a-high-throughput-platform-for-screening-salmonella-sppshigella

A High-throughput Platform for the Screening of <em>Salmonella</em> spp./<em>Shigella</em> spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Zhuhai International Travel Healthcare Center

Rotavirus is one of the major viral pathogens leading to diarrhea. Diagnosis has been conducted by either traditional cultural, serological methods or molecular biology techniques, which include RT-PCR and nucleic acid sequence-based amplification (NASBA). However, their differences regarding accuracy and sensitivity remain unknown. In this study, an in-house conventional RT-PCR assay and more importantly, an in-house real-time NASBA (RT-NASBA) were established, and compared with a commercial TaqMan RT-PCR assay. The results showed that all of these methods were able to detect and distinguish rotavirus from other diarrhea viruses with a 100% concordance rate during the course of an evaluation on 20 clinical stool samples. However, RT-NASBA was much quicker than the other two methods. More importantly, the limit of detection of RT-NASBA could reach seven copies per reaction and was one to two logs lower than that of conventional RT-PCR and TaqMan RT-PCR. These results indicate that this in-house assay was more sensitive, and thus could be used as an efficient diagnosis tool for rotavirus. To the best of our knowledge, this is the first direct comparison among three different assays for the detection of rotavirus. These findings would provide implication for the rational selection of diagnosis tool for rotavirus. 

Comparative detection of rotavirus RNA by conventional RT-PCR, TaqMan RT-PCR and real-time nucleic acid sequence-based amplification.

Rotaviruses, noroviruses and astroviruses are the major viral pathogens leading to diarrhea worldwide. Epidemiological investigations of outbreaks associated with these viruses have been impeded by the lack of methods for quick diagnosis and detection. In the current study, a multiplex real-time nucleic acid sequence-based amplification (RT-NASBA) system was developed for the simultaneous detection of rotavirus A/norovirus genogroup II/astrovirus. The specificity and sensitivity of the assay were compared with multiplex RT-PCR. The results showed that the multiplex RT-NASBA assay was established successfully, and robust signals could be observed in 10 minutes with high specificity. The limit of detection of the multiplex RT-NASBA assay was 7, 100, and 200 copies per reaction for rotavirus A, norovirus genogroup II, and astrovirus, respectively. The assay was thus 10 to 100 times more sensitive than multiplex RT-PCR. Clinical evaluation indicated that the assay was 100% concordant with multiplex RT-PCR and was reliable for the detection of both single infections and multiple infections in stool samples. To the best of our knowledge, this is the first multiplex RT-NASBA assay established for the detection of three major diarrhea-causing viruses. This assay provides a valuable platform for the rapid, specific, sensitive and simultaneous diagnosis of these pathogens, especially in resource-limited countries where expensive thermocycling equipment is not available. 

Rapid and simultaneous detection of three major diarrhea-causing viruses by multiplex real-time nucleic acid sequence-based amplification.

Human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) is a worldwide epidemic, with over 35 million people infected currently. Therefore, the development of a safe and effective HIV-1 vaccine is on top of the global health priority. In the past few years, there have been many promising advances in the prevention of HIV/AIDS, among which the RV144 Thai trial has been encouraging and suggests optimization of the current vaccine strategies or search for novel strategies. Here we reviewed the brief history of HIV-1 vaccine, analyzed key challenges existing now, and illustrated future research priority/directions for a therapeutic or prophylactic HIV-1 vaccine, with the hope of accelerating the speed of vaccine development. We believe that an effective HIV-1 vaccine, together with other prevention approaches, will bring an end to this epidemic in the near future. 

HIV vaccine research: the challenge and the way forward.

Norovirus is an important pathogen which accounts for majority of the viral related acute gastroenteritis. Recently, a variant of genotype GII.17 was reported to be predominant over GII.4 and accounted for several acute gastroenteritis outbreaks in Asia. In the current study, the full genome of a norovirus strain ZHITHC-12 isolated during this outbreak period in China was identified and characterized. The viral genome was 7557 nucleotides in length and a phylogenetic analysis based on full length genome sequences indicated that ZHITHC-12 belonged to GII.17 genotype. A further phylogenetic analysis based on all available polymerase and capsid sequences showed that ZHITHC-12 was in Cluster III on both phylogenetic trees and grouped with other strains also isolated during 2013 to 2015. Moreover, homology modeling analysis based on GII norovirus capsid 5BSX template revealed that substitutions, mutations, and more importantly, deletions and insertions, occurred at or near the putative epitopes and histo-blood group antigen (HBGA) binding sites in its protruding P2 domain, which might confer new antigenic or biological properties for this novel variant. In summary, the first full genome and capsid protein structure of a novel norovirus GII.17 variant isolated in China was extensively characterized. The data would be helpful not only for the epidemiology study, but also for the diagnostic tool development and effective vaccine design in the future. 

Complete nucleotide sequence analysis of the norovirus GII.17: A newly emerging and dominant variant in China, 2015.

Rapid and sensitive diagnostic methods are needed to help physicians make faster and better treatment decision for patients suffered from diarrhea. In the present study, a probe-free and sensitive RT-PCR combined high resolution melting analysis (HRMA) assay was established successfully for the detection of four major diarrhea-causing pathogens. The lower limit of detection of the assay were 10(0), 10(2), 10(0) and 10(3) copies/reaction for rotaviruses group A, astroviruses serotype 1, noroviruses genogroup II, and sapoviruses genegroup I, respectively, which were 1000-fold, 10-fold, 1000-fold and 10-fold more sensitive than conventional RT-PCR assay developed in parallel and comparable to or higher than commercially available real-time RT-PCR assay. Blinded sample evaluation showed that the assay was 100% concordant to both conventional RT-PCR and commercial real-time RT-PCR, indicating high reliability of the new assay. Therefore, the assay could provide a valuable platform for the probe-free and sensitive diagnosis of these pathogens. 

Probe-free and sensitive detection of diarrhea-causing pathogens using RT-PCR combined high resolution melting analysis.

Salmonella spp./Shigella spp. are often associated with food poisoning and fecal-oral transmission of acute gastroenteritis that requires strict monitoring, especially among people who would handle food and water. In 2014, the National Health and Family Planning Commission of the P. R. China issued a national standard protocol (recommendatory) for the screening of Salmonella spp./Shigella spp.. However, its performance has not been fully studied. Whether it was suitable for use in our laboratory was still unknown. In the current study, the new protocol was first verified by various experiments and then its clinical performance was evaluated in about 20,000 stool samples over a three-year period. Verification results showed that the new protocol was highly specific and reproducible. Sensitivity (as defined as the lower limit of detection) of the new protocol at the PCR step was 10CFU/mL and 10CFU/mL for Salmonella spp. and Shigella spp., while that at the guided culture step was 10CFU/mL and 10CFU/mL, respectively. The large scale clinical evaluation indicated that the new protocol could increase the positivity rate by two fold and decrease the workload/median turnaround time significantly. In conclusion, the protocol was verified and evaluated and was proven to be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

Verification and large scale clinical evaluation of a national standard protocol for Salmonella spp./Shigella spp. screening using real-time PCR combined with guided culture.

The cross border movement of populations would potentially increase the transmission and spread of various diseases including HIV/AIDS, which needed strict surveillance. In the current study, a total of 2,961,530 specimens were collected between 2012 and 2016 from the entry-exit populations at frontier ports of China for epidemiology and molecular epidemiology study. Results showed that HIV-1 prevalence rate among these populations was significantly higher than that of other general populations in China (p < .001). Epidemiological investigation indicated that most of the HIV-1 infected participants were male and young general population, in contrast to intravenous drug users as revealed by previous studies. There were significantly more female, Chinese, and migrant labors in southeastern ports than in northwestern ports (p < .05). Molecular epidemiology study revealed that three subtypes C/BC, CRF01_AE and B dominated, with the emerging of novel unique recombinant forms and drug-resistant viruses among this population. Overall, the results suggests and calls for the shift of HIV-1 prevention projects' focus and the continuous monitoring of HIV-1 molecular epidemiology among entry-exit populations. These findings are useful for the generation of evidence-based biomedical and behavioral prevention programs to HIV/AIDS.

High HIV-1 prevalence and viral diversity among entry-exit populations at frontier ports of China, 2012-2016: A cross-sectional molecular epidemiology study.

Article	Year
Comparative detection of rotavirus RNA by conventional RT-PCR, TaqMan RT-PCR and real-time nucleic acid sequence-based amplification. Journal of virological methods\| PubMed ID: 25433218	2015
Rapid and simultaneous detection of three major diarrhea-causing viruses by multiplex real-time nucleic acid sequence-based amplification. Archives of virology\| PubMed ID: 25559674	2015
HIV vaccine research: the challenge and the way forward. Journal of immunology research\| PubMed ID: 25861656	2015
Complete nucleotide sequence analysis of the norovirus GII.17: A newly emerging and dominant variant in China, 2015. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases\| PubMed ID: 26687061	2016
Probe-free and sensitive detection of diarrhea-causing pathogens using RT-PCR combined high resolution melting analysis. Biologicals : journal of the International Association of Biological Standardization\| PubMed ID: 27461241	2016
Verification and large scale clinical evaluation of a national standard protocol for Salmonella spp./Shigella spp. screening using real-time PCR combined with guided culture. Journal of microbiological methods\| PubMed ID: 29262293	2018
High HIV-1 prevalence and viral diversity among entry-exit populations at frontier ports of China, 2012-2016: A cross-sectional molecular epidemiology study. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases\| PubMed ID: 30086369	2018

Hai-Bo Wang

Biography

JoVE Journal Publications

Other Publications