Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry.

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

Quantitative microbial risk assessment to estimate the risk of diarrheal diseases from fresh produce consumption in India.

This study estimates illness (diarrhea) risks from fecal pathogens that can be transmitted via fecal-contaminated fresh produce. To do this, a quantitative microbial risk assessment (QMRA) framework was developed in National Capital Region, India based on bacterial indicator and pathogen data from fresh produce wash samples collected at local markets. Produce wash samples were analyzed for fecal indicator bacteria (Escherichia coli, total Bacteroidales) and pathogens (Salmonella, Shiga-toxin producing E. coli (STEC), enterohemorrhagic E. coli (EHEC)). Based on the E. coli data and on literature values for Cryptosporidium and norovirus, the annual mean diarrhea risk posed by ingestion of fresh produce ranged from 18% in cucumbers to 59% in cilantro for E. coli O157:H7, and was <0.0001% for Cryptosporidium; for norovirus the risk was 11% for cucumbers and up to 46% for cilantro. The risks were drastically reduced, from 59% to 4% for E. coli O157:H7, and from 46% to 2% for norovirus for cilantro in post-harvest washing and disinfection scenario. The present QMRA study revealed the potential hazards of eating raw produce and how post-harvest practices can reduce the risk of illness. The results may lead to better food safety surveillance systems and use of hygienic practices pre- and post-harvest.

Genome sequence of vanilla distortion mosaic virus infecting Coriandrum sativum.

The 9573-nucleotide genome of a potyvirus was sequenced from a Coriandrum sativum plant from India with viral symptoms. On analysis, this virus was shown to have greater than 85 % nucleotide sequence identity to vanilla distortion mosaic virus (VDMV). Analysis of the putative coat protein sequence confirmed that this virus was in fact VDMV, with greater than 91 % amino acid sequence identity. The genome appears to encode a 3083-amino-acid polyprotein potentially cleaved into the 10 mature proteins expected in potyviruses. Phylogenetic analysis confirmed that VDMV is a distinct but ungrouped member of the genus Potyvirus.

A polymerase chain reaction-based method for the detection of hepatitis A virus in produce and shellfish.

Outbreaks of gastroenteritis that are suspected to be of viral origin are on the rise. Thus, there is a need for regulatory agencies entrusted with food safety to develop adequate techniques for the detection of viruses in foods. We have established a general procedure for the detection of hepatitis A virus (HAV) in shellfish that, with minor modifications, is also applicable to fresh produce such as cilantro. Total RNA was isolated from shellfish or cilantro, followed by isolation of poly(A)-containing RNA. Because HAV genomic RNA contains a poly(A) tail, the isolation of poly(A)-containing RNA also enriches HAV genomic RNA. Reverse transcription was used to convert the RNA to cDNA, and then amplification was carried out by polymerase chain reaction (PCR). Reamplification with internal primers was used to improve the quality and the quantity of amplified DNA, allowing for post-PCR analysis such as sequence identification of the viral strain. With this procedure, multiple samples could be analyzed in four working days by a single trained individual. The nominal sensitivity of detection of the procedure was 0.15 TCID50 (50% tissue culture infective dose) per 0.62 g of tissue with a test virus. The direct RNA isolation protocol avoided pitfalls associated with whole-virus purification procedures by replacing virus precipitation steps involving polyethylene glycol and Procipitate with phenol extraction. The method is straightforward and reliable. We successfully used this procedure to detect naturally occurring HAV in clams involved in a gastroenteritis outbreak, as well as in cilantro artificially contaminated with a test virus.

A simple method to screen cilantro and parsley for fecal indicator viruses.

A protocol has been developed to process cilantro and parsley samples for male-specific coliphages. Coliphage recovery depended on the duration of peptone rinsing, and whether the products were intact or cut. After 60 min of rinsing with 0.1% peptone, 78% of spiked coliphages were recovered from intact cilantro samples, and 60% of the spiked coliphages were recovered from cut cilantro samples. The protocol was field tested on a limited scale using cilantro and parsley samples from six retail outlets using enrichment-based and quantitative coliphage assays. Of the 18 retail cilantro and parsley samples that were analyzed, 50% (9 of 18) of the cilantro samples were positive for male-specific coliphages using the enrichment-based assay compared to 39% (7 of 18) of the parsley samples. Using the quantitative coliphage assay, only 28% (5 of 18) of the cilantro samples were positive, and none of the parsley samples were positive. The number of male-specific coliphages ranged between 1 and 11 plaque-forming units per 10 g of cilantro samples. None of the samples was positive for Escherichia coli. The results suggest that simplified male-specific coliphage screening of herb samples is possible and that male-specific coliphages be used along with conventional bacteriological indicators to screen produce for presence of fecal contamination.