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Hand, foot and mouth disease (HFMD) is a serious public health problem, and coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) are two of the major causative pathogens, in addition to enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). A simple and rapid reverse transcription recombinase-aided amplification assay (RT-RAA) was developed for the detection of CVA10 and CVA6 in this study. The analytical sensitivity for detection of CVA10 and CVA6 at 95% probability by probit regression analysis was 35 copies per reaction and 38 copies per reaction, respectively, with 100% specificity. Compared with commercial RT-qPCR assays, when testing 455 fecal specimens, the kappa value of the RT-RAA assay for CVA10 and CVA6 was 0.920 (p < 0.001) and 0.952 (p < 0.001), respectively. Moreover, four samples that were positive for CVA10 and five that were positive for CVA6 by RT-RAA but negative by RT-qPCR were further determined to be true positives. These results demonstrate that the proposed RT-RAA assays are very valuable tools for the detection of CVA10 and CVA6 and have potential for use in resource-limited settings.

In this study, a rapid reverse-transcription recombinase aided amplification (RT-RAA) assay was developed to detect respiratory syncytial virus (RSV) subgroups A and B, respectively. The reaction was performed at 39°C in less than 30min. The analytical sensitivities of RSVA and RSVB at 95% probability by probit regression analysis were 38copies per reaction and 35 copies per reaction, respectively, and no cross reactions with other related respiratory viruses were observed. The RT-RAA assay was further utilized to detect and subgroup 306 clinical specimens and the results showed that 79(25.82%, 79/306) samples were positive for RSV, of those 16(20.25%, 16/79) were identified as RSVA and 63(79.75%, 63/79) were RSVB, which is completely consistent with the results obtained by RSV RT-qPCR assay. In conclusion, the developed RAA assay will be of benefit as a faster, sensitive and specific alternative tool for detection of RSV., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Respiratory Pathogen 13 Detection Kit (13× kit) is able to simultaneously detect 11 respiratory viruses, Mycoplasma pneumoniae (MP) and Chlamydia in a single reaction. Using 572 Nasopharyngeal aspirates collected from hospitalized children, the clinical performance of 13× kit for detecting 11 respiratory viruses was evaluated in comparison with a routinely used 2-tube multiplex reverse transcription PCR assay (2-tube assay) at provincial Centers for Disease Control and Prevention in China. The clinical performance of 13× kit for detecting MP and Chlamydia was evaluated by commercial real-time quantitative PCR (qPCR) kits or sequencing. For tested viruses, the assay concordance was 95.98% and the kappa coefficient was 0.89. All the MP and Chlamydia positive samples detected by 13× kit were confirmed as true positives. The utilization of the 13× kit in clinical settings will be helpful for doctors to assess clinical outcome according to virus type or multiple infections, and to limit the use of antibiotics., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Human rhinoviruses (HRVs) have long been recognized as the cause of more than one-half of acute viral upper respiratory illnesses, and they are associated with more-serious diseases in children, such as asthma, acute otitis media and pneumonia. A rapid and universal test for of HRV infection is in high demand. In this study, a reverse transcription genome exponential amplification reaction (RT-GEAR) assay targeting the HRV 5' untranslated region (UTR) was developed for pan-HRV detection. The reaction was performed in a single tube in one step at 65 °C for 60 min using a real-time fluorometer (Genie(®)II; Optigene). The RT-GEAR assay showed no cross-reactivity with common human enteroviruses, including HEV71, CVA16, CVA6, CVA10, CVA24, CVB5, Echo30, and PV1-3 or with other common respiratory viruses including FluA H3, FluB, PIV1-4, ADV3, RSVA, RSVB and HMPV. With in vitro-transcribed RNA containing the amplified regions of HRV-A60, HRV-B06 and HRV-C07 as templates, the sensitivity of the RT-GEAR assay was 5, 50 and 5 copies/reaction, respectively. Experiments to evaluate the clinical performance of the RT-GEAR assay were also carried out with a panel of 143 previously verified samples, and the results were compared with those obtained using a published semi-nested PCR assay followed by sequencing. The tested panel comprised 91 HRV-negative samples and 52 HRV-positive samples (18 HRV-A-positive samples, 3 HRV-B-positive samples and 31 HRV-C-positive samples). The sensitivity and specificity of the pan-HRVs RT-GEAR assay was 98.08 % and 100 %, respectively. The kappa correlation between the two methods was 0.985. The RT-GEAR assay based on a portable Genie(®)II fluorometer is a sensitive, specific and rapid assay for the universal detection of HRV infection.

Background: Existing 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., Methods: A 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., Results: The 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., Conclusions: In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for the detection of respiratory virus infections.

Multiplex reverse transcription-polymerase chain reaction (mRT-PCR) is currently available in virus detection and defined as the simultaneous amplification of two or more DNA/RNA targets in a single reaction vessel. In this study, we attempted to modify the conventional mRT-PCR technique on a basis of GenomeLab Genetic Analysis System (GeXP). Initially, we optimized the analytical validation of the GeXP analyzer and its design of workflow and simultaneously detected eight arboviruses that related to epidemic encephalitis by verifying the specificity of mRT-PCR with Japanese encephalitis virus(JEV) cell cultures and positive strains identified previously and determining the sensitivity with in vitro-transcribed RNA of serial dilutions. The GeXP system after optimization could amplify the specific fragments related to the viruses and exposed specifically a total of 13 target genes out of eight types of arboviruses at the level of 10(2) copies/microL, and the findings suggest that the novel protocol we developed can be high-throughput and highly specific and sensitive as well as quickness in screening of the encephalitis viruses, and is promising in detection of encephalitis-associated viruses for molecular epidemiological studies.

A GeXP based multiplex RT-PCR assay was developed to simultaneously detect twelve different respiratory viruses types/subtypes including influenza A virus, influenza B virus, influenza A virus sH1N1, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, human rhinovirus, human metapneumovirus, adenovirus, respiratory syncytial virus A, respiratory syncytial virus B and human bocavirus. Twelve sets of specific primers were designed based on the conserved sequences of available respiratory-virus sequence database. The specificity of the multiplex system was examined by positive specimens confirmed previously. The sensitivity to detect twelve respiratory viruses simultaneously was 10(3) copies/microL. Twenty four clinical specimens were further detected by this novel assay and the results were compared with that of the real-time RT-PCR. These results showed that this novel assay based on GeXP is a fast, sensitive, and high throughput test for the detection of respiratory virus infections.

A multiplex RT-PCR assay based on GeXP system was developed in order to detect simultaneously human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) and other coxsackieviruses (CVA4, 5, 9 and 10, CVB1, 3 and 5). Enterovirus detection was performed with a mixture of 12 pairs of oligonucleotide primers including one pair of published primers for amplifying all known pan-enterovirus genomes and eleven primer pairs specific for detection of the VP1 genes of EV71, C A16, CVA4, CVA5, CVA9, CVA10, CVB1, CVB3 and CVB5, respectively. The specificity of multiplex RT-PCR system was examined using enterovirus cell cultures and positive strains identified previously from hand-foot-and-mouth disease (HFMD) patients. Serial dilution of titrated EV71 and C A16 cell cultures and in vitro transcripted RNA of enterovirus VP1 regions were used to detect the sensitivity of the multiplex RT-PCR system. The limit of detection for this multiplex RT-PCR system was 10(0.5) TCID50/microL for EV71 and C A16 cell cultures and 1000 copies for in vitro transcripted RNA of nine viruses per assay. This multiplex RT-PCR assay is a rapid, sensitive and specific assay for the diagnosis of common enterovirus infection in cases of HFMD outbreak and is also potentially useful for molecular epidemiological investigation.

To develop and optimize a simultaneous detection method of RotavirusA, Norovirus GI, GII, Sapovirus, human astrovirus, enteric adenoviruses and HBoV2 with GenomeLab GeXP analysis system. The sensitivity was verified to be 10(4) copies/microL with plasmids containing the viral targets in triplicate on different days, and no cross-reaction with enterovirus71, human Parechovirus and PicobirnavirusII was observed. Finally, we successfully developed a high throughout, rapid and maneuverable multiplex RT-PCR assay for simultaneous detection of seven viruses related with viral gastroenteritis, which provide a novel method for the molecular diagnosis of diarrhea-associated virus.

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., (Copyright 2010 Elsevier B.V. All rights reserved.)

A simple, rapid and sensitive colorimetric Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) method was established to detect human influenza A H1N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the HA gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for one and half hour. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP assay was validated by cross-reaction with different swine and human influenza virus including human seasonal influenza A /H1N1 A /H3N2, influenza B and swine A /H1N1. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of human influenza A H1N1 HA gene. The assay was further evaluated with 30 clinical specimens with suspected pandemic influenza A H1N1 virus infection in parallel with RT-PCR detection and 26 clinical specimens with seasonal influenza virus infection. Our results showed that the RT-LAMP was able to achieve a sensitivity of 60 RNA copies with high specificity, and detection rate was comparable to that of the RT-PCR with the clinical samples of pandemic influenza A H1N1 infection. The RT-LAMP reaction with HNB could also be measured at 650nm in a microplate reader for quantitative analysis. Thus, we concluded that this colorimetric RT-LAMP assay had potential for the rapid screening of the human influenza A H1N1 virus infection in National influenza monitoring network laboratories and sentinel hospitals of provincial and municipal region in China.

In this study, we established a rapid and sensitive multiplex Real-time reverse transcription polymerase chain reaction (mrtRT-PCR) to simultaneously detect the novel human influenza A H1N1 virus, human seasonal influenza A H1N1 and H3N2. This assay had three pairs of primer to target the conserved regions of the HA gene for each of the HA types including novel H1N1, seasonal H1N1 and seasonal H3N2, and one pair of primer designed to detect the internal control-RnaseP gene. This assay was performed in one-step in one tube. To validate the specificity of the multiplex Real-time RT-PCR assay, different human influenza virus including human seasonal influenza A H1N1 and H3N2, human influenza B and reference A/California/07/2009 (H1N1) sw1 was tested. To evaluate the sensitivity of the assay, serial dilutions of RNA from in vitro transcription of the novel human influenza A H1N1 HA gene was tested. Finally this assay was evaluated with clinical samples from 54 fever patients diagnosed with novel influenza A H1N1 or seasonal H1/H3 or HB infection either by real-time PCR recommended by the WHO or HI assay by the National Influenza Center. Our results showed that the assay could achieve a sensitivity of 20 RNA copies of novel influenza A H1N1 with high specificity and could detect potential mixed co-infection. In conclusion, this multiplex real-time RT-PCR assay combines both rapidity and sensitivity for not only detecting the novel human influenza A H1N1 virus, but also monitoring the human seasonal influenza A H1N1 and H3N2 simultaneously.

A simple and sensitive Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method was established to provide a new alternative for clinical diagnosis of Avian influenza A H5N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the target for amplification of nucleic acid under isothermal conditions. In current study, fifty-one experimentally infected animal specimens and viral cultures that had been tested were analyzed by RT-LAMP for NA gene and HA gene, respectively. The amplification process of LAMP was monitored in real-time by the addition of SYBR Green dye. Meanwhile, the result showed high correlation between nested PCR and RT-LAMP. The specificity of the RT-LAMP assay was confirmed by restriction enzyme digestion analysis and sequencing of the amplified product. When the sensitivity of this assay was tested by serial 10-fold dilutions of RNA molecules from specimens, it was found that the RT-LAMP method achieved theoretically a sensitivity of 10 RNA molecules. Thus, we concluded that the RT-LAMP assay has potential usefulness for rapid detection of the Avian influenza A H5N1 virus.