Xue-Jun Ma

Visual detection of pandemic influenza A H1N1 virus 2009 by reverse-transcription loop-mediated isothermal amplification with hydroxynaphthol blue dye.

A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of pandemic influenza A H1N1 virus infection. The reaction was performed in one step in a single tube at 65 degrees C for 60 min with the addition of hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was approximately 60 copies, and no cross-detection was observed. The assay was evaluated further with 50 clinical specimens diagnosed clinically with seasonal influenza or pandemic influenza A H1N1 virus infection. RT-LAMP with HNB dye was demonstrated to be a sensitive and easy assay for rapid detection of pandemic influenza A H1N1 virus.

Long persistence of EV71 specific nucleotides in respiratory and feces samples of the patients with Hand-Foot-Mouth Disease after recovery

BackgroundEV71 is associated with the fatal cases of brain stem encephalitis during large HFMD outbreaks from 1998 to 2008. EV71 may continuously shed from upper respiratory tracts and feces of HFMD patients for relatively long time after recovery. However, the persistence of viruses in the patients secretions and excretions is not clear.MethodsSerial throat swabs and feces of 34 definitely diagnosed patients, including 30 mild cases and 4 severe cases, were traced and collected with the interval of 2 to 4 days for up to 32 and 48 days, respectively, and tested by a nested RT-PCR.ResultsThe EV-71 specific sequences were identified by a Nested RT-PCR in all specimens of 0-4 days, and 5-8 days. The positive rates of EV71 in throat swabs dropped markedly to 42.86% during 9-12 days, and maintained at 20-30% during 13-24 days, while that in feces reduced to 71.43% during 9-12 days, and maintained roughly 20% till 37-40 days. EV71 nucleotide of 36.36% cases disappeared simultaneously both in throats and feces, 39.39% cases showed longer persistence of EV71 nucleotides in feces, and 21.21% were longer in throats. The longest duration of shedding observed was 24 days for throat swabs and 42 days for fecal specimens.ConclusionsEV71 shedding from respiratory tract may continue for nearly four weeks after onset, but its excretion through feces can persist more than five weeks.

Simultaneous Detection of Seven Enteric Viruses Associated with Acute Gastroenteritis by a Multiplexed Luminex-Based Assay

ABSTRACT Rapid and broad diagnostic methods are needed for the identification of viral agents of gastroenteritis. In this study, we used Luminex xMAP technology to develop a multiplexed assay for the simultaneous identification of major enteric viral pathogens, including rotavirus A (RVA), noroviruses (NoVs) (including genogroups GI and GII), sapoviruses (SaV), human astrovirus (HAstV), enteric adenoviruses (EAds), and human bocavirus 2 (HBoV2). The analytical sensitivity allowed detection of 103 (EAds, HBoV2, and RVA) and 104 (NoV GI and GII, SaV, and HAstV) copies per reaction mixture. Compared to conventional PCR, the Luminex-based assay yielded greater than 75% sensitivity and 97% specificity for each virus, and the kappa correlation for detection of all viruses ranged from 0.75 to 1.00. In conclusion, this multiplexed Luminex-based assay provides a potentially rapid, high-throughput, and maneuverable diagnostic tool for major viral pathogens associated with gastroenteritis.

Simultaneously typing nine serotypes of enteroviruses associated with hand, foot, and mouth disease by a GeXP analyzer-based multiplex reverse transcription-PCR assay.

ABSTRACT Hand, foot, and mouth disease (HFMD) is a contagious enteroviral disease occurring primarily in young children and caused by enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and other serotypes of coxsackievirus and echovirus. In this study, a GeXP analyzer-based multiplex reverse transcription (RT)-PCR assay (GeXP assay) consisting of chimeric primer-based PCR amplification with fluorescent labeling and capillary electrophoresis separation was developed to simultaneously identify nine serotypes of enteroviruses associated with HFMD in China, including EV71, CVA16, CVA4, -5, -9, and -10, and CVB1, -3, and -5. The RNAs extracted from cell cultures of viral isolates and synthetic RNAs via in vitro transcription were used to analyze the specificity and sensitivity of the assay. The GeXP assay detected as little as 0.03 tissue culture infective dose (TCID50) of EV71 and CVA16, 10 copies of panenterovirus, EV71, CVA16, CVB1, and CVB5, and 100 copies of 10 (including panenterovirus) premixed RNA templates. A total of 180 stool specimens collected from HFMD patients and persons suspected of having HFMD were used to evaluate the clinical performance of this assay. In comparison with the results of conventional methods, the sensitivities of the GeXP assay for detection of panenterovirus, EV71, and CVA16 were 98.79% (163/165), 91.67% (44/48), and 91.67% (33/36), respectively, and the specificities were 80.00% (12/15), 98.48% (130/132), and 100% (144/144), respectively. The concordance of typing seven other serotypes of enteroviruses with the results of conventional methods was 92.59% (25/27). In conclusion, the GeXP assay is a rapid, cost-effective, and high-throughput method for typing nine serotypes of HFMD-associated enteroviruses.

The development of a GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of sixteen human respiratory virus types/subtypes

BackgroundExisting standard non-molecular diagnostic methods such as viral culture and immunofluorescent (DFA) are time-consuming, labor intensive or limited sensitivity. Several multiplex molecular assays are costly. Therefore, there is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens.MethodsA GeXP-based multiplex RT-PCR assay (GeXP assay) was developed to detect simultaneously sixteen different respiratory virus types/subtypes. Seventeen sets of chimeric primers were used to initiate the RT-PCR, and one pair of universal primers was used for the subsequent cycles of the RT-PCR. The specificity of the GeXP assay was examined with positive controls for each virus type/subtype. The sensitivity was evaluated by performing the assay on serial ten-fold dilutions of in vitro-transcribed RNA of all RNA viruses and the plasmids containing the Adv and HBoV target sequence. GeXP assay was further evaluated using 126 clinical specimens and compared with Luminex xTAG RVP Fast assay.ResultsThe GeXP assay achieved a sensitivity of 20–200 copies for a single virus and 1000 copies when all of the 16 pre-mixed viral targets were present. Analyses of 126 clinical specimens using the GeXP assay demonstrated that GeXP assay and the RVP Fast assay were in complete agreement for 109/126 (88.51%) of the specimens. GeXP assay was more sensitive than the RVP Fast assay for the detection of HRV and PIV3, and slightly less sensitive for the detection of HMPV, Adv, RSVB and HBoV. The whole process of the GeXP assay for the detection of 12 samples was completed within 2.5u2009hours.ConclusionsIn conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for the detection of respiratory virus infections.

Visual Detection of High-Risk Human Papillomavirus Genotypes 16, 18, 45, 52, and 58 by Loop-Mediated Isothermal Amplification with Hydroxynaphthol Blue Dye

ABSTRACT A simple, rapid, sensitive, qualitative, colorimetric loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB) was established to detect high-risk human papillomavirus (HPV) genotypes 16, 18, 45, 52, and 58. All initial validation studies with the control DNA proved to be type specific. The colorimetric type-specific LAMP assay could achieve a sensitivity of 10 to 100 copies at 63°C for 65 min, comparable to that of real-time PCR. In order to evaluate the reliability of HPV type-specific LAMP, the assay was further evaluated with HPV DNAs from a panel of 294 clinical specimens whose HPV status was previously determined with a novel one-step typing method with multiplex PCR. The tested panel comprised 108 HPV DNA-negative samples and 186 HPV-DNA-positive samples of 14 genotypes. The results showed that the sensitivity of HPV type-specific LAMP for HPV types 16, 18, 45, 52, and 58 was 100%, 100%, 100%, 100%, and 100%, respectively, and the specificity was 100%, 98.5%, 100%, 98.8%, and 99.2%, respectively, compared with a novel one-step typing method with multiplex PCR. No cross-reactivity with other HPV genotypes was observed. In conclusion, this qualitative and colorimetric LAMP assay has potential usefulness for the rapid screening of HPV genotype 16, 18, 45, 52, and 58 infections, especially in resource-limited hospitals or rural clinics of provincial and municipal regions in China.

Detection of pandemic influenza A H1N1 virus by multiplex reverse transcription-PCR with a GeXP analyzer

A novel application of the GeXP genetic analysis system for the differential detection of pandemic influenza A H1N1 from seasonal influenza A H1N1 and H3N2 is described. The assay was evaluated using identified influenza viruses and clinical samples. The results indicate that the assay is both highly sensitive and specific for the detection of the pandemic influenza A H1N1 virus with a detection limit of 10 copies per reaction superior to that of assays in use currently. The assay is able to detect potential mixed infections. This technique has the potential to provide both a powerful method to enhance surveillance of influenza and a platform for investigating the differentiation of other similar pathogens.

Visual detection of human enterovirus 71 subgenotype C4 and Coxsackievirus A16 by reverse transcription loop-mediated isothermal amplification with the hydroxynaphthol blue dye☆

A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of human enterovirus 71 subgenotype C4 (EV71-C4) and Coxsackievirus A16 (CVA16) infection, respectively. The reaction was performed in one step in a single tube at 65°C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limits of the RT-LAMP assay were 0.33 and 1.58 of a 50% tissue culture infective dose (TCID(50)) per reaction based on 10-fold dilutions of a titrated EV71 or CVA16 strain, respectively. No cross-reaction was observed with Coxsackievirus A (CVA) viruses (CVA2, 4, 5, 7, 9, 10, 14, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3, 6, 11, and 19). The assay was further evaluated with 47 clinical stool specimens diagnosed previously with EV71, CVA16 or other human enterovirus infections. Virus isolates from stool samples were confirmed by virus neutralization testing and sequencing. RT-LAMP with HNB dye was demonstrated to be a sensitive and cost-effective assay for rapid visual detection of human EV71-C4 and CVA16.

Genotyping of 11 human papillomaviruses by multiplex PCR with a GeXP analyzer.

A new, rapid, and high‐throughput method was developed for simultaneous detection of 11 human papillomavirus (HPV) genotypes including nine high‐risk types (HPV16, 18, 31, 33, 35, 39, 52, 58, and 66) and two low‐risk types (HPV6 and 11) in a single tube by multiplex PCR based on a GenomeLab Gene Expression Profiler (GeXP) analyzer (GeXP‐PCR). Eleven sets of chimeric primers were used to initiate the PCR, and one pair of universal primers was used for the subsequent cycles of the PCR. The specificity of GeXP‐PCR for each HPV type was examined with clinical samples of single type HPV infection tested previously. The sensitivity of GeXP‐PCR was evaluated by performing the assay on serial 10‐fold dilutions of cloned PCR products. The GeXP‐PCR achieved a sensitivity of 100 copies when all of the 11 pre‐mixed plasmids containing HPV targets were present. Analyses of 124 clinical specimens using the GeXP‐PCR demonstrated that the GeXP‐PCR assay had comparable sensitivity and specificity to those of reported multiple PCR assay and an increased detection of HPV 11 in samples with mixed infections. In conclusion, the GeXP‐PCR is a fast, sensitive, and high throughput method for the detection of multiple HPV infections. J. Med. Virol. 84:957–963, 2012.

EV71 viral secretion by symptomatic Hand Foot and Mouth Disease patients and their asymptomatic close contacts

HFMD patients positive 16 16 32 negative 0 7 7 total 16 23 39 Hand-foot-and-mouth disease (HFMD) is a common febrile illness in young children caused by enteroviruses of the Picornaviridae family, in which the most common strains are Coxsackie A virus and Enterovirus 71 (EV71). HFMD outbreaks have been reported in Malaysia, Taiwan, Australia and Singapore. To investigate viral circulating characteristics of EV 71 of HFMD, the throat and anal swabs were collected from the 278 inpatients in three large hospitals in Fuyang city, China, including 203 mild, 72 severe and 3 fatal cases. High positive rates of EV71 specific fragments were identified in throats (237/278, 85.25%) and in feces (89/117, 74.13%). Only three out of 278 throat samples (1.08%) were CA16 positive by automatic platform for RNA extraction and nest RT-PCR. It strongly indicates that EV71 is the etiological agent for this HFMD outbreak. Extremely high circulations of EV71, but very low CA16 and other enteroviruses, possibly relate with more severe and fatal cases. Similar situations have been seen in some large outbreaks with more deaths in Taiwan and Malaysia. Out of 101 paired samples, 93 (92.10%) throat swabs showed EV71 positive, while 77 (76.20%) feces were positive. Among the 77 HFDM cases with EV71 positive in feces, 75 were positive in their throat swabs. Among 8 cases showing EV71 negative in throats, 6 were also negative in feces (Table 1). It indicates a causal relationship in identification of EV71 related sequences between throat and feces samples from HFMD patients. To explore the possible distribution of EV71 in the close contacts, throat swabs from the healthy adults who took care of those children were collected simultaneously when sampling 39 sick children, including 17 mild (15 EV71 positive) and 22 severe but survival (17 EV71 positive) cases. Half (16/32, 50%) of the close contacts of EV71 positive children showed EV71 positive. All seven adults whose children were EV71 negative were also EV71 negative (Table 2). It indicates a wide distribution of EV71 in the upper respiratory tracts of the close contacts, possibly being potential infection source. To see the circulation of EV71 in healthy persons, throat swabs and feces samples were collected from three villages, Village A with a fatal case in which samples were taken after the patient died (one week after onset), Village B with a mild case in which samples were taken immediately after hospitalized (two days after onset) and Village C without HFMD. In Village A, EV71 nucleotides were