD with 2 recorded deaths in young children occurred in peninsular Malaysia in late 2005 and this was followed by a larger outbreak in Sarawak (Malaysian Borneo) with 6 reported fatalities in the early part of 2006.The current on-going outbreak of HFMD started in peninsular Malaysia in epidemiological week 12 of 2010.As with other HFMD outbreaks in Malaysia,both EV71 and CA16 were the main aetiological viruses isolated.In similarity with the HFMD outbreak in 2005,the isolation of CA16 preceded the appearance of EV71.Based on the VP 1 gene nucleotide sequences,4 sub-genogroups of EV71 (C1,C2,B3 and B4) co-circulated and caused the outbreak of hand,foot and mouth disease in peninsular Malaysia in 1997.Two sub-genogroups (C1 and B4) were noted to cause the outbreak in 2000 in both peninsular Malaysia and Sarawak.EV71 of sub-genogroup B5 with smaller contribution from sub-genogroup C1 caused the outbreak in 2003.In the 2005 outbreak,besides the EV71 strains of sub-genogroup C1,EV71 strains belonging to sub-genogroup B5 were isolated but formed a cluster which was distinct from the EV71 strains from the sub-genogroup B5 isolated in 2003.The four EV71 strains isolated from clinical specimens of patients with hand,foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to sub-genogroup B5.Phylogenetic analysis of the VP1 gene suggests that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia.Epidemiological and molecular data since 1997 show the recurrence of HFMD due to EV71 in Malaysia every 2 to 4 years.In each of the past outbreaks,more than one sub-genogroup of the virus co-circulate.     

82 例成人手足口病的临床特点分析

目的:总结82例成人手足口病的临床表现,实验室检查和流行病学特点,有利于做好手足口病的预防治疗.方法:对82例成人手足口病的临床表现和流行病学特点进行回顾性分析.结果:成人手足口病临床症状轻,预后好,但成人手足口病患者作为传染源,在临床中对手足口病的传播很有意义.结论:做好成人手足口病的诊断治疗和隔离,对预防控制手足口病有重要意义.     

A One-Step,Triplex, Real-Time RT-PCR Assay for the Simultaneous Detection of Enterovirus 71, Coxsackie A16 and Pan-Enterovirus in a Single Tube

The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD), which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71) and coxsackie A16 (CA16) are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs) in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001–0.04 TCID₅₀/ml, 0.02 TCID₅₀/ml, and 0.001 TCID₅₀/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD.     

A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log₁₀. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log₁₀. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log₁₀ lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log₁₀. Conversely, sensitivity was only 0.30 log₁₀ better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health.