Graduation Year

2003

Document Type

Thesis

Degree

M.S.P.H.

Degree Granting Department

Public Health

Major Professor

Boo H. Kwa, Ph.D.

Committee Member

Ann C. Debaldo, Ph.D.

Committee Member

Lillian M. Stark, Ph.D.

Keywords

Norovirus, HAV, Amplification, Ham, Enterovirus

Abstract

The Centers for Disease Control and Prevention estimated 23,000,000 cases of viral gastroenteritis caused by Norovirus in 2000, 40% of which were transmitted by food including: a variety of fresh produce, cake, deli meats, fruit salad, cheeses and ice. (CDC, 2003). An estimated 83,391 cases of Hepatitis A virus was reported in 2000, of which 5% was attributed to foodborne transmission (CDC, 2003). These figures underscore an urgent need for a method that can isolate virus from a variety of food matrices.

The aim of this study was to develop an overall assessment of the inhibitory effects of a variety of food matrices on Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Additionally, to compare a sequence specific hybridization probe amplification format to a non sequence specific SYBR Green format using the Roche LightCycler. The secondary aim was to evaluate the effectiveness of a food virus concentration and isolation protocol under development at the Florida Department of Health Bureau of Laboratories, Tampa.

Three food specimens consisting of prepackaged smoked ham, fresh cilantro, and Thompson's green grapes were seeded with three dilutions of poliovirus 3 (Sabin strain). A viral concentration procedure under development at the Florida Department of Health Bureau of Laboratories, Tampa was used to isolate the virus. Real Time RT-PCR was carried out on the Roche LightCycler in SYBR Green and Hybridization probe formats.

Spiking the virus-negative samples of each matrix with a dilution series of poliovirus 3 created post flocculation spikes. This post-flocculation dilution series amplification allowed a standard curve to be created unique to each food matrix. The flocculation and concentrations specimens were then amplified and the standard curves from the post-flocculation seed were used to calculate the loss associated with the concentration procedure.

This study reports significant differences (p<0.05) in recovery detected between the various matrices, and Real Time RT-PCR formats. The concentration protocol under development at the Florida Department of Health Bureau of Laboratories, Tampa, demonstrates a 12-78% recovery of seeded virus in a simulated “real world” virus contamination event among the various matrices.

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