Rapid detection of human rotavirus using colorimetric nucleic acid sequence-based amplification (NASBA)-enzyme-linked immunosorbent assay in sewage treatment effluent

A colorimetric nucleic acid sequence-based amplification-enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for rapid detection and identification of human rotavirus. Oligonucleotide primers targeting gene 9 encoding a serotype-specific antigen VP7 were selected and used for the amplification of viral RNA by the isothermal NASBA process, resulting in the accumulation of biotinylated RNA amplicons. Amplicons were hybridized with a specific amino-linked oligonucleotide probe covalently immobilized on microtiter plates. The DNA-RNA hybrids were colorimetrically detected by the addition of streptavidin-peroxidase conjugate and tetramethylbenzidine substrate. Using the NASBA-ELISA system, as little as 0.2 PFU (4 x 10(1) PFU ml(-1)) and 15 PFU (3 x 10(3) PFU ml(-1)) of rotavirus were detected within 6 h in spiked MQ water and sewage treatment effluent respectively. No interference was encountered in the amplification and detection of rotavirus in the presence of non-target RNA or DNA. Moreover, the presence of non-target bacteria and virus does not generate any non-specific signal, confirming the specificity of the developed NASBA-ELISA system and its effectiveness in specifically detecting rotavirus. The NASBA-ELISA system offers several advantages in terms of sensitivity, rapidity and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.     

Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.     

五种主要上呼吸道病毒多重RT-PCR 检测方法的建立

目的:建立甲型、乙型流感病毒、呼吸道合胞病毒A型、B型(RSV-A、RSV-B)和腺病毒(ADV)五种主要上呼吸道病毒的多重RT-PCR检测方法。方法:利用Primer premier5.0分别针对甲型流感病毒的M基因、乙型流感病毒的PB1基因、RSV-A和RSV-B的F基因及ADV的hexon基因设计五对特异性引物,对Mg2+、dNTP、引物浓度及退火温度等进行优化,建立同时检测甲型、乙型流感病毒、RSV-A、RSV-B和ADV的多重RT-PCR方法,并验证该检测方法的灵敏性。结果:所建立的五种病毒的多重RT-PCR方法可以同时或者分别扩增甲型、乙型流感病毒、RSV-A、RSV-B及ADV的141bp、635bp、525bp、377b和283bp基因片段,敏感度分别达到770PFU/ml、800PFU/ml、680PFU/ml、970PFU/ml和850PFU/ml,且五种病毒间无交叉反应。结论:所建立的多重RT-PCR方法可以迅速准确地检测甲型、乙型流感病毒、RSV-A、RSV-B和ADV,为五种病毒的检测提供了一种方便易行的方法。     

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.     

Real-time nucleic acid sequence-based amplification assay for detection of hepatitis A virus

A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5' noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.     

Simultaneous detection of waterborne viruses by multiplex real-time PCR

Norovirus, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus are all common causes of gastroenteritis. Conventional diagnoses of these causative agents are based on antigen detection and electron microscopy. To improve the diagnostic potential for viral gastroenteritis, internally controlled multiplex real-time polymerase chain reaction (PCR) methods have been recently developed. In this study, individual real-time PCRs were developed and optimized for specific detections of Norovirus genogroup I, Norovirus genogroup II, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus group B1. Subsequently, individual PCRs were combined with multiplex PCR reactions. In general, multiplex real-time PCR assays showed comparable sensitivities and specificities with individual assays. A retrospective clinical evaluation showed increased pathogen detection in 29% of samples using conventional PCR methods. Prospective clinical evaluations were detected in 123 of the 227 (54%) total samples used in the multiplex real-time PCR analysis. The Norovirus genogroup II was found most frequently (23%), followed by Rotavirus (20%), the Hepatitis A virus (4.5%), Coxsackievirus (3.5%), and Norovirus genogroup I (2.6%). Internally controlled multiplex real-time PCR assays for the simultaneous detection of Rotavirus, Coxsackievirus group B, the Hepatitis A virus, and Norovirus genogroups I and II showed significant improvement in the diagnosis of viral gastroenteritis.     

Simultaneous investigation of influenza and enteric viruses in the stools of adult patients consulting in general practice for acute diarrhea

ABSTRACT: BACKGROUND: Gastrointestinal symptoms are not an uncommon manifestation of an influenza virusinfection. In the present study, we aimed to investigate the presence of influenza viruses inthe stools of adult patients consulting their general practitioner for uncomplicated acutediarrhea (AD) and the proportion of concurrent infections by enteric and influenza viruses. METHOD: A case-control study was conducted from December 2010 to April 2011. Stool specimenswere collected and tested for influenza viruses A (seasonal A/H3N2 and pandemic A/H1N1)and B, and for four enteric viruses (astrovirus, group A rotavirus, human enteric adenovirus,norovirus of genogroups I - NoVGI - and genogroup II - NoVGII). RESULTS: General practitioners enrolled 138 cases and 93 controls. Of the 138 stool specimenscollected, 92 (66.7%) were positive for at least one of the four enteric viruses analysed and 10(7.2%) tested positive for one influenza virus. None of these 10 influenza positive patientsreported respiratory symptoms. In five influenza-positive patients (3.6%), we also detectedone enteric virus, with 4 of them being positive for influenza B (2 had co-detection withNoVGI, 1 with NoVGII, and 1 with astrovirus). None of the 93 controls tested positive forone of the enteric and/or other influenza viruses we investigated. CONCLUSIONS: In this study we showed that the simultaneous detection of influenza and enteric viruses is nota rare event. We have also reported, for the first time in general practice, the presence ofseasonal and pandemic influenza viruses in the stools of adult patients consulting foruncomplicated AD. A simultaneous investigation of enteric and influenza viruses in patientscomplaining of gastrointestinal symptoms could be useful for future studies to better identifythe agents responsible for AD.     

流感病毒的检测和分型技术研究进展

近年来,病毒性流感的不断流行和暴发已引起世界范围的广泛关注。为了更好地对流感病毒进行检测和分型,我们对目前流感病毒常用的血清学检测和分型方法、免疫荧光法、PCR方法、多重RT-PCR法、实时RT-PCR法、依赖核酸序列的扩增法、环介导等温扩增法、焦磷酸测序法、基因芯片技术分别进行了描述,并阐述了其优缺点。     

三重RT-PCR同步检测马铃薯多种病毒影响因素

根据病毒外壳蛋白区序列设计PVX、PVS特异性引物对,根据P1基因区序列设计PVA特异性引物对,应用三重RT-PCR同步检测马铃薯X病毒,马铃薯A病毒及马铃薯S病毒,分别得到562bp、255bp、182bp大小的扩增片段。试验从反转录反应、PCR反应及循环条件3方面讨论了试剂和循环条件对三重RT-PCR同步检测3种病毒的影响。结果表明反转录反应中dNTPs浓度、3种病毒下游引物浓度比例对整个反应影响较大;其次是PCR反应中MgC12浓度和退火温度;反转录时间,循环条件对RT-PCR影响较小。     

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。     

Rapid and sensitive detection of avian influenza virus subtype H7 using NASBA

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) is an isothermal technique allowing rapid amplification and detection of specific regions of nucleic acid from a diverse range of sources. It is especially suitable for amplifying RNA. A NASBA/ECL technique has been developed allowing the detection of RNA from avian influenza virus subtype H7 derived from allantoic fluid harvested from inoculated chick embryos and from cell cultures. Degenerate amplification primers and amplicon capture probes were designed enabling the detection of low and highly pathogenic avian influenza of the H7 subtype from the Eurasian and North American lineages and the Australian sub-lineage. The NASBA/ECL technique is specific for subtype H7 and does not cross-react with other influenza subtypes or with viruses containing haemagglutinin-like genes. The assay is 10- to 100-fold more sensitive than a commercially available antigen capture immunoassay system. The NASBA/ECL assay could be used in high throughput poultry screening programmes.     

Rapid and sensitive detection of Taura syndrome virus using nucleic acid-based amplification

Requiring only simple heating devices, isothermal nucleic acid-based amplification (NASBA) is a potential detection platform to be developed for on-site diagnosis of aquaculture pathogens. In this report, an NASBA assay has been developed for the Taura syndrome virus (TSV), one of the most devastating RNA virus pathogens for several penaeid shrimp species. The NASBA amplicons were detected by agarose gel electrophoresis and confirmed by Northern-blotting and dot-blotting analysis, using a biotinylated TSV-specific primer. The sensitivity of the TSV NASBA coupled with dot-blotting detection was approximately 5-fold less sensitive than that of the commercially available RT-nested, PCR-based IQ2000 TSV Detection and Prevention System that was also confirmed to be more sensitive than the RT-PCR-based TSV detection protocol recommended by the OIE (Office International des Epizooties). The specificity of the TSV NASBA reaction was substantiated by the results that RNA of non-target viruses did not generate any signals. Furthermore, a simple colorimetric microtiter plate assay employing TSV-specific capture and detection primers was developed as a simple alternative approach for the detection of NASBA amplicons. Taken together, the combination of the isothermal NASBA and colorimetric solid phase-based assays should allow sensitive, straightforward, and speedy on-site detection of TSV.     

Development of a multiplex RT-RPA assay for simultaneous detection of three viruses in cucurbits

Begomoviruses and criniviruses, vectored by whiteflies (Bemisia tabaci), are important threats to crops worldwide. In recent years, the spread of cucurbit leaf crumple virus (CuLCrV), cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV) on cucurbit crops has been reported to cause devastating crop losses in many regions of the world. In this study, a multiplex recombinase polymerase amplification (RPA) assay, an isothermal technique for rapid and simultaneous detection of DNA and RNA viruses CuLCrV, CYSDV and CCYV was developed. Highly specific and sensitive multiplex RPA primers for the coat protein region of these viruses were created and evaluated. The sensitivity of the multiplex RPA assay was examined using serially diluted plasmid containing the target regions. The results demonstrated that multiplex RPA primers have high sensitivity with a detection limit of a single copy of the viruses. The multiplex RPA primers were specific to the target as indicated by testing against other begomoviruses, potyviruses and an ilarvirus, and no nonspecific amplifications were noted. The primers simultaneously detected mixed infection of CCYV, CYSDV and CuLCrV in watermelon and squash crude extracts. This study is the first report of a multiplex RPA assay for simultaneous detection of mixed infection of DNA and RNA plant viruses.     

NASBA快速检测禽流感H5亚型病毒

采用建立的依赖核酸序列的扩增(Nucleicacidsequencebasedamplification,NASBA)对禽流感病毒3株H5亚型、1株H1、H3、H6亚型、3株禽流感H9亚型、5株不同宿主来源的新城疫病毒、鸭肝炎病毒、鸭瘟病毒、SPF鸡胚尿囊液及禽流感(H9)疫苗、新城疫疫苗、传染性法氏囊病疫苗、传染性支气管炎疫苗进行检测,结果NASBA(H5试剂)仅检测到禽流感病毒H5亚型,表明方法的特异性强。采用已知禽流感病毒A/Chicken/HK/1000/97(H5N1)的鸡胚尿囊液(ELD5010-7.5/mL),经10倍连续稀释,将经典的鸡胚病原分离法和NASBA进行比较,二种方法的灵敏度相当。用A/Chicken/HK/1000/97(H5N1)病毒人工感染SPF鸡、商品鸡,采用NASBA和病原分离法同时对人工感染鸡的粪拭子、血液进行了动态检测;采集感染死亡鸡的组织脏器,共检测了101个组织脏器,两种方法的符合率为90%(87/97)。     

Nucleic acid sequence based amplification for the rapid and sensitive detection of Salmonella enterica from foods

AIMS: The purpose of this study was to apply nucleic acid sequence-based amplification (NASBA) for the detection of Salmonella enterica serovar Enteritidis (S. Enteritidis) in representative foods. METHODS AND RESULTS: A previously reported primer and probe set based on mRNA sequences of the dnaK gene of Salmonella were used in this study. To test for possible food matrix inhibition and assay detection limits, 25-g samples of representative food commodities (fresh meats, poultry, fish, ready-to-eat salads and bakery products) were pre-enriched with and without S. Enteritidis inoculation. The NucliSens(R) Basic Kit, supplemented with enzymes from various other commercial sources, was used for RNA isolation, NASBA amplification and electrochemiluminescent (ECL) detection. The end point detection limit of the NASBA-ECL assay was equivalent to 101 CFU of S. Enteritidis per amplification reaction. When the assay was tested on noncontaminated foods, none of the food matrices produced false-positive results. Some of the food matrices inhibited the NASBA-ECL reaction unless the associated RNA was diluted 10-fold prior to amplification. CONCLUSIONS: For all food items tested, positive ECL signals were achieved after 18 h of pre-enrichment and subsequent NASBA at initial inoculum levels of 102 and 101 CFU per 25 g food sample. SIGNIFICANCE AND IMPACT OF THE STUDY: This rapid, semi-automated detection method has potential for use in the food, agricultural and public health sectors.