关键词: 蝗虫微孢子虫, 套式PCR, 检测

Abstract: 【Objective】The objective of this study is to develop a rapid, sensitive and specific nested-PCR method for detection of Paranosema locustae and provide effective means for the monitoring of locusts and biological control. 【Method】 According to the sequence of small subunit ribosomal RNA genes of microsporidian published in GenBank, two pairs of primers were designed and synthesized (P.L.-F1/P.L.-R1 and P.L.-F2/P.L.-R2), and a series of assays (involving concentrations of primer pairs, Tm value and the number of cycling) were conducted to optimize the nested-PCR. P. locustae treated with 0.2 mol·L^-1 KOH were used as template for nested-PCR. P.L.-F1 and P.L.-R1 were employed as the initial primers for the first amplification, which generated a 298 bp product, and P.L.-F2 and P.L.-R2 as the secondary primers for the final amplification, which generated a 242 bp fragment. Analytical sensitivity and reproducibility were assessed, respectively. The nested-PCR method was used to detect a series of dilution of P. locustae suspension, and to test the P. locustae suspension derived from Barkol Bei mountain in Hami region. At the same time, the P. locustae suspension was extracted and detected with the traditional microscope, and then the sensitivity of two methods was compared. 【Result】The nested PCR was established. The optimal parameters for the nested PCR reaction were performed in standard mixtures of 20 μL containing 2.0 μL of Buffer (10×), 0.3 μL of dNTPs (10 mmol·L^-1), 0.3 μL of Taq DNA polymerase (250 U), 0.3 μL of each primer (10 μmol·L^-1), 1 μL of DNA template (or the diluent of the first-round product was then diluted 10-fold with water) and 15.8 μL of double distilled water. Each PCR run was carried out in a thermal cycler. The conditions of the amplification were as follows: 1 cycle at 94℃ for 3 min, 35 cycles at 94℃ for 30 s, 60℃ (62℃ at secondary PCR) for 30 s, and 72℃ for 20 s and then 1 cycle at 72℃ for 10 min, with a final hold at 10℃. The result of two rounds PCR showed that the nested PCR assay could detect 12.2 spores/µL (1.07 pg DNA). A total of 240 locust samples from Bei mountain in Hami region were analyzed by nested PCR and microscope testing, respectively, the results showed that the positive detection rate was 23.3% and 3.8%, respectively. 【Conclusion】 The nested PCR mothed established in this study is more sensitivity than traditional microscope detection method. As well as, this assay can avoid human error, and more suitable for rapid and large-scale infection intensity survey.

Key words: Paranosema locustae, nested polymerase chain reaction, detection