Mengjie Yang

Tembusu Virus in Ducks, China

In China in 2010, a disease outbreak in egg-laying ducks was associated with a flavivirus. The virus was isolated and partially sequenced. The isolate exhibited 87%–91% identity with strains of Tembusu virus, a mosquito-borne flavivirus of the Ntaya virus group. These findings demonstrate emergence of Tembusu virus in ducks.

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 2.5 hours.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.

Etiology of Multiple Non-EV71 and Non-CVA16 Enteroviruses Associated with Hand, Foot and Mouth Disease in Jinan, China, 2009-June 2013.

Hand, foot, and mouth disease (HFMD) is an infectious disease caused by human enterovirus 71 (EV71), coxsackievirus A16 (CVA16) and other enteroviruses. It is of interest that other enteroviruses associated with HFMD in Jinan have been rarely reported. The aim of the present study is to detect and characterize the circulating serotypes of non-EV71 and non-CVA16 enteroviruses associated with HFMD in Jinan city, Shandong province, China. A total of 400 specimens were collected from clinically diagnosed HFMD cases in Jinan from January 2009 to June 2013. All specimens were infected with non-EV71 and non-CVA16 enteroviruses previously confirmed by RT-PCR or real-time PCR according to the protocols at that time. The GeXP-based multiplex RT-PCR assay (GeXP assay) was performed to investigate the pathogen spectrum of 15 enteroviruses (coxsackieviruses A4, A5, A6, A9, A10, A16; coxsackieviruses B1, B3, B5; Echoviruses 6, 7, 11, 13, 19 and EV71) infections associated with HMFD. For GeXP assay negative samples, reverse transcription nested PCR (nested RT-PCR) based on the 5’ -untranslated region (5’- UTR) sequence and phylogenetic analysis were conducted to further explore the etiology of multiple enteroviruses. The results showed that a total of twenty serotypes of enteroviruses (including EV71 and CVA16) were identified by GeXP assay and nested RT-PCR. The most circulating twelve serotypes of enteroviruses with HFMD in Jinan from 2009 to June 2013 were EV71, CVA16, CVA10, CVA6, CVA12, CVA2, Echo3, CVA4, CVA9, CVB1, CVB3 and Echo6. CVA10 and CVA6 were the most prevalent pathogens other than EV71 and CVA16 in Jinan and their most prevalent seasons were spring and summer, and a slight increase was observed in autumn and early winter. It should be noted that mixed-infections were identified by GeXP assay and the phylogenetic tree clearly discriminated the multiple pathogens associated with HFMD. Our results thus demonstrate that there was a clear lack of a reliable testing method for EV71 and CVA16 and multiple non-EV71 and non-CVA16 enteroviruses associated with HFMD were present in Jinan. The GeXP assay combined with nested RT-PCR based on 5’-UTR region could meet the need for the national surveillance of multiple enteroviruses or the investigation of epidemic outbreaks triggered by enteroviruses in the future.

Evaluation of a Direct Reverse Transcription Loop-Mediated Isothermal Amplification Method without RNA Extraction for the Detection of Human Enterovirus 71 Subgenotype C4 in Nasopharyngeal Swab Specimens

Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD) worldwide and has been associated with neurological complications which resulted in fatalities during recent outbreak in Asia pacific region. A direct reverse transcription loop-mediated isothermal amplification (direct RT-LAMP) assay using heat-treated samples without RNA extraction was developed and evaluated for the detection of EV71 subgenotype C4 in nasopharyngeal swab specimens. The analytical sensitivity and specificity of the direct RT-LAMP assay were examined. The detection limit of the direct RT-LAMP assays was 1.6 of a 50% tissue culture infective dose (TCID50) per reaction and no cross-reaction was observed with control viruses including Cosackievirus A (CVA) viruses (CVA2,4,5,7,9,10,14,16, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3,6,11, and 19). The direct RT-LAMP assay was evaluated and compared to both RT-LAMP and quantitative real-time PCR (qRT-PCR) in detecting EV71 infection with 145 nasopharyngeal swab specimens. The clinical performance demonstrated the sensitivity and specificity of direct RT-LAMP was reported to be 90.3% and 100% respectively, compared to RT-LAMP, and 86.83% and 100% respectively, compared to qRT-PCR. These data demonstrated that the direct RT-LAMP assay can potentially be developed for the point of care screening of EV71 infection in China.

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.

A high throughput multiplex PCR assay for simultaneous detection of seven aminoglycoside-resistance genes in Enterobacteriaceae

BackgroundThe aminoglycoside-resistance genes encoding aminoglycoside modifying enzymes and 16S rRNA methyltransferases are main factors contributing to increasing resistance to aminoglycosides. Characterization and distribution of antimicrobial resistance gene profiles provide important information on the potential difficulty of treatment of bacteria. Several molecular methods have been developed to investigate the prevalence of aminoglycoside-resistance genes. These existing methods are time-consuming, labor-intensive, expensive or limited sensitivity in the epidemiological investigation. Therefore, it is necessary to develop a rapid, less-costly and high throughput and sensitive method to investigate the distribution of antimicrobial resistance gene in clinical isolates.ResultsIn this study, we developed a GeXP analyzer-based multiplex PCR assay to simultaneously detect seven aminoglycoside-resistance genes, including aac(3)-II, aac(6′)-Ib, aac(6′)-II, ant(3″)-I,aph(3′)-VI,armA and rmtB, and to analyze the distribution of these genes in clinical Enterobacteriaceae isolates. Under optimized conditions, this assay achieved a limit-of-detection as low as 10 copies of each of the seven genes. The presented method was applied to analyze the distribution of aminoglycoside-resistance genes in 56 clinical Enterobacteriaceae isolates, and the results were compared with that of the conventional single PCR assay. Kappa values of the two methods for detecting each of the seven resistance genes were 0.831, 0.846, 0.810, 0.909, 0.887, 0.810 and 0.825, respectively.ConclusionThis GeXP assay is demonstrated to be a rapid, cost-effective and high throughput method with high sensitivity and specificity for simultaneously detecting seven common aminoglycoside-resistance genes.

A Novel Multiplex Tetra-Primer ARMS-PCR for the Simultaneous Genotyping of Six Single Nucleotide Polymorphisms Associated with Female Cancers

Background The tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) is a fast and economical means of assaying SNPs, requiring only PCR amplification and subsequent electrophoresis for the determination of genotypes. To improve the throughput and efficiency of T-ARMS-PCR, we combined T-ARMS-PCR with a chimeric primer-based temperature switch PCR (TSP) strategy, and used capillary electrophoresis (CE) for amplicon separation and identification. We assessed this process in the simultaneous genotyping of four breast cancer–and two cervical cancer risk–related SNPs. Methods A total of 24 T-ARMS-PCR primers, each 5′-tagged with a universal sequence and a pair of universal primers, were pooled together to amplify the 12 target alleles of 6 SNPs in 186 control female blood samples. Direct sequencing of all samples was also performed to assess the accuracy of this method. Results Of the 186 samples, as many as 11 amplicons can be produced in one single PCR and separated by CE. Genotyping results of the multiplex T-ARMS-PCR were in complete agreement with direct sequencing of all samples. Conclusions This novel multiplex T-ARMS-PCR method is the first reported method allowing one to genotype six SNPs in a single reaction with no post-PCR treatment other than electrophoresis. This method is reliable, fast, and easy to perform.

A Two-Tube Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Sixteen Human Respiratory Virus Types/Subtypes

There is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens. With an intended application in provincial Centers for Diseases Control and Prevention, in this study, we present a two-tube multiplex RT-PCR assay (two-tube assay) using automatic electrophoresis to simultaneously detect sixteen common respiratory viruses. The specificity and the sensitivity of the assay were tested. The assay could detect 20–200 copies per reaction when each viral type was assayed individually, 2000 copies with 9 premixed viral targets in the multiplexed assay in tube 1, and 200 copies with 8 premixed templates in tube 2. A total of 247 specimens were used to evaluate the two-tube assay, and the results were compared with those obtained from the Luminex xTAG RVP Fast assay. The discordant results were confirmed by sequencing or by the Seeplex RV15 ACE detection kit. There were no false positives, but six false negatives occurred with the two-tube assay. In conclusion, the two-tube assay is demonstrated to have great potential for routine surveillance of respiratory virus infection in China.