DB37T 3533-2019 驢騾馬皮張源性成分鑒定 實時熒光定性PCR法　

DB37Identificationofdonkey,mule/hinnyandhorsederivedmaterialsinleatherReal-timefluorescentPCRmet山東省市場監(jiān)督管理局發(fā)布I 1 1 1 1 1 1 2 2 3 3 3 3 3 3 4 4 4 4 5 71驢騾馬皮張源性成分鑒定實時熒光定性PCR法GB/T6682分析實驗室用水規(guī)格GB/T27403實驗室質(zhì)量控制規(guī)范食品分子生物學(xué)檢測根據(jù)核基因CKM(creatinekinaseofmuscle)為靶25.2材料5.3配制溶液濃鹽酸調(diào)節(jié)pH至6.4，轉(zhuǎn)移至100mL容量瓶中,加水定容至刻度，103.4KPa蒸汽壓（15.3.6細胞裂解液（LysisBuffer取1mol/LTr胺四乙酸二鈉（pH8.0）5mL，5.00.5mol/L乙二胺四乙酸二鈉（pH8.0），然后再加入4mLTritionX-100溶液（溫度60℃),轉(zhuǎn)35.3.11TE緩沖液：量取0.5mL106.4振蕩型恒溫金屬?。?℃~100℃。6.6低溫冰箱：-20℃~4℃。6.9容量瓶：10mL，50mL，18.2.7也可采用經(jīng)驗證等效的動物組織DNA提取試4特異探針，并加入內(nèi)標質(zhì)控體系。PCR反應(yīng)體系見附錄A中3000g離心1min，取出PCR9試驗數(shù)據(jù)處理9.2檢測結(jié)果分析和表述9.2.2在PCR反應(yīng)體系有效的情況下，當5A.1實時熒光定性PCR引物和探針序列AGCAGAGCAGGAAGGGAGTTAGACCACCAAATGCCCCTATC5’CY5-GCGAGGTCCCCTAGAAGAAGAACTCCCTC5’JOE-ATAGTTGAAGGTTCTGAAAATTTTTAGGCAAAGM-P5’FAM-ATAGTTGAAGCTTCTGAAAATTTTTAGGTAAAGCAGACCACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTTGTTAGACGAAGAGGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGA.2實時熒光PCR反應(yīng)體系加量(μL）220.10μmol/L1-3ng/μL21.0ng～100ngA.3對各種樣品檢測結(jié)果的判定6+-+-+++-----7B.1馬(Equuscaballus)PCR產(chǎn)物序列（Genbank:JQ0TTCCCGAATTCGCCTGCGACTCATCGTAGCATCGAGGGGAGAGGCTCTCTCTTTCCCGGCCTCTCTCTCCCCATCTCGCCGGATCTTTCAAGTCTCTGCTTATCTGTGGGAGCTGATAGTTGAAGCTTCTGAAAATTTTTAGGTAAAGCCTCCAGGATTTCTGAAATGGGAATACTTTTCATACCTTGTCACTGGCCCCCTGAAACTCCCTTCCTGCTCTGCTGB.2驢（Equusasinus）PCR產(chǎn)物序列(Genbank:HQ336263.1)驢（Equusasinus）PCR產(chǎn)物序列(GeTTCCCGAATTCGCCTGCGACTCATCGTAGCATCGAGGGGAGAGGCTCTCTCTTTCCCGGCCTCTCTCTCCCCATCTCGCTGGATCTTTTAAGTCTCTGCTTATCTGTGGGAGCTGATAGTTGAAGGTTCTGAAAATTTTTAGGCAAAGCCTCCAGGATTTCTGAAATGGGAATACTTTTCGTACCTTGTCACTGGCCCCCTGAAACTCCCTTCCTGCTCTGCTGTGCACTCCTCCTGCATATAGACCACCAAATGCCCCTATCTTATCAACACTTCCGGAAACTACTGTTGTTAGACGAAGACCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGAAGGTCTCAATCGCCGCGTCGCAGAAGATCTCAATCTCGGGAATCTCATAT