Biomedical subjects

Doris H D'Souza

Publications and source records attributed to Doris H D'Souza.

3 recordsLinked to original sources

PubMed2006

Persistence of caliciviruses on environmental surfaces and their transfer to food.

The noroviruses (NoV) are a common cause of human gastroenteritis whose transmission by foodborne routes is well documented. Fecally contaminated surfaces are likely to contribute to this foodborne transmission and to the propagation of viral disease outbreaks. The purpose of this study was to (i) investigate the stability of NoV on various food preparation surfaces; and (ii) evaluate the degree of virus transfer from these surfaces to a model-ready-to-eat (RTE) food. For the virus persistence experiments, stainless steel, formica and ceramic coupons were artificially contaminated with Norwalk virus (NV), the prototype genogroup I NoV; NV RNA; or feline calicivirus (FCV) F9 (a NoV surrogate), stored at ambient temperature for up to 7 d, and periodically assayed for detection. In the transfer experiments, stainless steel coupons were inoculated with NV or FCV F9 and allowed to dry for 10, 30 and 60 min, after which lettuce leaves were exposed to the surface of the coupons at various contact pressures (10, 100, and 1000 g/9 cm2). Virus recovery was evaluated by RT-PCR (for NV and NV RNA) or by plaque assay (for FCV F9) using Crandell Reese Feline Kidney (CRFK) cells. NV and FCV were detected on all three surfaces for up to 7 d post-inoculation; for FCV, there was an approximate 6 to 7-log10 drop in virus titer over the 7 d evaluation period. By contrast, when stainless steel was inoculated with purified NV RNA, RT-PCR detection was not possible beyond 24 h. Transfer of both NV and FCV from stainless steel surfaces to lettuce occurred with relative ease. This study confirms lengthy NoV persistence on common food preparation surfaces and their ease of transfer, confirming a potential role for environmental contamination in the propagation of viral gastroenteritis.

Calicivirus, Feline↗

PubMed2004

Multiplex nucleic acid sequence-based amplification for simultaneous detection of several enteric viruses in model ready-to-eat foods.

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10(-1) reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 10(0) to 10(2) reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods.

Animals↗

PubMed2002

Improved detection of human enteric viruses in foods by RT-PCR.

Human enteric viruses (including hepatitis A virus (HAV) and Norwalk-like viruses (NLVs)) are now recognized as common causes of foodborne disease. While methods to detect these agents in clinical specimens have improved significantly over the last 10 years, applications to food samples have progressed more slowly. In an effort to improve the sensitivity and speed of virus detection from non-shellfish food commodities by reverse transcription-polymerase chain reaction (RT-PCR), we (i) evaluated multiple RNA extraction methods; (ii) compared alternative NLV primer sets; and (iii) developed a one-step RT-PCR method. Hamburger and lettuce samples, processed for virus concentration using a previously reported filtration-extraction-precipitation procedure, were inoculated with HAV or NV. Several RNA extraction methods (guanidinium isothiocyanate, microspin column, QIAshredder Homogenizer, and TRIzol) and primer pairs were compared for overall RNA yield (microg/ml), purity (A(260)/A(280)), and RT-PCR limits of detection. The use of TRIzol with the QIAshredder Homogenizer (TRIzol/Shred) yielded the best RT-PCR detection limits (<1 RT-PCR amplifiable units/reaction for NV), and the NVp110/NVp36 primer set was the most efficient for detecting NV from seeded food samples. A one-step RT-PCR protocol using the TRIzol/Shred extraction method and the NVp110/NVp36 or HAV3/HAV5 primer sets demonstrated improved sensitivity (>10-fold) over the routinely used two-step method. HAV RNA was detected by RT-PCR at initial inoculum levels corresponding to <10 and <100 PFU per 300 microl sample concentrate (corresponding to 6 g food sample) for hamburger and lettuce, respectively. NV RNA was detected by RT-PCR at initial inoculum levels <5 and <50 RT-PCR amplifiable units per 300 microl concentrate (corresponding to 6 g food sample) for hamburger and lettuce, respectively. Residual RT-PCR inhibitors were effectively removed as evidenced by the ability to detect viral RNA in food concentrates without prior dilution. The methods reported here show promise for rapid, sensitive detection of human enteric viruses in foods.

DNA Primers↗