Innovative Methods for Removal of PCR Inhibitors for Quantitative Detection of Plesiomonas shigelloides in Oysters by Real-Time PCR
Titel:
Innovative Methods for Removal of PCR Inhibitors for Quantitative Detection of Plesiomonas shigelloides in Oysters by Real-Time PCR
Auteur:
Gu, Weimin Levin, Robert E.
Verschenen in:
Food biotechnology
Paginering:
Jaargang 22 (2008) nr. 1 pagina's 98-113
Jaar:
2008-01
Inhoud:
A quantitative assay for Plesiomonas shigelloides in pure culture and oysters based on the real-time polymerase chain reaction (Rti-PCR) was developed. The methodology involved the treatment of oyster tissue homogenates with formaldehyde, differential centrifugation, and treatment of samples with activated charcoal coated with Pseudomonas fluorescens. With seeded oyster tissue homogenates, without formaldehyde or coated charcoal treatments, the lowest level of detection for P. shigelloides was 1 × 107 genomic targets per gram of tissue, equivalent to 2.5 × 105 genomic targets per Rti-PCR. The addition of 4% formaldehyde to tissue homogenates reduced the minimum level of detection of P. shigelloides to 1 × 105 genomic targets per gram of tissue, equivalent to 2.5 × 103 genomic targets per real-time PCR. The treatment of tissue homogenates with only activated charcoal coated with P. fluorescens reduced the minimum level of detection of P. shigelloides to 1 × 104 genomic targets per gram, equivalent to 2.5 × 102 genomic targets per real-time PCR, without DNA purification or enrichment. The combination of adding 4.0% formaldehyde to oyster tissue homogenates and treating with coated charcoal reduced the level of detection of P. shigelloides to 1 × 103 genomic targets per gram, equivalent to 25 genomic targets per real-time PCR. The linear range of detection was from 1 × 103 to 1 × 106 genomic targets per gram without enrichment.
Tijdschrift beschrijving