Improved TaqMan real-time assays for detecting hepatitis A virus.

Rapid advancement in genomics and bioinformatics in recent years holds great promise for research and development in many disciplines including public health. For the detection of pathogens, methods based on nucleic acid amplification need to be re-evaluated periodically to ensure the validity of signature primers and probes as more and more outbreak strains are sequenced and collected into databases in public domains. In this study, a previous assay designed computationally for detecting hepatitis A virus (HAV) was re-examined. Alignment of 57 complete or near complete HAV genomes allowed identification of conserved sequences for developing new primers and TaqMan probes. Two sets of real-time reverse transcription PCR reagents were developed by targeting highly conserved regions with primers and probes having optimal melting temperatures and minimum secondary structures. These two assays had 10 to 1000 fold lower detection limits than the previous assay when tested using representative human HAV genotypes IA, IB, and IIIA. The better of the two improved assays had a detection limit of 3.7 × 10-2 to 6.6 × 10-2 TCID50 or less. The improved detection sensitivity was likely due to improvement in the following four areas: 1) The Gardner1 probe has a single nucleotide mismatch at the 5' end in all 19 strains of genotypes IIIA and IIIB. 2) For the Gardner1 forward primer, there is a mismatch corresponding to the 3' end of the oligonucleotides in two strains belonging to genotype IA. 3) The Gardner1 probe had a melting temperature of 66.2 °C, which is less than the optimum of 68-70 °C (Dorak, 2006). 4) The Gardner1 forward and reverse primers had high potential of forming primer dimers. The improved HAV detection assays developed in this study would support better food safety surveillance initiatives and response to disease outbreaks of viral food-borne illness.

Rapid detection and differentiation of human noroviruses using RT-PCR coupled to electrospray ionization mass spectrometry.

The goal of this study was to develop an assay for the detection and differentiation of noroviruses using RT-PCR followed by electrospray ionization mass spectrometry (ESI-MS). Detection of hepatitis A virus was also considered. Thirteen primer pairs were designed for use in this assay and a reference database was created using GenBank sequences and reference norovirus samples. The assay was tested for inclusivity and exclusivity using 160 clinical norovirus samples, 3 samples of hepatitis A virus and 3 other closely related viral strains. Results showed that the assay was able to detect norovirus with a sensitivity of 92% and a specificity of 100%. Norovirus identification at the genogroup level was correct for 98% of samples detected by the assay and for 75% of a subset of samples (n = 32) compared at the genotype level. Identification of norovirus genotypes is expected to improve as more reference samples are added to the database. The assay was also capable of detecting and genotyping hepatitis A virus in all 3 samples tested. Overall, the assay developed here allows for detection and differentiation of noroviruses within one working day and may be used as a tool in surveillance efforts or outbreak investigations.

Development of a custom 16S rRNA gene library for the identification and molecular subtyping of Salmonella enterica.

The use of 16S rRNA gene sequencing within the regulatory workflow may help to reduce the time and labor involved in the identification and differentiation of Salmonella enterica isolates. However, a comprehensive, standardized reference library is needed in order to use this method with regulatory samples. The goal of this project was to acquire 16S rRNA partial and full gene sequences for a variety of S. enterica isolates and to use these sequences to build a custom 16S rRNA reference library. A total of 535 S. enterica isolates representing over 100 serotypes and 5 subspecies were selected for 16S rRNA partial gene sequencing (~500 bp) and 66 isolates representing 32 serotypes and 2 subspecies were selected for 16S rRNA full gene sequencing (~1500 bp). PCR, sequencing, and automated sequence assembly and editing were carried out using the MicroSEQ ID Microbial Identification System (Applied Biosystems). High quality sequences were obtained for 94.4% and 95.5% of the isolates sequenced over the partial and full genes, respectively. These sequences did not show sufficient divergence to reliably differentiate serotypes; however, they could be differentiated using 16S rRNA sequence typing based on intragenomic heterogeneity. A total of 83 unique 16S sequence types were obtained for use in the partial gene library and 58 unique 16S sequence types were obtained for entry into the full gene library. Preliminary sequencing results with one isolate analyzed in replicate were promising, with consistent matches to a specific 16S type in the custom library. The result of this study is a custom S. enterica 16S rRNA type library for potential use in the identification of isolates at the species, subspecies, and molecular subtype level. Further work will include validating the method for parameters such as exclusivity, sensitivity, and reproducibility.

Detection and Identification of Salmonella enterica, Escherichia coli, and Shigella spp. via PCR-electrospray ionization mass spectrometry: isolate testing and analysis of food samples.

An assay to identify the common food-borne pathogens Salmonella, Escherichia coli, Shigella, and Listeria monocytogenes was developed in collaboration with Ibis Biosciences (a division of Abbott Molecular) for the Plex-ID biosensor system, a platform that uses electrospray ionization mass spectroscopy (ESI-MS) to detect the base composition of short PCR amplicons. The new food-borne pathogen (FBP) plate has been experimentally designed using four gene segments for a total of eight amplicon targets. Initial work built a DNA base count database that contains more than 140 Salmonella enterica, 139 E. coli, 11 Shigella, and 36 Listeria patterns and 18 other Enterobacteriaceae organisms. This assay was tested to determine the scope of the assay's ability to detect and differentiate the enteric pathogens and to improve the reference database associated with the assay. More than 800 bacterial isolates of S. enterica, E. coli, and Shigella species were analyzed. Overall, 100% of S. enterica, 99% of E. coli, and 73% of Shigella spp. were detected using this assay. The assay was also able to identify 30% of the S. enterica serovars to the serovar level. To further characterize the assay, spiked food matrices and food samples collected during regulatory field work were also studied. While analysis of preenrichment media was inconsistent, identification of S. enterica from selective enrichment media resulted in serovar-level identifications for 8 of 10 regulatory samples. The results of this study suggest that this high-throughput method may be useful in clinical and regulatory laboratories testing for these pathogens.

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The evaluation of a PCR-based method for identification of Salmonella enterica serotypes from environmental samples and various food matrices.

The most commonly used method for serotyping Salmonella spp. is based on the Kaufmann-White scheme, and is composed of serological reactions using antibodies to LPS agglutinins. The multiplex PCR used in this investigation was established by Kim et al. to serotype the 30 most common clinical Salmonella serotypes, as determined by CDC. The PCR assay consists of two five-plex reactions and a single two-plex PCR reaction, based on six genetic loci from Salmonella enterica serotype Typhimurium and four loci from S. enterica serotype Typhi. In this investigation, we further evaluated the method for serotyping Salmonella spp. using a reference collection, environmental samples collected from a Mid-Atlantic region tomato farm study, four food matrices spiked with different Salmonella serotypes and a proficiency test. The PCR assay was first evaluated using DNA isolated from pure cultures of isolates obtained from various clinical and environmental samples, and then DNA isolated from broth cultures of food matrices of "Red round" and Roma tomatoes, Romaine lettuce, green onions and Serrano peppers spiked with serotypes Newport, Typhimurium, Javiana and Saintpaul, respectively. The results showed that the PCR assay correctly serotyped Salmonella spp. from the clinical, environmental, spiked food matrices, and proficiency test samples. These findings are significant because the PCR assay was successful in the identification of Salmonella in the spiked samples in a broth culture containing other non-salmonella organism. This method may be a useful resource for the food safety community.