基于线粒体DNA-COI序列的禾谷孢囊线虫和菲利普孢囊线虫双重PCR检测

doi:10.3864/j.issn.0578-1752.2016.08.007

Abstract

Abstract: 【Objective】 The specific primers for the duplex-PCR reaction of Heterodera avenae and H. filipjevi were designed and a synchronous detection system was established for these two species. The work will provide technical supports for rapid and accurate diagnosis of cereal cyst nematodes (CCN) in wheat field of China and benefit to the integrate management strategies.【Method】The forward primers HaF8 and HfF9 respectively specific to H. avenae and H. filipjevi together with the common reverse primer HafR8 were designed according to the comparisons of mtDNA-COI sequences from 24 populations of 10 plant parasitic nematode species. The concentrations of primers and the annealing temperatures for duplex-PCR reaction were optimized and a high efficient detection system for these two CCN species was developed. The established system was applied to identify the CCN species of samples collected from wheat fields in Huanghuai region of China. 【Result】 The specific primers HaF8/HafR8 and HfF9/HafR8 obtained in this study could rapidly detect H. avenae and H. filipjevi in one PCR reaction with high specificity. The PCR product for H. avenae amplified with primers HaF8/HafR8 was 200 bp, for H. filipjevi with primers HfF9/HafR8 was 320 bp. The band sizes were easily distinguished. The optimization showed that the duplex-PCR detection system had a high specificity and amplification efficiency with the annealing temperature of primers HaF8/HfF9/HafR8 at 58℃. In addition, the detection system could identify H. avenae and H. filipjevi simultaneously from all detected samples with a high efficiency, when the concentration of HaF8﹕HfF9﹕HafR8 was 0.24﹕0.16﹕0.4 μmol·L^-1. The sensitivities of the duplex-PCR system detection for single cyst of H. avenae and H. filipjevi were both 1/2 000 000, but for single second stage juvenile were 1/640 and 1/1 280, respectively. The duplex-PCR system was used to identify the species of 14 CCN samples collected from wheat fields in Huanghuai region of China. The results showed that the 200 bp band was amplified from 8 samples, indicating the species is H. avenae. The 320 bp band was amplified from 4 samples, indicating the species is H. filipjevi. However, both the band 200 and 320 bp were only amplified from 2 samples assuming a mixture of H. avenae and H. filipjevi occurred. The results demonstrated that the duplex-PCR detection system could be successfully applied in rapid diagnosis of the complex infection situation of CCN in fields. 【Conclusion】 The established duplex-PCR system with the specific primers designed on basis of mtDNA-COI sequences is a reliable and simple technique for simultaneously rapid detecting H. avenae and H. filipjevi in field samples on wheat.

Key words: Heterodera avenae, Heterodera filipjevi, duplex-PCR, COI, specificity

NIU Wen-wen, WANG Xuan, LI Hong-mei, JU Yu-liang, WAN Wen-wen. Duplex-PCR Detection for Heterodera avenae and Heterodera filipjevi Based on mtDNA-COI Sequences[J].Scientia Agricultura Sinica, 2016, 49(8): 1499-1509.

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Duplex-PCR Detection for Heterodera avenae and Heterodera filipjevi Based on mtDNA-COI Sequences

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