Establishment of nested PCR assay for detection of Thelohanellus wuhanensis in allogynogenetic crucian carp

Abstract:

The nested polymerase chain reaction (nested PCR) was developed to amplify a DNA segment of the Thelohanellus wuhanensis, which caused disease in allogynogenetic crucian carp [Carassius auratus gibelio(♀)×Cyprinus carpio var singuonensis♂)]. The DNA of Thelohanellus wuhanensis was extracted as a template to be amplified. The first PCR amplification reacted with the primers: 5'-CTGCGGACGGCTCAGTAAATCAGT-3' and 5'-CCAGGACATCTTAGGGCATCACAGA-3'. The length of the first PCR amplified DNA was 1584 bp.The second nested PCR amplification reacted with the primers: 5'-ACCCACTTCTGTGGCCTTTC-3' and 5'-AATCCGAC CTACAACGCTGG-3'. The length of the second nested PCR amplified DNA was 853 bp.After optimizing the annealing temperature, the time of extension, the amplification cycle number, the concentration of dNTP, magnesium and template, the DNA detected sensitivity of nested PCR was 10000 times higher than that of conventional PCR. The DNA detected quantity of nested PCR was only 10 fg. The established nested PCR could be used for the microquantity detection of the Thelohanellus wuhanensis and provided a more sensitive technique of disease diagnosis and molecular epidemiological survey.