CN105219860A - 一种结核分枝杆菌KatG耐药突变位点检测试剂盒 - Google Patents
一种结核分枝杆菌KatG耐药突变位点检测试剂盒 Download PDFInfo
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Abstract
本发明公开了一种结核分枝杆菌KatG耐药突变位点检测试剂盒。通过与结核分枝杆菌标准株H37Rv序列（NC_000962.3）的比对，发明人从临床分离的耐异烟肼结核分枝杆菌katG基因中发现了未报道的2个突变位点：294GGG和296ATC。同时，该耐药株katG基因有且仅有此2个突变位点。通过克隆质粒及MIC值测定证实katG基因294GGG，296ATC，590AGG，690GCC位点的突变可以导致显著异烟肼耐药。其中590AGG，690GCC位点突变的发生是在质粒克隆过程中自发发生的，经过生物信息学分析，这两个位点突变也未有研究报道。针对这4种突变位点，发明人研发结核分枝杆菌katG耐药突变位点检测试剂盒，如PCR-荧光探针法，为耐异烟肼结核患者的早期诊断提供分子检测基础。
Description
技术领域
本发明涉及一种结核分枝杆菌KatG耐药突变位点检测试剂盒，特别涉及一种结核分枝杆菌KatG耐药突变位点检测试剂盒。
背景技术
结核病是一种严重危害人类健康的慢性呼吸道传染病，其病原体是结核分枝杆菌，是人类所知的最古老的一种传染病。结核分枝杆菌（Mycobacteriumtuberculosis,MTB）的耐药性问题已成为新世纪结核病控制的难题之一。随着抗结核药物的广泛使用，以及结核分枝杆菌染色体的突变，导致结核耐药问题日益严重。
异烟肼（Isoniazid,INH）是结核化疗方案中的一线药物之一，可通过氧依赖途径抑制结核分枝杆菌胞壁酸的合成，从而起到杀菌作用。目前已发现结核分枝杆菌对异烟肼耐药涉及多个基因变化：katG,inhA,kasA,ndh,及oxyR-ahpC基因连接区。而研究证明，katG突变导致的结核分枝杆菌触酶-过氧化物酶活性降低或缺失可以解释90%以上的INH耐药。最普遍的katG突变是发生在第315位密码子，而其它位点密码子突变造成INH耐药也有陆续报道。因此，建立方便快捷经济的katG突变检测方法，对实现早期迅速对结核患者的结核分枝杆菌异烟肼耐药鉴定非常重要。
发明内容
本发明的目的在于提供一种检测结核分枝杆菌新型KatG耐药突变位点的试剂盒。
通过与结核分枝杆菌标准株H37Rv序列（NC_000962.3）的比对，发明人从临床分离的耐异烟肼结核分枝杆菌katG基因中发现了未报道的2个突变位点：294GGG和296ATC。同时，该耐药株katG基因有且仅有此2个突变位点。通过克隆质粒及MIC值测定证实katG基因294GGG，296ATC，590AGG，690GCC位点的突变可以导致显著异烟肼耐药。其中590AGG，690GCC位点突变的发生是在质粒克隆过程中自发发生的，经过生物信息学分析，这两个位点突变也未有研究报道。针对这4种突变位点，发明人研发结核分枝杆菌katG耐药突变位点检测试剂盒，如PCR-荧光探针法，为耐异烟肼结核患者的早期诊断提供分子检测基础。
本发明的有益效果是：
本发明的结核分枝杆菌KatG耐药突变位点检测试剂盒，为耐异烟肼结核患者的早期诊断提供分子检测基础。有利于对结核患者进行个性化治疗，达到更好的治疗效果。
具体实施方式
下面结合实验，进一步说明本发明的技术方案。
结核分枝杆菌的药敏实验
参照结核分枝杆菌药敏实验标准比例法。从培养平板或者培养管中挑选适量的菌体移到磨菌器底部，搅动均匀成为乳酪状，以0.5%Tween80生理盐水溶解后，将菌悬液与标准麦氏比浊管比浊，即配成1mg/ml的菌悬液。静置片刻，使菌液中的颗粒或菌块沉淀后，将其依旧用0.5%Tween80生理盐水倍比稀释，稀释为两种浓度，分别为：10-2mg/ml，10-4mg/ml的菌悬液。将上述两个浓度的菌悬液0.1ml接种于含药及对照培养基斜面或表面。接种时，使菌液尽可能均匀分散于培养基表面。37℃培养，4-6周观察结果。计算耐药百分比。耐药百分比=含药培养基上生长菌落数/对照培养基生长菌落数*100%，>1％者为耐药，<1％为敏感。本实验所使用的抗结核药物及浓度为：异烟肼，0.2μg/mL；利福平，40μg/mL；链霉素，4μg/mL；乙胺丁醇，2μg/mL。
质粒构建
质粒载体为pMV306整合质粒（pMV361的衍生质粒）。pMV306克隆质粒电转入耻垢分枝杆菌（MS）后，不能自主复制。根据位点特异性重组原理，pMV306克隆质粒会被整合入的耻垢分枝杆菌基因组中，外源基因从而被高效表达，可用卡那霉素平板来筛选重组子。本实验通过基因克隆方法，把临床耐异烟肼结核分枝杆菌MtbM35的katG基因以及katG野生型基因分别克隆在pMV306载体上。所使用的酶切位点为：HindIII和KpnI。
质粒电转
分别把pMV306-MtbM35katG和pMV306-katGwt质粒电转入耻垢分枝杆菌（非条件致病菌，能够进行高频率的外源性DNA转化），用含异烟肼8μg/mL及卡那霉素40μg/mL的抗性平板筛选电转子，得到克隆菌株。
MIC实验
分别表达MtbM35katG基因以及katG野生型基因的克隆菌株，用7H9液体培养基过夜培养，制备成1mg/ml菌悬液。菌悬液稀释10倍后取10μL菌液，加入含不同异烟肼药物浓度梯度的7H9液体培养基中。37℃培养4周，肉眼观察菌液是否浑浊，判断MIC值。本实验所用异烟肼药物浓度梯度为：0，1，2，4，8，16，32，64，128，256，512μg/mL。
PCR-荧光探针法
PCR-荧光探针法是在PCR基础上加入一种与靶基因序列互补的荧光双标记的寡核苷酸探针，探针的5’端标记荧光报告基团，3’端标记荧光淬灭基团。利用TaqDNA聚合酶和根据靶序列设计的特异性引物、探针，特异识别靶序列区域，在TaqDNA聚合酶的作用下，能将已跟靶序列互补配对的探针酶切降解，使报告基团和淬灭基团分离，从而产生荧光信号，进而对SNPs进行很好的区分，并通过荧光扩增曲线客观判断突变类型的存在。
实验结果
临床耐异烟肼结核分枝杆菌MtbM35耐药表型
临床耐异烟肼结核分枝杆菌MtbM35katG基因测序
附测序序列：
GTGCCCGAGCAACACCCACCCATTACAGAAACCACCACCGGAGCCGCTAGCAACGGCTGTCCCGTCGTGGGTCATATGAAATACCCCGTCGAGGGCGGCGGAAACCAGGACTGGTGGCCCAACCGGCTCAATCTGAAGGTACTGCACCAAAACCCGGCCGTCGCTGACCCGATGGGTGCGGCGTTCGACTATGCCGCGGAGGTCGCGACCATCGACGTTGACGCCCTGACGCGGGACATCGAGGAAGTGATGACCACCTCGCAGCCGTGGTGGCCCGCCGACTACGGCCACTACGGGCCGCTGTTTATCCGGATGGCGTGGCACGCTGCCGGCACCTACCGCATCCACGACGGCCGCGGCGGCGCCGGGGGCGGCATGCAGCGGTTCGCGCCGCTTAACAGCTGGCCCGACAACGCCAGCTTGGACAAGGCGCGCCGGCTGCTGTGGCCGGTCAAGAAGAAGTACGGCAAGAAGCTCTCATGGGCGGACCTGATTGTTTTCGCCGGCAACTGCGCGCTGGAATCGATGGGCTTCAAGACGTTCGGGTTCGGCTTCGGCCGGGTCGACCAGTGGGAGCCCGATGAGGTCTATTGGGGCAAGGAAGCCACCTGGCTCGGCGATGAGCGTTACAGCGGTAAGCGGGATCTGGAGAACCCGCTGGCCGCGGTGCAGATGGGGCTGATCTACGTGAACCCGGAGGGGCCGAACGGCAACCCGGACCCCATGGCCGCGGCGGTCGACATTCGCGAGACGTTTCGGCGCATGGCCATGAACGACGTCGAAACAGCGGCGCTGATCGTCGGCGGTCACACTTTCGGTAAGACCCATGGCGCCGGCCCGGCCGATCTGGTCGGCCCCGAACCCGAGGCTGCTCCGCTGG G GCAGAT C GGCTTGGGCTGGAAGAGCTCGTATGGCACCGGAACCGGTAAGGACGCGATCACCAGCGGCATCGAGGTCGTATGGACGAACACCCCGACGAAATGGGACAACAGTTTCCTCGAGATCCTGTACGGCTACGAGTGGGAGCTGACGAAGAGCCCTGCTGGCGCTTGGCAATACACCGCCAAGGACGGCGCCGGTGCCGGCACCATCCCGGACCCGTTCGGCGGGCCAGGGCGCTCCCCGACGATGCTGGCCACTGACCTCTCGCTGCGGGTGGATCCGATCTATGAGCGGATCACGCGTCGCTGGCTGGAACACCCCGAGGAATTGGCCGACGAGTTCGCCAAGGCCTGGTACAAGCTGATCCACCGAGACATGGGTCCCGTTGCGAGATACCTTGGGCCGCTGGTCCCCAAGCAGACCCTGCTGTGGCAGGATCCGGTCCCTGCGGTCAGCCACGACCTCGTCGGCGAAGCCGAGATTGCCAGCCTTAAGAGCCAGATCCGGGCATCGGGATTGACTGTCTCACAGCTAGTTTCGACCGCATGGGCGGCGGCGTCGTCGTTCCGTGGTAGCGACAAGCGCGGCGGCGCCAACGGTGGTCGCATCCGCCTGCAGCCACAAGTCGGGTGGGAGGTCAACGACCCCGACGGGGATCTGCGCAAGGTCATTCGCACCCTGGAAGAGATCCAGGAGTCATTCAACTCCGCGGCGCCGGGGAACATCAAAGTGTCCTTCGCCGACCTCGTCGTGCTCGGTGGCTGTGCCGCCATAGAGAAAGCAGCAAAGGCGGCTGGCCACAACATCACGGTGCCCTTCACCCCGGGCCGCACGGATGCGTCGCAGGAACAAACCGACGTGGAATCCTTTGCCGTGCTGGAGCCCAAGGCAGATGGCTTCCGAAACTACCTCGGAAAGGGCAACCCGTTGCCGGCCGAGTACATGCTGCTCGACAAGGCGAACCTGCTTACGCTCAGTGCCCCTGAGATGACGGTGCTGGTAGGTGGCCTGCGCGTCCTCGGCGCAAACTACAAGCGCTTACCGCTGGGCGTGTTCACCGAGGCCTCCGAGTCACTGACCAACGACTTCTTCGTGAACCTGCTCGACATGGGTATCACCTGGGAGCCCTCGCCAGCAGATGACGGGACCTACCAGGGCAAGGATGGCAGTGGCAAGGTGAAGTGGACCGGCAGCCGCGTGGACCTGGTCTTCGGGTCCAACTCGGAGTTGCGGGCGCTTGTCGAGGTCTATGGCGCCGATGACGCGCAGCCGAAGTTCGTGCAGGACTTCGTCGCTGCCTGGGACAAGGTGATGAACCTCGACAGGTTCGACGTGCGCTGA（SEQIDNO：1）。
MSkatGM35测序
附测序序列：
GTGCCCGAGCAACACCCACCCATTACAGAAACCACCACCGGAGCCGCTAGCAACGGCTGTCCCGTCGTGGGTCATATGAAATACCCCGTCGAGGGCGGCGGAAACCAGGACTGGTGGCCCAACCGGCTCAATCTGAAGGTACTGCACCAAAACCCGGCCGTCGCTGACCCGATGGGTGCGGCGTTCGACTATGCCGCGGAGGTCGCGACCATCGACGTTGACGCCCTGACGCGGGACATCGAGGAAGTGATGACCACCTCGCAGCCGTGGTGGCCCGCCGACTACGGCCACTACGGGCCGCTGTTTATCCGGATGGCGTGGCACGCTGCCGGCACCTACCGCATCCACGACGGCCGCGGCGGCGCCGGGGGCGGCATGCAGCGGTTCGCGCCGCTTAACAGCTGGCCCGACAACGCCAGCTTGGACAAGGCGCGCCGGCTGCTGTGGCCGGTCAAGAAGAAGTACGGCAAGAAGCTCTCATGGGCGGACCTGATTGTTTTCGCCGGCAACTGCGCGCTGGAATCGATGGGCTTCAAGACGTTCGGGTTCGGCTTCGGCCGGGTCGACCAGTGGGAGCCCGATGAGGTCTATTGGGGCAAGGAAGCCACCTGGCTCGGCGATGAGCGTTACAGCGGTAAGCGGGATCTGGAGAACCCGCTGGCCGCGGTGCAGATGGGGCTGATCTACGTGAACCCGGAGGGGCCGAACGGCAACCCGGACCCCATGGCCGCGGCGGTCGACATTCGCGAGACGTTTCGGCGCATGGCCATGAACGACGTCGAAACAGCGGCGCTGATCGTCGGCGGTCACACTTTCGGTAAGACCCATGGCGCCGGCCCGGCCGATCTGGTCGGCCCCGAACCCGAGGCTGCTCCGCTGG G GCAGAT C GGCTTGGGCTGGAAGAGCTCGTATGGCACCGGAACCGGTAAGGACGCGATCACCAGCGGCATCGAGGTCGTATGGACGAACACCCCGACGAAATGGGACAACAGTTTCCTCGAGATCCTGTACGGCTACGAGTGGGAGCTGACGAAGAGCCCTGCTGGCGCTTGGCAATACACCGCCAAGGACGGCGCCGGTGCCGGCACCATCCCGGACCCGTTCGGCGGGCCAGGGCGCTCCCCGACGATGCTGGCCACTGACCTCTCGCTGCGGGTGGATCCGATCTATGAGCGGATCACGCGTCGCTGGCTGGAACACCCCGAGGAATTGGCCGACGAGTTCGCCAAGGCCTGGTACAAGCTGATCCACCGAGACATGGGTCCCGTTGCGAGATACCTTGGGCCGCTGGTCCCCAAGCAGACCCTGCTGTGGCAGGATCCGGTCCCTGCGGTCAGCCACGACCTCGTCGGCGAAGCCGAGATTGCCAGCCTTAAGAGCCAGATCCGGGCATCGGGATTGACTGTCTCACAGCTAGTTTCGACCGCATGGGCGGCGGCGTCGTCGTTCCGTGGTAGCGACAAGCGCGGCGGCGCCAACGGTGGTCGCATCCGCCTGCAGCCACAAGTCGGGTGGGAGGTCAACGACCCCGACGGGGATCTGCGCAAGGTCATTCGCACCCTGGAAGAGATCCAGGAGTCATTCAACTCCGCGGCGCCGGGGAACATCAAAGTGTCCTTCGCCGACCTCGTCGTGCTCGGTGGCTGTGCCGCCATAGAGAAAGCAGCAAAGGCGGCTGGCCACAACATCACGGTGCCCTTCACCCCGGGCCGCACGGATGCGTCGCAGGAACAAACCGACGTGGAATCCTTTGCCGTGCTGGAGCCCA G GGCAGATGGCTTCCGAAACTACCTCGGAAAGGGCAACCCGTTGCCGGCCGAGTACATGCTGCTCGACAAGGCGAACCTGCTTACGCTCAGTGCCCCTGAGATGACGGTGCTGGTAGGTGGCCTGCGCGTCCTCGGCGCAAACTACAAGCGCTTACCGCTGGGCGTGTTCACCGAGGCCTCCGAGTCACTGACCAACGACTTCTTCGTGAACCTGCTCGACATGGGTATCACCTGGGAGCCCTCGCCAGCAGATGACGGGACCTACCAGGGCAAGGATGGCAGTGGCAAGGTGAAGTGG G CCGGCAGCCGCGTGGACCTGGTCTTCGGGTCCAACTCGGAGTTGCGGGCGCTTGTCGAGGTCTATGGCGCCGATGACGCGCAGCCGAAGTTCGTGCAGGACTTCGTCGCTGCCTGGGACAAGGTGATGAACCTCGACAGGTTCGACGTGCGCTGA（SEQIDNO：2）。
上述两个测序序列中，下划线+倾斜的核酸为突变位点。
INH的MIC值测定
KatG结核分枝杆菌KatG耐药突变位点检测试剂盒
在确定上述突变位点之后，通过现有技术即可制备得到其检测试剂盒。如PCR-荧光探针试剂盒，该试剂盒中主要原材料有：TaqDNA聚合酶、引物、Taqman-MGB探针、10×buffer、dNTPs、MgCl2、甜菜碱、DMSO、阴性对照和阳性对照等。试剂盒中阳性对照来源于人工构建的含靶序列片段的大肠杆菌工程菌DNA，无感染活性，无潜在的生物安全危险。阴性对照为超纯水。
也可以采用其他公开的SNP检测方法原理，设计出对应的检测试剂盒。
<110>中山大学
<120>一种结核分枝杆菌KatG耐药突变位点检测试剂盒
<130>
<160>2
<170>PatentInversion3.5
<210>1
<211>2223
<212>DNA
<213>结核分枝杆菌
<400>1
gtgcccgagcaacacccacccattacagaaaccaccaccggagccgctagcaacggctgt60
cccgtcgtgggtcatatgaaataccccgtcgagggcggcggaaaccaggactggtggccc120
aaccggctcaatctgaaggtactgcaccaaaacccggccgtcgctgacccgatgggtgcg180
gcgttcgactatgccgcggaggtcgcgaccatcgacgttgacgccctgacgcgggacatc240
gaggaagtgatgaccacctcgcagccgtggtggcccgccgactacggccactacgggccg300
ctgtttatccggatggcgtggcacgctgccggcacctaccgcatccacgacggccgcggc360
ggcgccgggggcggcatgcagcggttcgcgccgcttaacagctggcccgacaacgccagc420
ttggacaaggcgcgccggctgctgtggccggtcaagaagaagtacggcaagaagctctca480
tgggcggacctgattgttttcgccggcaactgcgcgctggaatcgatgggcttcaagacg540
ttcgggttcggcttcggccgggtcgaccagtgggagcccgatgaggtctattggggcaag600
gaagccacctggctcggcgatgagcgttacagcggtaagcgggatctggagaacccgctg660
gccgcggtgcagatggggctgatctacgtgaacccggaggggccgaacggcaacccggac720
cccatggccgcggcggtcgacattcgcgagacgtttcggcgcatggccatgaacgacgtc780
gaaacagcggcgctgatcgtcggcggtcacactttcggtaagacccatggcgccggcccg840
gccgatctggtcggccccgaacccgaggctgctccgctggggcagatcggcttgggctgg900
aagagctcgtatggcaccggaaccggtaaggacgcgatcaccagcggcatcgaggtcgta960
tggacgaacaccccgacgaaatgggacaacagtttcctcgagatcctgtacggctacgag1020
tgggagctgacgaagagccctgctggcgcttggcaatacaccgccaaggacggcgccggt1080
gccggcaccatcccggacccgttcggcgggccagggcgctccccgacgatgctggccact1140
gacctctcgctgcgggtggatccgatctatgagcggatcacgcgtcgctggctggaacac1200
cccgaggaattggccgacgagttcgccaaggcctggtacaagctgatccaccgagacatg1260
ggtcccgttgcgagataccttgggccgctggtccccaagcagaccctgctgtggcaggat1320
ccggtccctgcggtcagccacgacctcgtcggcgaagccgagattgccagccttaagagc1380
cagatccgggcatcgggattgactgtctcacagctagtttcgaccgcatgggcggcggcg1440
tcgtcgttccgtggtagcgacaagcgcggcggcgccaacggtggtcgcatccgcctgcag1500
ccacaagtcgggtgggaggtcaacgaccccgacggggatctgcgcaaggtcattcgcacc1560
ctggaagagatccaggagtcattcaactccgcggcgccggggaacatcaaagtgtccttc1620
gccgacctcgtcgtgctcggtggctgtgccgccatagagaaagcagcaaaggcggctggc1680
cacaacatcacggtgcccttcaccccgggccgcacggatgcgtcgcaggaacaaaccgac1740
gtggaatcctttgccgtgctggagcccaaggcagatggcttccgaaactacctcggaaag1800
ggcaacccgttgccggccgagtacatgctgctcgacaaggcgaacctgcttacgctcagt1860
gcccctgagatgacggtgctggtaggtggcctgcgcgtcctcggcgcaaactacaagcgc1920
ttaccgctgggcgtgttcaccgaggcctccgagtcactgaccaacgacttcttcgtgaac1980
ctgctcgacatgggtatcacctgggagccctcgccagcagatgacgggacctaccagggc2040
aaggatggcagtggcaaggtgaagtggaccggcagccgcgtggacctggtcttcgggtcc2100
aactcggagttgcgggcgcttgtcgaggtctatggcgccgatgacgcgcagccgaagttc2160
gtgcaggacttcgtcgctgcctgggacaaggtgatgaacctcgacaggttcgacgtgcgc2220
tga2223
<210>2
<211>2223
<212>DNA
<213>结核分枝杆菌
<400>2
gtgcccgagcaacacccacccattacagaaaccaccaccggagccgctagcaacggctgt60
cccgtcgtgggtcatatgaaataccccgtcgagggcggcggaaaccaggactggtggccc120
aaccggctcaatctgaaggtactgcaccaaaacccggccgtcgctgacccgatgggtgcg180
gcgttcgactatgccgcggaggtcgcgaccatcgacgttgacgccctgacgcgggacatc240
gaggaagtgatgaccacctcgcagccgtggtggcccgccgactacggccactacgggccg300
ctgtttatccggatggcgtggcacgctgccggcacctaccgcatccacgacggccgcggc360
ggcgccgggggcggcatgcagcggttcgcgccgcttaacagctggcccgacaacgccagc420
ttggacaaggcgcgccggctgctgtggccggtcaagaagaagtacggcaagaagctctca480
tgggcggacctgattgttttcgccggcaactgcgcgctggaatcgatgggcttcaagacg540
ttcgggttcggcttcggccgggtcgaccagtgggagcccgatgaggtctattggggcaag600
gaagccacctggctcggcgatgagcgttacagcggtaagcgggatctggagaacccgctg660
gccgcggtgcagatggggctgatctacgtgaacccggaggggccgaacggcaacccggac720
cccatggccgcggcggtcgacattcgcgagacgtttcggcgcatggccatgaacgacgtc780
gaaacagcggcgctgatcgtcggcggtcacactttcggtaagacccatggcgccggcccg840
gccgatctggtcggccccgaacccgaggctgctccgctggggcagatcggcttgggctgg900
aagagctcgtatggcaccggaaccggtaaggacgcgatcaccagcggcatcgaggtcgta960
tggacgaacaccccgacgaaatgggacaacagtttcctcgagatcctgtacggctacgag1020
tgggagctgacgaagagccctgctggcgcttggcaatacaccgccaaggacggcgccggt1080
gccggcaccatcccggacccgttcggcgggccagggcgctccccgacgatgctggccact1140
gacctctcgctgcgggtggatccgatctatgagcggatcacgcgtcgctggctggaacac1200
cccgaggaattggccgacgagttcgccaaggcctggtacaagctgatccaccgagacatg1260
ggtcccgttgcgagataccttgggccgctggtccccaagcagaccctgctgtggcaggat1320
ccggtccctgcggtcagccacgacctcgtcggcgaagccgagattgccagccttaagagc1380
cagatccgggcatcgggattgactgtctcacagctagtttcgaccgcatgggcggcggcg1440
tcgtcgttccgtggtagcgacaagcgcggcggcgccaacggtggtcgcatccgcctgcag1500
ccacaagtcgggtgggaggtcaacgaccccgacggggatctgcgcaaggtcattcgcacc1560
ctggaagagatccaggagtcattcaactccgcggcgccggggaacatcaaagtgtccttc1620
gccgacctcgtcgtgctcggtggctgtgccgccatagagaaagcagcaaaggcggctggc1680
cacaacatcacggtgcccttcaccccgggccgcacggatgcgtcgcaggaacaaaccgac1740
gtggaatcctttgccgtgctggagcccagggcagatggcttccgaaactacctcggaaag1800
ggcaacccgttgccggccgagtacatgctgctcgacaaggcgaacctgcttacgctcagt1860
gcccctgagatgacggtgctggtaggtggcctgcgcgtcctcggcgcaaactacaagcgc1920
ttaccgctgggcgtgttcaccgaggcctccgagtcactgaccaacgacttcttcgtgaac1980
ctgctcgacatgggtatcacctgggagccctcgccagcagatgacgggacctaccagggc2040
aaggatggcagtggcaaggtgaagtgggccggcagccgcgtggacctggtcttcgggtcc2100
aactcggagttgcgggcgcttgtcgaggtctatggcgccgatgacgcgcagccgaagttc2160
gtgcaggacttcgtcgctgcctgggacaaggtgatgaacctcgacaggttcgacgtgcgc2220
tga2223
Claims (5)
1.一种结核分枝杆菌KatG耐药突变位点检测试剂盒，其特征在于：其包括可以检测出KatG294GGG，296ATC，590AGG，690GCC4个点突变中至少一个试剂。
2.根据权利要求1所述的KatG结核分枝杆菌KatG耐药突变位点检测试剂盒，其特征在于：其包括可以检测出KatG294GGG，296ATC两个点突变位点的试剂。
3.根据权利要求1所述的KatG结核分枝杆菌KatG耐药突变位点检测试剂盒，其特征在于：其包括可以检测出KatG294GGG，296ATC，590AGG，690GCC4个点突变的试剂。
4.根据权利要求1所述的KatG结核分枝杆菌KatG耐药突变位点检测试剂盒，其特征在于：可以检测出KatG294GGG，296ATC，590AGG，690GCC4个点突变的试剂相互独立。
5.根据权利要求1所述的KatG结核分枝杆菌KatG耐药突变位点检测试剂盒，其特征在于：可以检测出KatG294GGG，296ATC，590AGG，690GCC4个点突变试剂为PCR-荧光探针。
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| CN101144099A (zh) * | 2006-09-11 | 2008-03-19 | 中山大学达安基因股份有限公司 | 检测结核分枝杆菌及其耐药基因突变的方法和试剂盒 |
| CN104419710A (zh) * | 2013-09-04 | 2015-03-18 | 上海市肺科医院 | 一种结核分枝杆菌的耐药新基因及其应用 |
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| CN101144099A (zh) * | 2006-09-11 | 2008-03-19 | 中山大学达安基因股份有限公司 | 检测结核分枝杆菌及其耐药基因突变的方法和试剂盒 |
| CN104419710A (zh) * | 2013-09-04 | 2015-03-18 | 上海市肺科医院 | 一种结核分枝杆菌的耐药新基因及其应用 |
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| CN111394484A (zh) * | 2020-03-27 | 2020-07-10 | 上海市临床检验中心 | 一种以异源菌株模拟的结核分枝杆菌核酸检测质控品 |
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