T-CVMA 160-2024 豬鏈球菌與副豬格拉菌雙重TaqMan實(shí)時(shí)熒光PCR檢測(cè)方法

T/CVMA160—2024豬鏈球菌與副豬格拉菌雙重TaqMan實(shí)時(shí)熒光PCR檢測(cè)方法DetectionmethodofduplextheTaqManreal-timePCRassayforStreptococcussuisandGlaesserellaparasuis2024-5-8發(fā)布2024-5-8實(shí)施中國(guó)獸醫(yī)協(xié)會(huì)發(fā)布I12規(guī)范性引用文件NY/T541獸醫(yī)診斷樣品采集、保存與運(yùn)輸技術(shù)規(guī)范實(shí)時(shí)熒光PCR：實(shí)時(shí)熒光聚合酶鏈?zhǔn)椒磻?yīng)（Real-timepolymerasechainCt值：每個(gè)反應(yīng)管內(nèi)的熒光信號(hào)達(dá)到設(shè)定的閾值所經(jīng)歷的循環(huán)數(shù)（CyclethreshoDNA：脫氧核糖核苷酸（DeoxyriTAMRA：6-羧基四甲基若丹明（Carboxytetramethylrhodamine）BHQ：無(wú)熒光淬滅基團(tuán)（Blackholequencher）DEPC：焦碳酸二乙酯（DiethylpddH2O：雙蒸水（Distillation-DistillationH2O）gdh：谷氨酸脫氫酶（glucosedehydrogenase）infB：翻譯起始因子（translationinitiationfactorIF2-IF2alphaandIF2be5試劑和耗材25.1試劑5.1.5酚/氯仿/異戊醇混合液按5.1.9PCR試劑及DNA凝膠回收試20μL、200μL和1000μL）、冰箱（2℃~8℃和?20℃以下）、組織勻漿器、混勻器。8細(xì)菌核酸的提取38.2豬鼻/口拭子、唾液、關(guān)節(jié)液及環(huán)境樣品DNA提取重懸，水浴鍋沸水中煮10min，冰浴5min，12000r/min離心1min，取上清作為PCR擴(kuò)增模板。8.3臨床組織樣品（扁桃體、肺臟等）DNA提取8.3.1用無(wú)菌剪刀和鑷子剪取典型病變組織0.5g~1.0g，消化液II，反復(fù)吹吸混勻，置55℃,水浴30min~60min。9.1反應(yīng)體系雙重TaqMan實(shí)時(shí)熒光定量PCR反應(yīng)體系見(jiàn)9.2反應(yīng)程序雙重TaqMan實(shí)時(shí)熒光定量PCR反應(yīng)程序見(jiàn)9.3結(jié)果判定9.3.1質(zhì)控標(biāo)準(zhǔn)當(dāng)所有陽(yáng)性對(duì)照的FAM和TAMRA檢測(cè)通道均有典型的S型擴(kuò)增曲線，陰性對(duì)照的FAM和TAMRA9.3.2結(jié)果判定45A.1豬鏈球菌gdh基因片段參考序列（GenBankAccessionNo.AM946016.CACCTGCAAGGTAGTCTGTACCAAGGTCGTATTTTTCAGCAGTTTCTTTGTGGCCATGATGTCTTTAAGACGGCCGTCTACTTCTTCACGAGTCCATGACAAGCGTGACTCATTTCAAGGGCAGATACAGCTACACCACCAGCGTTGGCAGCTTTTGCGAGTAACGCCATTTTCCTTGTAGACTTTGATGGCATCAAGGTCAGATGGCATGCACAGTACACGCCATTTTTTACAAGGGCAGCAGCTTGTTTGCCGTTGATCTCATTTTGAGTATGGAAGGGCAATATCAGCCTTGCCATCGTAGTTCCATACAGAACCTTTGAAGTACGATTTTTCTGCAGCGTATTCTGTCAAACGAGCGCGGCGTTTTTCTTTGATGTCCACCAAAAGTCGATACCAGTTTCGTCAATGATGTAACCATTTGAGTCTGAAACAGAAATAAAAGTTCAGTCGCTTTTTGAACAGCATATTGGGCAACGTTACCAGAACCTGAGATAAGGTCTTTGAAGGATTTACCGTTTGCTGCCAACATGTTATCAGTGAAGTAAACCAAACCGTGTTGCTTCTGGGCGGATCAATGAACCACCGAAGCCAAGAGGTTTACCAGTCAAGACACAAACTGGCGGAGGCGTTTGTATTGACCGTACATGTAACCGATCTCACGACCACCGACCACCAGCAGGGACGTCAAGTGAAGGTCCGATGTGTTTTTGCAATTCAGTCATGAAGCTAAGCGCATGATTTCAGCATCAGTTTTTCCTTTAGGATCAAAGTCTGAACCACCTTTACATTGGAAGACCAGTCAAGACGTTTTTGAAGATTTGCTCAAAACCGAGGAACTTCAAGATGGATTGGTTTACAGTTGGGTGGAAGCGAAGACCGCCTTTATAAGGACCTACAGCTGAGTTGAACGGTAGCCACGGTTGACTTGAACATTTCCATCTTTATCTGTCCATGGAACACGGGATACGCTCAGGCTCAACGATACGAGCCAAGATGTTTTCTTCGATGTATTCTGGAAACAGGCTCAAGTGTAGAGAAGAGCTCTTCTACAGCTTGGAGGAATTCTGTTTCATCGGGCTTTGACTGCTTCAAAAGAAGCTTGGATGTAAGCTTTGGCATTTGACAT-3'注：下劃線部分為連入pMD-18T載體中的包含豬鏈球菌gdh目標(biāo)檢測(cè)片段部分，粗體為引物和探針A.2副豬格拉菌infB基因片段參考序列（GenBankAccessionNo.CP071489.1）5＇-GTTACGGACTTCTGAAACGTCGTCTTTGAAACGACGGAAATTACCACGTTATCACGTAATACACGGATTGGGTTGTTACGTTTGACGATACCTTCATACAACCTGCAATTGCACCAAATTTCGGGTGACGGAAGACATTACGCACTATTTCTTGTTTAAATTCAGGTTGTAACATACCGCTCATTGCTGCTTTGATTTCGTTTAATAGCTCATAAATGATTGAATAATAACGTAAATCAATGTTTTCGCTTTCGATAATGCGACCTGCACGGACGTTAAAGCCAAGTACAATAGCGTTAGACGCTGCCGCTAAGGTTGCATCCGTAATACCACCTACGCCAGAGCCAACTACTTTCACTTTTACTTCTGCCGTTGAAAGCAAAGATTGAACAATCGCTTCTACAGAGCCTTGTACGTCTGCTTTCACAATCACGTTCAACATCGCCTTCCGCCATATTGCTAAACATATTTTCAAGTTTCGCTTTTTGTTGGCGAGCCAAACATCACGGAATTTACCTTGACGGTATAACGCCACTTCACGCGCTTTTTTCTCATCACGGTCGCTTCATCACCTGCGGCTGGTACGCCTGAAAGTCCTAATACTTCCACAGGAATTGATGGG6GGCTGATTCAATGTCTTTACCATTTTCATCACGCATTGCACGAACACGGCCATATTCAAAGTACGATGTCGCCTTTGTTTAACGTACCTGATTGAACAAGGATAGTTGCAACTGGCCTTTATCGAGGTAAGATTCGATAACCACACCGCTTGCCATACCCTCTTTCACTGCACTTAAATACTTCCGATTGAAGAAGAATGGCTTCAAGTAAGTCGTCAATCCCCATTCCTTTTTTACAGGAACAAATTGAACATCACCACCGAATTTCTCAGAAATCACTTCGTGTTGTAATAACTTTCTACACGCTCTAGGTTTGCTTCTGGTTTATCAATTTTGTTTACCGCAACCACAGCTGCTTTCGCGTGTTGGATTGCTTCAATGGTTTGTGGCATTACGCCATCGTCAGCTAAGAACAACGATATCCGTTGCTTTCGCACCACGCGCACGCATTGAGGTAAATGGTGTATCTAAGAAGGTAATCATCTTACCGTCGTCGGTTTCAACGTGATATGCACCGATATGTAATACCGCCAGCTTCACCTGCTGCTACTTTCGCTTTACGGATATAATCTAATAA注：下劃線部分為連入pMD-18T載體中的包含副豬格拉菌infB基因目標(biāo)增，PCR擴(kuò)增產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳檢測(cè)后，用DNA凝膠回收試劑盒回收PCR產(chǎn)物，連接至pMD18-T載體構(gòu)建重組質(zhì)粒pMD-SS光度計(jì)檢測(cè)2種陽(yáng)性質(zhì)粒濃度，并按下面公式將濃度換算成拷貝數(shù)，DNA為豬鏈球菌和副豬格拉菌雙重TaqMan熒光PCR檢測(cè)方法的陽(yáng)性對(duì)照使A.4陰性對(duì)照的制備滅菌生理鹽水：稱(chēng)取0.9g氯化鈉，溶解在7gdhGdh-RGdh-ProbeinfB-FinfB-RinfB-Probe8Tris-HCl（pH值8.0）終濃度為10mmol/L；EDTA（pH值8.0）終濃度為250mmol/L；SDS終濃度為200ug/mL；NaCl終濃度為100mmol/L。杯中加入約400mlddH2O均勻混合；將溶液定容到509表D.1雙重TaqMan實(shí)時(shí)熒光PCR反應(yīng)體體積(μL）終濃度(μM）2×AnimalDetectionU+ProbeqPCRSuperPreSsuis探針（FAM標(biāo)記）Gparasuis探針（TAMRA標(biāo)記）25.0注：1.此反應(yīng)體積以Animal