Development of a diagnostic scar marker for Vibrio shilonii caused acute hepatopancreatic necrosis disease in whiteleg shrimp

Hoang Tan Quang, Pham Thi Diem Thi, Tran Thuy Lan, Nguyen Duc Huy, Nguyen Duy Quynh Tram, Nguyen Thi Thu Lien


Background: In a previous report, we showed that Vibrio shilonii was found on whiteleg shrimp (Litopenaeus vannamei) with acute hepatopancreatic necrosis disease in Thua Thien Hue province, Vietnam. This study was performed to develop a diagnostic molecular marker generated by random amplified polymorphic DNA (RAPD) for V. shilonii rapid detection.

Methods: Pathogen Vibrio spp. were isolated from shrimps and fishes, and were identified by 16S rRNA sequencing. Genetic diversity of Vibrio strains was analysis by RAPD technique. Specific PCR product for V. shilonii was cloned and sequenced. SCAR marker was developed from specific PCR product.

Result: Twenty random primers were evaluated for RAPD to identify DNA polymorphisms between Vibrio species. The random primer OPN-06 generated a 468-bp DNA fragment specific for V. shilonii. This was then converted into a sequence-characterized amplified region (SCAR) marker designated N6-441.

Conclusion: Specific primers (Vshi-441F/Vshi-441R) amplified a unique DNA fragment in all V. shilonii isolates but not in the other Vibrio spp. This PCR assay showed significantly sensitive to the target DNA and reliably for the amplification the V. shilonii genome.

Keywords: AHPND; RAPD; SCAR; Vibrio shilonii; Vietnam

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