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Title: AP4 method for two-tube nested PCR detection of AHPND isolates of Vibrio parahaemolyticus
Authors: Sirintip Dangtip
Ratchanok Sirikharin
Piyachat Sanguanrut
Siripong Thitamadee
Kallaya Sritunyalucksana
Suparat Taengchaiyaphum
Rapeepat Mavichak
Porranee Proespraiwong
Timothy W. Flegel
Thailand National Center for Genetic Engineering and Biotechnology
Mahidol University
Aquatic Animal Health Research Center
Keywords: Agricultural and Biological Sciences
Issue Date: 1-Nov-2015
Citation: Aquaculture Reports. Vol.2, (2015), 158-162
Abstract: © 2015 The Authors. Our previous work on the mechanism of virulence for the unique isolates of Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis disease (VPAHPND)revealed that it was mediated by a binary Pir-like toxin 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 of ToxA, a one-step PCR detection method for VPAHPNDwas introduced in June 2014 but had the limitation that 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 culture to allow bacterial growth before extraction of template DNA. Here, we describe the AP4 (abbreviation of AHPND detection version 4) method, a two-tube nested PCR method that targets the tandem genes ToxA and ToxB, including the 12 bp spacer that separates them on pVA plasmid. Testing of the method revealed that it gave 100% positive and negative predictive values for VPAHPNDusing a panel of 104 bacterial isolates 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-step AP3 (10 pg total DNA template) method, and it could detect VPAHPNDin experimentally challenged shrimp by 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 or samples preserved in alcohol).
ISSN: 23525134
Appears in Collections:Scopus 2011-2015

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