A sensitive nested reverse transcriptase PCR assay to detect viable cells of the fish pathogen Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.)

Article Abstract:

A nested reverse transcriptase (RT) polymerase chain reaction (PCR) assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between one and 10 cells. Total RNA extracted from cultured bacteria, Atlantic salmon kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. After DNase treatment, extracted RNA was amplified by both RT PCR and PCR by employing primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 249-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain.

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Reverse transcriptase PCR detection of astrovirus, hepatitis A virus, and poliovirus in experimentally contaminated mussels: comparison of several extraction and concentration methods

Article Abstract:

Reverse transcriptase polymerase chain reaction (RT-PCR) was used in assessing four methods of extraction and three methods of concentration of three enteric viruses from mussels. The mussels were contaminated by immersion in seawater with astrovirus, hepatitis A virus and poliovirus. Results showed that more RT-PCR-positive samples were obtained with the glycine solution and borate buffer extraction methods than with the saline beef extraction method. Detoxification of the samples by Sephadex LH20 filtration significantly diminished the efficiency of RT-PCR virus detection.

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Quantification of hepatitis A virus in shellfish by competitive reverse transcription-PCR with coextraction of standard RNA

Article Abstract:

An internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription-polymerase chain reaction (RT-PCR) is used in quantifying hepatitis A virus (HAV) in experimentally contaminated mussels. The standard RNA is deposited directly into the sample and is used as an extraction control and as quantification tool. The RT-PCR assay offers a reliable method for the routine determination of HAV genome copies in shellfish specimens. The assay can be automated by using DNA enzyme immunoassay to save time and increase reliability and accuracy.

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