我国外来入侵生物福寿螺种类的多重PCR鉴别方法

Multiplex PCR detection of alien invasive apple snails introduced to China

为研发能快速、准确地鉴别在我国为害严重的3种福寿螺——小管福寿螺Pomacea canaliculata、斑点福寿螺P. maculata和新发现入侵种Pomacea sp.的PCR技术,基于其线粒体全基因组序列,通过比对分析找到种间差异区段,分别设计并筛选了上述3种福寿螺的特异性引物对,建立了多重PCR检测方法,对其特异性和灵敏度进行评价。结果表明,所设计的引物特异性强,能从不同地理种群的小管福寿螺、斑点福寿螺和Pomacea sp.中分别扩增出1 238、901和571 bp的特异性条带,阴性对照无条带;退火温度为60℃时,32个扩增循环的检测灵敏度可达1 ng样品DNA量。所构建的多重PCR体系可应用于福寿螺不同部位组织碎片、不同性别及不同发育阶段样品的鉴别,具有准确快速、灵敏高效的特点,可用于植保检疫及食品安全部门福寿螺种类的检测。

To develop fast and accurate PCR-based diagnostic methods for three apple snail species, i.e. Pomacea canaliculata, P. maculata and a newly discovered species Pomacea sp., which are damaging crops seriously in China, the species-specific primers for the three species based on the divergent sequences of their whole mitochondrial genomes were designed and screened, and a multiplex PCR was established and the specificity and sensitivity of the primers were assessed. The results showed that the primers were of high specificity, which were able to amplify 1 238, 901, and 571 bp fragments from P. canaliculata, P. maculata, and Pomacea sp., respectively. No amplicon was observed from the negative control. The detective limitation was 1 ng genomic DNA of any of P. canaliculata, P. maculata, and Pomacea sp. in the PCR assay at 60℃ annealing temperature for 32 cycles. This multiplex PCR with species-specific primers could be applied to detecting tissue fragments and samples of both sexes at various developmental stages for assisting species detection of apple snails in plant protection, plant quarantine and food security.