一种蛹虫草菌种的快速检测方法

摘要

本发明涉及一种蛹虫草菌种的快速检测方法，包括下列步骤：(1)菌株或子实体基因组DNA的提取；(2)蛹虫草菌种或其子实体特异DNA片段的获得；(3)特异PCR扩增引物的获得；(4)与蛹虫草子实体形成有关的分子标记的PCR扩增；(5)琼脂糖凝胶电泳检测。该检测方法具有检测时间短，准确性高，提高效率，减少损失等优点；在试管菌种生产阶段进行检测，可检测出50％不能正常出菇的菌株，从而可以在实际生产中减少50％不出子实体的损失。

主权项

一种蛹虫草菌种的快速检测方法，包括下列步骤：(1)菌株或子实体基因组DNA的提取将子实体及菌丝，放入‑20℃冰箱冻存，用CTAB法提取基因组DNA，DNA稀释至50ng/μL，‑20℃冰箱贮藏备用；(2)蛹虫草菌种或其子实体特异DNA片段的获得对应457bp的DNA片段的PCR扩增引物对序列为：2F：5′CCGTCATCTGCAAGAGGAGACAAT3′；2R：5′GCTTCCAATACGTTCCATGAAC CCT3′；对应1031bp的DNA片段的PCR扩增引物对序列为：3F：：5′TTCCTTCGCTTCCCTGAGTTCTGT3′；3R：5′GGCAGACATCGTACC TGGTCAAAT3′；对应819bp的DNA片段的PCR扩增引物对序列为：1F：5′TCAACAATCCTCCATTTCCTCCAGC3′；1R：5′CTACATGATTGACTCCGGGCTCATTG3′；应用以上引物分别对蛹虫草菌种或其子实体DNA进行扩增；其扩增得到的DNA片段的序列分别是：2F/2R引物对所扩增的DNA序列为：5‑CCGTCATCTGCAAGAGGAGACAATATTGCAGATCGCCGCAAAGTGCTTCGAAGACAAAGAAAAGGAGAACAATTCGACAAAATACTCCAAGCCTCAATCGACCCTTCATTTCACGGAGCCGCAACGAGCCTTGCCACAGAAGAGAAGCAACAGCAAGCAGGCCGGAGACCGCGCAAAACGACCATTGAATGCTTTCATCGCATTCAGAAGTGAGTGCAGAATATTAATTTCCCTCGAGAACAAATCTAACTCCAATGATTAAAAGGCTACTACGTGAAACTATTTCCAGAGTCGCAACAAAAGGCGGCGTCCGGCTTCCTCACGACACTCTGGAATAAAGACCCGTTCAGAAATCGCTGGGCAATGATCGCGAAGGTATATACTTTCATTCGCGATGAGATGGGAAAGAAGAGGGCTCCCCTCTCCTCATTTCTGATGGCTGCTTGTCCGGCAATGGACATTTTACCTCCTGATGAGTACCTTCAGGTTCTGGGCTGGGCAGTCGAAGATACCGAACAAGGTACCAAGATGCTTGTTCAGACGCTTGTCCAAATACAAGTCATGGTACAATATGCACATTATCTTCAAGGATCCATGATCCAACTCTATTCGCCGACCTCACGATCGTCCGCCCAGCGCACCAATGACATGATCCGCCACCGCCAAGTCCTCGTCCGGAGCGCCTTGCCCCCcAAGTATAAGGGAATCCACATGATAATGACAGCCGCCGAGATGCCAGCCATGGCGCCATATGTATTTTGATAGCCAGACTTGTTGATCCAAGGGTTTGTCTCGAAGGAAAGGAAAAATCCGAATAGTGCTGTACAGATCTGTGTTAGCGCAGGGTTCATGGAACGTATTGGAAGC‑3；3F/3R引物对所扩增的DNA序列为：5‑TTCAGCTTCAGTCCGTTCTGGGCCGAGGACGAGTGCATTGTAGATTCAGAATCTGCCATCGACTCAATCCGATGCCATGCTTTACGAGTTTCTCTGGAAAAGAACAGGGGAAGGGAAGAGAAACTACCGTGCCTGCAGGGTACGCGGTCTCTGGTCGGTTTGATATTTTGTCGCTAACCAAGTGCACCTAGATGTACTAGAAGATGTCATCTACGCGATCGAGAGCCTACTATGGAACCCCCGAGACGACAACATTATAATAAACCGAATAGTTGCGCTCGGCGTAGAAGAGACGATCGACCCCGTCCTGCTGGTCAAAGAAGCGATCGCCCACTGGTACATTAGCGCAGTGCCAATCTCAACCCGAGATCCTCACCATGGCTTGCCGCCGGACGCAGAGATAGAGCAGGAGGACGGCGCGACAATACTGAGATGGAATCGACAATATGCAGCTGAGAAACACGAAATAGCGAACCTCGGTCTCATAGCAATGCTCTTGACATCTGAGACATGGTCCCGTCCTGAAAGCCCAACTTTGCGGTTAGGGACATTAGTGGCAAACGCCGCCACTGCAATTTTATTCGGAGCTTTCATGATTATACCGAAAATTAGCATCAGAGAATCTTCAAATTTGCTGGTATCAGTCCTGAAAGATAACGAAGCGATGCGATTATATCTGAGAACCACCTGGCAATTGGCTCAAGATGGCGCTTCAAAATACACACACTCGCCGTCCGCTGAGTTCGGAACAACTACATCTGAGGTTAAAATTGATCATGCTGGAAAGCGACTCTTGAATAAGGTTGGACAAGAGAACTGGCCTGTGGCGTCGCTTTGGCATTCTAGTCGAAAGACCCCCGGTTCTGCTTGGAATAAATTCATGAGGAATACACAGCAACCATATTTCCCCTGCAAGAAAATCAAAACTGAGATTCGATATCGACTTCCAAGCTCTTGTCTCTCTTTAGCCGAGCCTTGGGAGGTATATTATTCTGAGCTGCGCTCCAAATTTGACCAGGTACGATGTCTGCC‑3；1F/1R引物对所扩增的DNA序列为：5‑ATGGATCTGCAACTGGATCGGACCAGACTCCAAGATCAGGAGCTTTGGGATGCCGTCAGCGTCGGCATCAACCCATTTGTGAAAGTTCTCAGTATCGACGGTCTCATCTACCGTTCCTTGACTGTGAAAACAAAAAGATCTTTGGCTGAAAATTTCGTGTAAGGACACATCCATGGCGACATTTGGGCGGCGCGCTAACTTTGCCAAGCAAACACGTACAAGAACCAGTCATGTTTTGTCGCGATGGTTTTGGCCCAGACAGATACTTCCTTGGAAATCCACGACATTTGATGTCCGGCACTGGCATCATTCTAGCTGCCGCGGAAGTAGAGCCTATTTGGTTTGAACAACCAGGCTTGAAGTACCAAAAGGCGACCATGTGTCAGTCGTCGCTTCCACGCAAACACATCAAGATCCCGAGGCCACCCAATGCGTATATTCTATACCGCAGAGACCGACATACGCTTGTCAAGAAAAGCGAGCCACATTTGTCGAACAATGAAGTTTGTAAGAGCAACTGTACGCCCCCTAAAGATAAAGTTACTGACCATTCATAGCGCAAGTGCTTGGTAAAGCTTGGAATGCAGAACCTCCAGAGGTGCGACAACGCTACAAGGAAATGTCGGAGAGGATCAAGAAGGCTCTCCTCGAACGGCATCCCCAATATCAATATCAACCTCGCAAACCCTCTGAGCGGAAGCGACGCCGCAAGCGCACTCAGGATACCGAATCTCAAAGCAGTGAGACTACGTCGGCAACGCCAGACAGCACCATTATGACAGGAAGCGAGCTTTCAATGAGCCCGGAGTCAATCATGTAG‑3；(3)特异PCR扩增引物的获得以上述扩增得到的三条DNA片段的序列为基础，分别设计457bp，1031bp及819bp的特异PCR扩增引物，引物对的序列如下：457bp特异DNA片段的PCR扩增引物对序列为：457bpF：5′ATGGAACACAGATCGAGCGACACT3′；457bpR：5′ATATACCTTCGCGATCATTGCCCAG3′；1031bp特异DNA片段的PCR扩增引物对序列为：1031bpF：5′TTCAGCTTCAGTCCGTTCTGGACA3′；1031bpR：5′GGCAGACATCGTACCTGGTCAAAT3′；819bp特异DNA片段的PCR扩增引物对序列为：819bpF：5′ATGGATCTGCAACTGGATCGGACCA3′；819bpR：5′CTACATGATTGACTCCGGGCTCATTG3′；(4)与蛹虫草子实体形成有关的分子标记的PCR扩增457bpF/R引物对特异扩增DNA片段的序列是：5‑ATGGAACACAGATCGAGCGACACTGAGCATCAAGAGCTGCTTAGGCGGCTCTCAACCGTCTCTTCACATACAATTCTCCGTCATCTGCAAGAGGAGACAATATTGCAGATCGCCGCAAAGTGCTTCGAAGACAAAGAAAAGGAGAACAATTCGACAAAATACTCCAAGCCTCAATCGACCCTTCATTTCACGGAGCCGCAACGAGCCTTGCCACAGAAGAGAAGCAACAGCAAGCAGGCCGGAGACCGCGCAAAACGACCATTGAATGCTTTCATCGCATTCAGAAGTGAGTGCAGAATATTAATTTCCCTCGAGAACAAATCTAACTCCAATGATTAAAAGGCTACTACGTGAAACTATTTCCAGAGTCGCAACAAAAGGCGGCGTCCGGCTTCCTCACGACACTCTGGAATAAAGACCCGTTCAGAAATCGCTGGGCAATGATCGCGAAGGTATAT‑3；1031bpF/R引物对特异扩增DNA片段的序列是：5‑TTCAGCTTCAGTCCGTTCTGGGCCGAGGACGAGTGCATTGTAGATTCAGAATCTGCCATCGACTCAATCCGATGCCATGCTTTACGAGTTTCTCTGGAAAAGAACAGGGGAAGGGAAGAGAAACTACCGTGCCTGCAGGGTACGCGGTCTCTGGTCGGTTTGATATTTTGTCGCTAACCAAGTGCACCTAGATGTACTAGAAGATGTCATCTACGCGATCGAGAGCCTACTATGGAACCCCCGAGACGACAACATTATAATAAACCGAATAGTTGCGCTCGGCGTAGAAGAGACGATCGACCCCGTCCTGCTGGTCAAAGAAGCGATCGCCCACTGGTACATTAGCGCAGTGCCAATCTCAACCCGAGATCCTCACCATGGCTTGCCGCCGGACGCAGAGATAGAGCAGGAGGACGGCGCGACAATACTGAGATGGAATCGACAATATGCAGCTGAGAAACACGAAATAGCGAACCTCGGTCTCATAGCAATGCTCTTGACATCTGAGACATGGTCCCGTCCTGAAAGCCCAACTTTGCGGTTAGGGACATTAGTGGCAAACGCCGCCACTGCAATTTTATTCGGAGCTTTCATGATTATACCGAAAATTAGCATCAGAGAATCTTCAAATTTGCTGGTATCAGTCCTGAAAGATAACGAAGCGATGCGATTATATCTGAGAACCACCTGGCAATTGGCTCAAGATGGCGCTTCAAAATACACACACTCGCCGTCCGCTGAGTTCGGAACAACTACATCTGAGGTTAAAATTGATCATGCTGGAAAGCGACTCTTGAATAAGGTTGGACAAGAGAACTGGCCTGTGGCGTCGCTTTGGCATTCTAGTCGAAAGACCCCCGGTTCTGCTTGGAATAAATTCATGAGGAATACACAGCAACCATATTTCCCCTGCAAGAAAATCAAAACTGAGATTCGATATCGACTTCCAAGCTCTTGTCTCTCTTTAGCCGAGCCTTGGGAGGTATATTATTCTGAGCTGCGCTCCAAATTTGACCAGGTACGATGTCTGCC‑3；819bpF/R引物对特异扩增DNA片段的序列是：5‑ATGGATCTGCAACTGGATCGGACCAGACTCCAAGATCAGGAGCTTTGGGATGCCGTCAGCGTCGGCATCAACCCATTTGTGAAAGTTCTCAGTATCGACGGTCTCATCTACCGTTCCTTGACTGTGAAAACAAAAAGATCTTTGGCTGAAAATTTCGTGTAAGGACACATCCATGGCGACATTTGGGCGGCGCGCTAACTTTGCCAAGCAAACACGTACAAGAACCAGTCATGTTTTGTCGCGATGGTTTTGGCCCAGACAGATACTTCCTTGGAAATCCACGACATTTGATGTCCGGCACTGGCATCATTCTAGCTGCCGCGGAAGTAGAGCCTATTTGGTTTGAACAACCAGGCTTGAAGTACCAAAAGGCGACCATGTGTCAGTCGTCGCTTCCACGCAAACACATCAAGATCCCGAGGCCACCCAATGCGTATATTCTATACCGCAGAGACCGACATACGCTTGTCAAGAAAAGCGAGCCACATTTGTCGAACAATGAAGTTTGTAAGAGCAACTGTACGCCCCCTAAAGATAAAGTTACTGACCATTCATAGCGCAAGTGCTTGGTAAAGCTTGGAATGCAGAACCTCCAGAGGTGCGACAACGCTACAAGGAAATGTCGGAGAGGATCAAGAAGGCTCTCCTCGAACGGCATCCCCAATATCAATATCAACCTCGCAAACCCTCTGAGCGGAAGCGACGCCGCAAGCGCACTCAGGATACCGAATCTCAAAGCAGTGAGACTACGTCGGCAACGCCAGACAGCACCATTATGACAGGAAGCGAGCTTTCAATGAGCCCGGAGTCAATCATGTAG‑3；(5)琼脂糖凝胶电泳检测。