| CPC C12Q 1/689 (2013.01) [C12Q 1/6806 (2013.01); C12Q 2600/158 (2013.01); C12Q 2600/16 (2013.01)] | 1 Claim |
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1. An assay to detect Vibrio parahaemolyticus (V. parahaemolyticus) in shrimp, the method comprising:
amplifying a fragment of a pirA gene and a pirB gene of V. parahaemolyticus using a multiplex real-time polymerase chain reaction (PCR) method, wherein the step of amplifying comprises: contacting a nucleic acid sample obtained from one or more shrimp with a first, second, third, fourth, fifth, sixth, seventh, and eighth oligonucleotide primer,
wherein the first oligonucleotide primer is a forward direction oligonucleotide primer adapted for PCR amplification of a fragment of the pirA gene of V. parahaemolyticus with the second oligonucleotide primer and specifically binds a region of the pirA gene of V. parahaemolyticus, and wherein the second oligonucleotide primer is a reverse direction oligonucleotide primer adapted for PCR amplification of a fragment of the pirA gene of V. parahaemolyticus with the first oligonucleotide primer and specifically binds a region of the pirA gene of V. parahaemolyticus,
wherein the third oligonucleotide primer is a forward direction oligonucleotide primer adapted for PCR amplification of a fragment of the pirB gene of V. parahaemolyticus with the fourth oligonucleotide primer and specifically binds a region of the pirB gene of V. parahaemolyticus, and wherein the fourth oligonucleotide primer is a reverse direction oligonucleotide primer adapted for PCR amplification of a fragment of the pirB gene of V. parahaemolyticus with the third oligonucleotide primer and specifically binds a region of the pirB gene of V. parahaemolyticus; and
wherein the fifth oligonucleotide primer is a forward direction oligonucleotide primer adapted for PCR amplification of a fragment of a 16S rRNA gene of V. parahaemolyticus with the sixth oligonucleotide primer and specifically binds a region of the 16S rRNA gene of V. parahaemolyticus, and wherein the sixth oligonucleotide primer is a reverse direction oligonucleotide primer adapted for PCR amplification of a fragment of the 16S rRNA gene of V. parahaemolyticus with the fifth oligonucleotide primer and specifically binds a region of the 16S rRNA gene of V. parahaemolyticus;
wherein the seventh oligonucleotide primer is a forward direction oligonucleotide primer adapted for PCR amplification of a fragment of a beta actin gene or a 18S rRNA gene of shrimp with the eighth oligonucleotide primer and specifically binds a region of the beta actin gene or the 18S rRNA gene of shrimp, and wherein the eighth oligonucleotide primer is a reverse direction oligonucleotide primer adapted for PCR amplification of a fragment of the beta actin gene or the 18S rRNA gene of shrimp with the seventh oligonucleotide primer and specifically binds a region of the beta actin gene or the 18S rRNA gene of shrimp; and
detecting in real time the presence or absence of an amplified double stranded DNA fragment of the pirA gene, and the pirB gene, and 16S rRNA gene of V. parahaemolyticus, and the 18S rRNA gene of shrimp in the nucleic acid sample,
wherein the first oligonucleotide primer consists of the sequence consisting of SEQ ID NO: 8, the second oligonucleotide primer consists of the sequence consisting of SEQ ID NO: 9, the third oligonucleotide primer consists of the sequence consisting of SEQ ID NO: 10, and the fourth oligonucleotide primer consists of the sequence consisting of SEQ ID NO: 11, the fifth oligonucleotide primer consists of the sequence consisting of SEQ ID NO: 12; the sixth oligonucleotide primer consists of the sequence consisting of SEQ ID NO: 13, the seventh oligonucleotide primer consists of the sequence consisting of SEQ ID NO: 14, and the eighth oligonucleotide primer consists of the sequence consisting of SEQ ID NO: 15.
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