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Multiplex real-time quantitative polymerase chain reaction (mRT-qPCR) assay is commonly used to detect respiratory viruses, however, the sensitivity is limited for most reports. A panel of locked nucleic acid based multiplex closed one-tube nested real-time PCR (mOTNRT-PCR) assay consisting of five separate internally controlled RT-qPCR assays was developed for detection of 14 respiratory viruses. The sensitivity and reproducibility of mOTNRT-PCR panel were evaluated using plasmid standards and the specificity was evaluated using clinical samples. The clinical performance of mOTNRT-PCR panel was further evaluated with 468 samples collected from patients with an acute respiratory infection and compared with individual real-time PCR (RT-qPCR) assay. The analytical sensitivities of mOTNRT-PCR panel ranged from 2 to 20 copies/reaction, and no cross-reaction with common respiratory viruses was observed. The coefficients of variation of intra-assay and inter-assay were between 0.35% and 8.29%. Totally 35 clinical samples detected by mOTNRT-PCR assay panel were missed by RT-qPCR and confirmed true positive by sequencing of nested PCR products. The mOTNRT-PCR assay panel provides a more sensitive and high-throughput method for the detection of 14 respiratory viruses., (© 2020 Wiley Periodicals, Inc.)

The capability of high-throughput sequencing (HTS) for detection of known and unknown viruses timely makes it a powerful tool for public health emergency response. Third-generation sequencing (TGS) offers advantages in speed and length of detection over second-generation sequencing (SGS). Here, we presented the end-to-end workflows for both Oxford Nanopore MinION and Pacbio Sequel on a viral disease emergency event, along with Ion Torrent PGM as a reference. A specific pipeline for comparative analysis on viral genomes recovered by each platform was assembled, given the high errors of base-calling for TGS platforms. All the three platforms successfully identified and recovered at least 85% Norovirus GII genomes. Oxford Nanopore MinION spent the least sample-to-answer turnaround time with relatively low but enough accuracy for taxonomy classification. Pacbio Sequel recovered the most accurate viral genome, while spending the longest time. Overall, Nanopore metagenomics can rapidly characterize viruses, and Pacbio Sequel can accurately recover viruses. This study provides a framework for designing the appropriate experiments that are likely to lead to accurate and rapid virus emergency response.

Objective: Unbiased next generation sequencing (NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences., Methods: A viral Sequence Independent Targeted Amplification (VSITA) approach using a set of non-ribosomal and virus-enriched octamers (V8) was developed and compared with traditionally used random hexamers (N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR (qPCR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis., Results: A minimum 30 hexamers matching to viral reference sequences (sense and antisense) were selected from a dataset of random 4,096 (46) hexamers (N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480 (30 × 42) octamers (V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted cDNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin., Conclusion: The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples., (Copyright © 2018 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. 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.)

Loop-mediated isothermal amplification (LAMP) is a novel in vitro nucleic acid amplification method conducted under isothermal conditions with the advantages of high specificity, sensitivity, rapidity and easy detection. Since it was established in 2000, it has been widely applied in various fields of analytical science including the diagnosis of a variety of pathogens, identification of embryo sex, detection of genetically modified organisms and cancer gene identification. Additionally, significant progress has been made in the optimization of the LAMP method, such as accelerated reactions, simplified sample processing, the realization of multiplex amplification, and the enhanced specificity of reaction and detection methods. LAMP technology also shows much potential to be adopted as part of point-of-care testing platforms by the micromation, automation and integration with other technologies such as Lab-on-a-Chip and digital nucleic acid amplification. This review summarizes the latest advances in the LAMP technique and its applications in developing point-of-care testing platforms.

In this study, a novel resequencing pathogen microarray (RPM)-based multi-pathogen detection assay was developed to simultaneously detect 14 rotaviruses, 7 caliciviruses, 8 astroviruses, 28 enteroviruses, and 16 rare diarrhea viruses in patients with diarrhea syndrome. The specificity of the assay was examined using confirmed virus-positive specimens, and the sensitivity was evaluated by serial ten-fold dilutions of in vitro transcribed RNA. RPM assay could detect and differentiate virus types/subtypes at 20-2000 copies/microL. The detection threshold of RPM was determined by adjusting the reference concentration, and the detection steps were optimized to type Enterovirus. The nucleic acids of 10 stool samples from patients with unexplained diarrhea were screened, and 6 of them showed positive results. The RPM results were further verified by singleplex PCR followed by sequencing, and no difference was found between the two assays. In conclusion, we have established a high-throughput RPM assay with high specificity and sensitivity, which demonstrates a great potential for the identification of pathogens in patients with unexplained diarrhea and the management of emerging epidemic.

Acute respiratory tract infections (ARTIs) are widely distributed among the population, mainly caused by respiratory viruses. ARTIs are responsible for significant morbidity and mortality among the elderly and infants or young children, causing a serious economic burden. The rapid and accurate identifi cation of a pathogen will provide a guideline for the clinical diagnosis and therapy. Multiplex polymerase chain reaction (PCR) technologies combine the rapidness and high sensitivity of PCR with high through put, thus achieving the capability of detecting multiple pathogens simultaneously. The commercial kits based on these multiplex PCR methods allow to detect more than twelve respiratory viruses simultaneous ly, reaching the comparable sensitivities and specificities to those of real-time PCR. The recent progress of novel multiplex PCR assays and their principles as well as applications in respiratory virus diagnosis were reviewed in this paper.

Resequencing Pathogen Microarray (RPM) is a new pathogen detection and identification technology based on DNA microarray. In order to apply RPM in the detection of unexplained infection and as a result, to improve the emergency response capacity, a new RPM-based respiratory pathogens detection assay was developed to simultaneously detect 19 common respiratory viruses, 9 influenza A viruses (Flu A),11 human rhinoviruses(HRV), 28 enteroviruses and 18 rare respiratory viruses. The specificity of multiplex system was examined by confirmed positive specimens for 16 common respiratory virus. The sensi-tivity was evaluated by serial ten-fold dilutions of plasmids or in vitro-transcribed RNA. RPM could detect and differentiate 16 virus types/subtypes at 10 - 1 000 copies/reaction level. Nucleic acids of 8 throat swabs with unexplained respiratory tract infections were pooled and detected by the new assay. The RPM result was verified by common PCR followed by sequencing as well as PLEX-ID (Abbott). Except for a false-positive of PIV1, no difference among the three assays was found. These results indicate the assay based on the new RPM is a highly sensitive, high throughput test for the detection of respiratory virus infections, which is significant for the management of emergent and epidemic infectious disease.

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.

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 10(3) (EAds, HBoV2, and RVA) and 10(4) (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.

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.