产毒碳黑曲霉菌双重荧光定量聚合酶链式反应检测技术的建立与应用

Establishment and application of dual fluorescence quantitative polymerase chain reaction for the detection of toxin-producing Aspergillus carbonarius

中文关键词:  产毒碳黑曲霉  双重荧光定量PCR  快速检测

英文关键词:toxin-producing Aspergillus carbonarius  dual fluorescence quantitative polymerase chain reaction  rapid detection

中文摘要:

      目的 建立检测产毒碳黑曲霉菌的聚酮合酶(polyketide synthases, PKS)基因和非核糖体肽合成酶(nonribosomal peptide synthetase, NRPS)基因的双重荧光定量聚合酶链式反应(polymerase chain reaction, PCR)方法。方法 以产毒碳黑曲霉菌PKS和NRPS相对保守基因片段作为靶点，分别设计并筛选出特异性引物和探针对, 在优化双重荧光定量PCR反应条件后，建立双重荧光PCR反应标准曲线, 随后验证该方法的特异性、灵敏度和重复性, 最后用该方法对80份实际样品和人工侵染不同浓度产毒碳黑曲霉菌孢子的玉米、花生及大豆样品进行检测。结果 特异性实验结果表明, 该双重荧光PCR对黄曲霉、寄生曲霉等常见产毒真菌均无非特异性反应，仅特异性扩增产毒碳黑曲霉菌的PKS基因和NRPS基因。灵敏度结果显示，该方法检测下限可达1×102 copies/μL。方法重复性佳, 批内和批间变异系数均小于1%。模拟样本检测结果表明, 双重荧光定量PCR在3种人工侵染样品中最低均可检出1×103 spores/mL的孢子。结论 本研究建立的方法具有较高的灵敏性和准确性, 能够为产赭曲霉毒素A的产毒碳黑曲霉菌的早期监测和防控提供技术支撑。

英文摘要:

      Objective To realize the rapid detection of toxin-producing Aspergillus carbonarius, and to establish a dual fluorescence quantitative polymerase chain reaction (qPCR) method for detecting the polyketide synthases (PKS) gene and nonribosomal peptide synthetase (NRPS) gene of Aspergillus carbonarius. Methods Taking the relatively conserved gene fragments of PKS and NRPS from toxin-producing Aspergillus carbonarius as targets, specific primer-probe sets were designed and screened respectively. After optimizing the reaction conditions of dual qPCR, the standard curve of the assay was established. Subsequently, the specificity, sensitivity, and repeatability of this method were verified. Finally, this method was used to test 80 actual samples and corn, peanut and soybean samples artificially inoculated with different concentrations of toxin-producing Aspergillus carbonarius spores. Results The specificity test results demonstrated that this dual qPCR assay exhibited no non-specific amplification with common toxin-producing fungi such as Aspergillus flavus and Aspergillus parasiticus, but specifically amplified the PKS and NRPS genes of toxin-producing Aspergillus carbonarius. The sensitivity assay showed that the limit of detection of this method could reach 1×102 copies/μL. And the method presented excellent repeatability, with the intra- and inter-batch coefficients of variation both less than 1%. In the simulated sample test, this dual qPCR could detect spores at a minimum concentration of 1×103 spores/mL in all 3 kinds of artificially infected samples. Conclusion The established dual fluorescence quantitative PCR has high sensitivity and accuracy, it can provide technical support for the early monitoring and control of ochratoxin A-toxin-producing Aspergillus carbonarius.