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

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.

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

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.

Rapid and simultaneous detection of three major diarrhea-causing viruses by multiplex 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.

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

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.

[Preparation of a 96-microwell plate DNA diagnostic chip for detection of foodborne bacteria and its application in an incident of food poisoning].

OBJECTIVE: To develop a 96-microwell plate DNA diagnostic chip for simultaneous detection of 9 major foodborne bacteria. METHODS: Type-specific PCR primers labeled with biotin and oligonucleotide probes were designed according to the conservative genes of 9 major foodborne bacteria Staphylococcus aureus, Salmonella spp., Escherichia coli O157:H7 (Stx1 and Stx2), Shigella spp., Listeria monocytogenes, Bacillus cereus, Yersinia enterocolitica, Vibrio cholerae and Vibrio parahaemolyticus. A one-tube multiplex PCR system for simultaneous amplification of these bacteria was established, and the DNA probes were spotted and immobilized in the wells of the plate in 5x5 array format. Stable hybridization system between PCR products and oligonucleotide probes in the microwell was established after condition optimization. Alkaline phosphatase-conjugated streptavidin and NBT/BCIP were used to detect the hybridized PCR products. RESULTS: Twenty standard bacteria strains were used to validate the 96 microwell plate DNA diagnostic chip and highly specific and stable experiment results were obtained. Using this chip assay, the causal pathogen Staphylococcus aureus was identified within 12 h after the sampling from an incident of food poisoning, and the result was consistent with that obtained using conventional bacterial culture and biochemical identification. CONCLUSION: The novel 96 microwell plate DNA diagnostic chip allows rapid, accurate, automated and high-throughput bacterial detection and is especially valuable for quick response to such public health emergencies as food poisoning.

[One-step multiplex RT-PCR for rapid screening of type A, B and novel A (H1N1) influenza viruses].

OBJECTIVE: To developed a multiplex RT-PCR assay for simultaneous screening of type A, B and novel A (H1N1) influenza viruses. METHODS: Two pairs of universal primers in were designed for amplifying the M gene and NS gene of type A and B influenza viruses, respectively. A pair of specific primers of HA gene was designed to detect novel A (H1N1) influenza virus. A one-step method was used to establish the multiplex RT-PCR system. A blinded experiment was carried out to validate the accuracy of this assay in comparison with the results of real-time fluorescence RT-PCR. The clinical practicability and efficacy of this assay was also evaluated. RESULTS: The RT-PCR products were analyzed using agarose gel electrophoresis, which yielded distinct bands of the target fragments without non-specific reactions, suggesting the high efficiency and specificity of the multiplex RT-PCR. Blinded study of 50 samples demonstrated a concordance rate of 100%. CONCLUSION: This multiplex RT-PCR assay allows one-step simultaneous detection of type A, B and novel A (H1N1) influenza viruses rapidly and accurately, and provides a valuable low-cost screening technique for influenza epidemic monitoring and early diagnosis.