e-journal

AP4 method for two-tube nested PCR detection of AHPND isolates ofVibrio parahaemolyticus

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Our previous work on the mechanism of virulence for the unique isolates of Vibrio parahaemolyticus thatcause acute hepatopancreatic necrosis disease (VPAHPND)revealed that it was mediated by a binary Pir-liketoxin pair ToxA and ToxB. These toxins are located on the pVA plasmid, a plasmid carried by AHPND-causing strain of V. parahaemolyticus with a size of approximately 69 kbp. Using the coding sequences ofToxA, a one-step PCR detection method for VPAHPNDwas introduced in June 2014 but had the limitationthat attempts to adapt it into a nested PCR protocol were unsuccessful. As a result, low levels of VPAHPNDin shrimp or other samples could not be detected without first preparing an enrichment broth cultureto allow bacterial growth before extraction of template DNA. Here, we describe the AP4 (abbreviation ofAHPND detection version 4) method, a two-tube nested PCR method that targets the tandem genes ToxAand ToxB, including the 12 bp spacer that separates them on pVA plasmid. Testing of the method revealedthat it gave 100% positive and negative predictive values for VPAHPNDusing a panel of 104 bacterialisolates including 51 VPAHPNDisolates and 53 non-AHPND isolates, the latter including 34 isolates of V.parahaemolyticus and 19 isolates of other bacteria found in shrimp ponds, including other Vibrio species.The AP4 nested PCR method was 100 times more sensitive (100 fg total DNA template) than the one-stepAP3 (10 pg total DNA template) method, and it could detect VPAHPNDin experimentally challenged shrimpby 6 h post immersion (n = 2/3), while AP3 could not detect is until 12 h post immersion (n = 1/3). Thus,the AP4 method may be useful in detecting VPAHPNDisolates in samples where target material is limited(e.g., small tissue quantity or archived DNA) and enrichment cannot be employed (i.e., frozen samples orsamples preserved in alcohol).

Keywords:Early mortality syndromeAcute Hepatopancreatic Necrosis Disease(AHPND)Marine shrimpToxinPCR method

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Informasi Detail

Judul Seri

Aquaculture Reports

No. Panggil

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Penerbit

: Elsevier., 2015

Deskripsi Fisik

Aquaculture Reports 2 (2015) 158–162

Bahasa

English

ISBN/ISSN

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Klasifikasi

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Tipe Isi

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Tipe Media

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Tipe Pembawa

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Edisi

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Subjek

KIMIA

Info Detail Spesifik

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Pernyataan Tanggungjawab

Wati/Agus

Versi lain/terkait

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