SCAR-PCR Rapid Molecular Detection Technology of Heterodera zeae

Corn cyst nematode (Heterodera zeae) is one of the sedentary semi-endoparasitic nematode of Heterodera spp., infects the roots of various gramineae crops. The occurrence and spread of H. zeae will pose a serious threat to corn yield production. The objective of this study is to establish the rapid and accurate molecular detection system for H. zeae from the related cyst nematode species, which will lay a technical foundation for the monitoring, early warning and prevention and control of H. zeae.

A total of 20 cyst nematode populations of H. zeae related species were collected as the nematode samples, from Henan, Hebei, Gansu, Shandong, Hunan, Guangxi and Beijing. Thirteen random primers containing 10 bases were selected, and RPAD technique was used to analyze the polymorphism of the tested nematode samples. The specific RPAD fragment was screened and transformed to SCAR-PCR primers of H. zeae. PCR was used to test the accuracy of specific primers for H. zeae and the stability, sensitivity and effectiveness of the detection technology system.

By comparative analysis of the RAPD results, one specific fragment of 468 bp was produced by primer OPA03. The fragment was recovered for sequencing. According to the fragment sequencing information, a pair of specific primers HzF1/HzR1 was designed. The specific primers HzF1 and HzR1 specificity test results showed that, one 393 bp specific fragment was amplified from H. zeae, no target bands were amplified in the other 16 populations of 5 related Heterodera spp. species (H. avenae, H. filipjevi, H. glycines, H. elachista and H. schachtii) and 4 populations of other species (Aphelenchoides besseyi, Ditylenchus destructor, Pratylenchus neglectus, P. coffeae). Furthermore, the 393 bp specific fragment was also amplified from the 6 related Heterodera spp. species mixed DNA. Meanwhile, there were no target bands when the mixed population without H. zeae. The system realized the accurate and stable detection of H. zeae, and the sensitivity and practical range of the established rapid detection technology system were tested. The results showed that the detection system was sensitive to single cyst and single 2nd stage juvenile (J2) of H. zeae. The minimum detection thresholds were 1/2 000 of single cyst or 1/80 of single J2, respectively.

The SCAR-PCR rapid molecular detection technique established in this study can be used for the rapid detection of single samples and mixed populations of H. zeae. It has sensitive detection ability for both the cysts and the J2 of H. zeae, with strong primer specificity, convenient and stable detection method and high sensitivity.