Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp (Penaeus monodon) disease caused by Vibrio parahaemolyticus (VP) since 2013 in Bangladesh. The aim of this work was to evaluate a PCR and RT-PCR techniques as rapid methods for detecting V. parahaemolyticus AHPND-positive P. monodon using genetic markers. Healthy and diseased shrimp (P. monodon) samples were collected from three monitoring stations. The samples were enriched in TCBS plates and DNA extraction from the cultured bacteria. DNA quantifications, PCR amplification, RT-PCR, and gene sequencing were done for the detection of V. parahaemolyticus AHPND-positive P. monodon. The sequence of PCR amplicons showed 100% identity and significant alignment with V. parahaemolyticus. The primers used provided high specificity for V. parahaemolyticus in PCR detection compared with another Vibrio species. In the PCR, amplification resulted positive amplicons, whereas, non-AHPND isolates showed no amplicons. Neighbor-joining methods indicated that all genes evolved from a common ancestor and clades have different traits with very low genetic distance and low variability. The pairwise alignment scores of atpA, tox, blaCARB, 16S rRNA, and pirA genes were 100.0, 98.90, 98.89, 95.53, and 41.42, respectively. The RT-qPCR exposed variable expression levels for all genes in the AHPND-positive strain. Homology analysis and distance matrix exhibited all genes to have the lowest similarity and most divergence, offering the highest specificity. In this study, the expression and variability of target genes confirmed the presence of V. parahaemolyticus in all sampling sites. The results suggested that PCR amplification, RT-qPCR, and gene sequencing can be used for the rapid detection of V. parahaemolyticus in AHPND-positive P. monodon that may lead to subsequent prevention and treatment research in the future for managing this disease.

急性肝胰腺坏死病（AHPND）是一种新出现的虾类（斑节对虾）疾病，由副溶血性弧菌（VP）自2013年在孟加拉国引起。这项工作的目的是评估 PCR 和 RT-PCR 技术作为使用遗传标记检测副溶血性弧菌AHPND 阳性斑节对虾的快速方法。从三个监测站采集了健康和患病虾（斑节对虾）样本。样品在 TCBS 板中富集并从培养的细菌中提取 DNA。DNA 定量、PCR 扩增、RT-PCR 和基因测序用于检测副溶血性弧菌AHPND 阳性斑节对虾. PCR 扩增子序列与副溶血性弧菌具有 100% 的同一性和显着的比对。与另一种弧菌相比，所使用的引物在 PCR 检测中对副溶血性弧菌具有高特异性。在 PCR 中，扩增产生阳性扩增子，而非 AHPND 分离株没有扩增子。邻接法表明，所有基因都是从一个共同的祖先进化而来的，进化枝具有不同的特征，遗传距离非常低，变异性低。atp A、tox、blaCARB、16S rRNA 和pir的成对比对分数A 基因分别为 100.0、98.90、98.89、95.53 和 41.42。RT-qPCR 暴露了 AHPND 阳性菌株中所有基因的可变表达水平。同源性分析和距离矩阵显示所有基因具有最低的相似性和最大的分歧，提供最高的特异性。在这项研究中，靶基因的表达和变异性证实了所有采样点都存在副溶血性弧菌。结果表明，PCR 扩增、RT-qPCR 和基因测序可用于快速检测AHPND 阳性斑节对虾中的副溶血性弧菌，这可能导致未来对该病的防治研究。