CN103882140A - 结核分枝杆菌快速诊断试剂盒 - Google Patents

结核分枝杆菌快速诊断试剂盒 Download PDF

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CN103882140A
CN103882140A CN201410137858.2A CN201410137858A CN103882140A CN 103882140 A CN103882140 A CN 103882140A CN 201410137858 A CN201410137858 A CN 201410137858A CN 103882140 A CN103882140 A CN 103882140A
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mycobacterium tuberculosis
pcr
rv3881c
primers
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李剑男
郑敬彤
王放
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Jilin University
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    • C12Q1/686—Polymerase chain reaction [PCR]

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Abstract

本发明涉及一种结核分枝杆菌快速诊断试剂盒，属于医学检测试剂盒领域。反应体系组成，10×PCR缓冲液2μl，10mmol/L dNTP 1μl，Taq DNA聚合酶0.5μl，Mg2+ 0.5μl，5%甘油2μl，IS6110 0.2μl，Rv3881c 1μl，双蒸水10.8μl。本发明在常规PCR技术的基础之上，运用多重PCR技术，即在同一反应中加入2对引物并分别扩增，得到结核分枝杆菌不同的特异性DNA片段。此技术既保留了常规PCR的特异性和敏感性，又减少了操作步骤和试剂用量。因此多重PCR技术在微生物检测中更适用于临床，拥有更好的发展前景。

Description

结核分枝杆菌快速诊断试剂盒
技术领域
本发明属于医学检测试剂盒领域。
背景技术
近年来,结核分枝杆菌感染人数有所增加，临床症状和X 线表现越发不典型，难以诊断，而且多数结核分枝杆菌对抗结核药物耐药现象严重，因此正确鉴定结核分枝杆菌及其毒力对于疾病诊断和治疗至关重要。随着分子生物学技术的进展,现临床已经应用一些分枝杆菌特异性的DNA 片断并进行PCR, 可对结核分枝杆菌分枝杆菌特异性鉴别，不仅提高了特异性和灵敏度，且操作快速、简便。然而，传统的临床微生物学检测中都要经过菌株的分离及罗氏培养和抗酸染色等步骤，结果需七周才能得到实验结果且特异性敏感性较低，不仅费时而且费力。
发明内容
本发明提供一种结核分枝杆菌快速诊断试剂盒，以解决临床鉴定结核分枝杆菌及诊断肺结核病中特异性敏感性弱和费力费时的问题，并能检测出菌株毒力，为临床治疗及用药提供参考依据。
本发明采取的技术方案是：包括下列反应体系，适合样本2μl：
    10×PCR缓冲液        2μl 
10mmol/L dNTP       1μl
  Taq DNA聚合酶      0.5μl 
Mg2+               0.5μl 
5%甘油              2μl 
IS6110             0.2μl 
Rv3881c             1μl
双蒸水            10.8μl；
所述IS6110（genebank ID: 885372，Location：NC_000962.3）是含有1361bp的核苷酸和28bp的反向末端重复序列，属于插入序列IS3 家族的一个成员， 
引物F1 CGTCGCTAGTGCATTGTCATAG； 
R1 TACTACGACCACTCAACCGGGAG ；
         扩增序列长度621bp
   所述Rv3881c（genebank ID 886214，Location： NC_000962.3）是EspB蛋白的编码基因，EspB在结核分枝杆菌中分子量为55 kDa，分泌出来后其C端被切除，分子量变为48 kDa； 
引物F2 CCTGCTGAGAACCCTTGGACT；
           R2 GCTGGCGCTGGTGACATT；
      扩增序列长度139bp。
本发明在常规PCR技术的基础之上，运用多重PCR技术，即在同一反应中加入2对引物并分别扩增，得到结核分枝杆菌不同的特异性DNA片段。此技术既保留了常规PCR的特异性和敏感性，又减少了操作步骤和试剂用量。因此多重PCR技术在微生物检测中更适用于临床，拥有更好的发展前景。
具体实施方式
包括下列基因、引物及反应体系：
（一）基因及引物
 （1）IS6110（genebank ID: 885372，Location：NC_000962.3）是含有1361bp的核苷酸和28bp的反向末端重复序列，属于插入序列IS3 家族的一个成员，它只存在于结核分支杆菌复合群中〔3〕，结核分支杆菌大多数分离株拷贝数高，它是目前PCR法鉴定结核分枝杆菌复合群的通用基因，
引物F1 CGTCGCTAGTGCATTGTCATAG； 
R1 TACTACGACCACTCAACCGGGAG ；
         扩增序列长度621bp
   （2）Rv3881c（genebank ID 886214，Location： NC_000962.3）是EspB蛋白的编码基因，EspB在结核分枝杆菌中分子量为55 kDa，分泌出来后其C端被切除，分子量变为48 kDa，EspB抗原富含丙胺酸和甘氨酸，EspB所在的基因组区域Rv3866-Rv3881c是一个毒力基因簇，在该区域中有若干个毒力基因，EspB具有较高的诊断应用价值，EspB同样是结核分枝杆菌ESX-1分泌系统的重要组成部分， EspB的分泌是分枝杆菌在巨噬细胞中增殖和抑制吞噬体成熟所必需的，敲除EspB基因的突变菌株几乎丧失毒力；
引物F2 CCTGCTGAGAACCCTTGGACT；
           R2 GCTGGCGCTGGTGACATT；
      扩增序列长度139bp；
    （二）反应体系组成，适合样本2μl；
  10×PCR缓冲液        2μl                
10mmol/L dNTP       1μl
  Taq DNA聚合酶      0.5μl 
Mg2+               0.5μl         
5%甘油              2μl 
IS6110             0.2μl  
Rv3881c             1μl
双蒸水            10.8μl。
应用例：
（1）临床痰液样本的预处理
样本要求
用无菌5ml 玻璃管取受检者肺深部咳出痰液1～3 毫升，密闭，即为标本，或直接抽取外周血2ml(抗凝)，标本可立即用于测试，也可保存于-20℃待测，保存期为6 个月，标本运送应采用0℃冰壶；
痰液中加入4 倍体积的4% NaOH，摇匀，室温下放置30 分钟左右液化，取0.5ml 至1.5ml 离心管中，再加入0.5ml 4%NaOH 室温放置10 分钟；
（2）样本中结核分枝杆菌DNA提取
15000rpm离心5分钟，去上清，沉淀加无菌生理盐水1ml打匀，15000rpm 离心5分钟；再重复洗涤一次，沉淀直接加50μl DNA 提取液充分混匀，沸水浴10 分钟(误差不超过1 分钟), 转至4℃静置6-8 小时以保证充分裂解，10000 rpm 离心5 分钟, 取上清液2μl做PCR 反应；
（3）PCR的扩增
取单管单人份PCR 反应管若干，加入反应体系所需试剂，后直接加入处理后样品，混匀，放入PCR仪，如ABI Prism 7000，ABI GeneAmp 5700；
PCR扩增条件如下：
预变性：   95℃5min；
变性：      95℃45s；
复性：      59℃50s；
延伸：      72℃1min；
循环次数：  30次；
延伸：      72℃10min
（4）PCR产物的电泳及结果观察
PCR产物用１％琼脂凝胶电泳、溴化乙锭（EB，终浓度为0.5μg/ml染色后，在紫外线台上观察。
（5）检测结果的解释
若菌株为结核分枝杆菌，则能扩增出621bp片段；若菌株致病性强，则能扩增出139bp片段。

Claims (1)

1.一种结核分枝杆菌快速诊断试剂盒，其特征在于包括下列反应体系，适合样本2μl：
    10×PCR缓冲液        2μl 
10mmol/L dNTP       1μl
  Taq DNA聚合酶      0.5μl 
Mg2+               0.5μl 
5%甘油              2μl 
IS6110             0.2μl 
Rv3881c             1μl
双蒸水            10.8μl；
所述IS6110（genebank ID: 885372，Location：NC_000962.3）是含有1361bp的核苷酸和28bp的反向末端重复序列，属于插入序列IS3 家族的一个成员， 
引物F1 CGTCGCTAGTGCATTGTCATAG； 
R1 TACTACGACCACTCAACCGGGAG ；
         扩增序列长度621bp
   所述Rv3881c（genebank ID 886214，Location： NC_000962.3）是EspB蛋白的编码基因，EspB在结核分枝杆菌中分子量为55 kDa，分泌出来后其C端被切除，分子量变为48 kDa； 
引物F2 CCTGCTGAGAACCCTTGGACT；
           R2 GCTGGCGCTGGTGACATT；
      扩增序列长度139bp。
CN201410137858.2A 2014-04-08 2014-04-08 结核分枝杆菌快速诊断试剂盒 Pending CN103882140A (zh)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104611420A (zh) * 2015-01-04 2015-05-13 南京爱必梦生物材料有限公司 一种结核菌检测试剂盒
CN104988217A (zh) * 2015-06-24 2015-10-21 吉林大学 一种检测结核分枝杆菌生物被膜产膜能力的方法
CN105586429A (zh) * 2016-03-11 2016-05-18 石河子大学 一种快速诊断结核病潜伏感染的引物及试剂盒
WO2017092483A1 (zh) * 2015-12-02 2017-06-08 北京市结核病胸部肿瘤研究所 通过检测游离核酸诊断结核病的试剂盒及其应用

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104611420A (zh) * 2015-01-04 2015-05-13 南京爱必梦生物材料有限公司 一种结核菌检测试剂盒
CN104988217A (zh) * 2015-06-24 2015-10-21 吉林大学 一种检测结核分枝杆菌生物被膜产膜能力的方法
WO2017092483A1 (zh) * 2015-12-02 2017-06-08 北京市结核病胸部肿瘤研究所 通过检测游离核酸诊断结核病的试剂盒及其应用
CN105586429A (zh) * 2016-03-11 2016-05-18 石河子大学 一种快速诊断结核病潜伏感染的引物及试剂盒

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