Detection and quantification of hepatitis A virus in seawater via real-time RT-PCR

Abstract

A real-time RT-PCR method utilizing SYBR Green chemistry was developed to detect and enumerate hepatitis A virus (HAV) in ocean water. Ocean water samples were taken at the Tijuana River mouth (Tijuana, Mexico) and Imperial Beach pier (1.4 km north of the Tijuana River mouth in San Diego, California) following four separate rain events. A total of eight samples were collected, one from each location, each consisting of 4 l of ocean water. Using conventional RT-PCR and primers based on the conserved sequence at the VP3-VP1 genes of HAV, a 247 bp cDNA was amplified from six out of eight rain event water samples. HAV cDNA (confirmed by sequence analysis) was cloned into a TOPO vector (Invitrogen, Carlsbad, CA), and four primer sets were designed for application in SYBR Green real-time RT-PCR. The water samples were shown to contain inhibitors that affected real-time RT-PCR amplifications, however diluting the cDNA solution enabled successful amplification. Using real-time RT-PCR, HAV could be detected in all eight samples. Depending on the rain event, the viral load in these samples varied from 90 to 3523 copies of HAV/L of ocean water near the mouth of the Tijuana River, and 347 to 2656 copies/l near the Imperial Beach pier. The sensitivity, quantitative ability and the high throughput nature of SYBR Green real-time RT-PCR will be useful in monitoring HAV contamination in seawater.