CN104630328A - 肺炎支原体23S rRNA 2064位点A:G突变检测特异性引物和探针 - Google Patents

肺炎支原体23S rRNA 2064位点A:G突变检测特异性引物和探针 Download PDF

Info

Publication number
CN104630328A
CN104630328A CN201310550193.3A CN201310550193A CN104630328A CN 104630328 A CN104630328 A CN 104630328A CN 201310550193 A CN201310550193 A CN 201310550193A CN 104630328 A CN104630328 A CN 104630328A
Authority
CN
China
Prior art keywords
probe
primer
rrna
sequence
mycoplasma pneumoniae
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201310550193.3A
Other languages
English (en)
Other versions
CN104630328B (zh
Inventor
杜君卿
李杨霞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
JIANGSU MOLE BIOSCIENCE CO Ltd
Original Assignee
JIANGSU MOLE BIOSCIENCE CO Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by JIANGSU MOLE BIOSCIENCE CO Ltd filed Critical JIANGSU MOLE BIOSCIENCE CO Ltd
Priority to CN201310550193.3A priority Critical patent/CN104630328B/zh
Publication of CN104630328A publication Critical patent/CN104630328A/zh
Application granted granted Critical
Publication of CN104630328B publication Critical patent/CN104630328B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明公开一种用于肺炎支原体23SrRNA2064位点A:G突变的特异性引物和探针，属于生物技术领域，核苷酸序列包括上游引物MPF序列为5’GGTGTAACCATCTCTTGA3’，下游引物MPR序列为5’CCTGATCAATATTAAGCTACAG3’和探针2064P序列为5’FAMCGGGGTCTCTCCGTCCBHQ13’。

Description

肺炎支原体23S rRNA 2064位点A:G突变检测特异性引物和探针
 
技术领域
本发明是一种用于检测肺炎支原体23S rRNA 2064位点A:G突变的特异性引物和探针。
 
背景技术
肺炎支原体（Mycoplasma pneumoniae，MP）是一种常见的呼吸道病原体，通常导致轻微的上呼吸道感染，如喉咙痛、咽喉炎和气管炎，其引起的肺炎占非细菌性肺炎的50%，还可引起肺外并发症，累及一个或多个系统、器官，如皮肤、胃肠道、心血管、骨骼肌肉和肾脏，严重时可致中枢和外周神经系统病变，甚至死亡。通过飞沫以气溶胶形式传播，存留在鼻、喉、气管和痰液中，密切接触可能引起肺炎支原体的暴发。肺炎支原体无细胞壁，对影响细胞壁合成的抗生素如青霉素等不敏感，但是对影响细菌蛋白合成的抗生素如大环内酯类、喹诺酮类等敏感。大环内酯类是治疗肺炎支原体感染的首选药物，可结合于肺炎支原体核糖体50S亚基的转肽酶中心与肽输出通道之间的部分，通过机械阻塞通道而抑制肽链的延伸，从而达到抑制蛋白合成的目的，其结合位点由23S rRNA结构域Ⅴ的核苷酸构成，其中2064位点是主要的组成部分。近年来，随着大环内酯类抗生素的广泛使用，临床分离出了耐药菌株，提示肺炎支原体耐药现象的存在。国内外的研究表明，产生耐药的肺炎支原体在23S rRNA序列上发生点突变，其中最常见的是2064位点A到G的突变。
目前，临床上对肺炎支原体耐药性的检查，主要是依靠肺炎支原体的分离及药敏实验，由于肺炎支原体分离较为困难，所以灵敏度较低，而且耗时耗力，不利于及时指导用药。本发明采用的荧光PCR技术，操作简便、检测迅速、检出率高，通过一次试验即可明确是否有耐药肺炎支原体的感染，有利于对疾病进行及时诊断及合理选择治疗方案，与传统分离培养检测技术相比，提高了检出效率、降低了漏检率。可对多种临床样本进行高效可靠的筛查，以及对临床病情和用药效果进行监测，指导合理用药。
发明内容
本发明的目的在于提供一种用于检测肺炎支原体23S rRNA 2064位点A:G突变的引物和探针。
基于上述目的，本发明采用以下技术方案：
用于检测肺炎支原体23S rRNA 2064位点A:G突变的特异性引物和探针序列包括：上游引物MPF序列为5’ GGTGTAACCATCTCTTGA 3’，下游引物MPR序列为5’ CCTGATCAATATTAAGCTACAG 3’和探针2064P序列为 5’FAM CGGGGTCTCTCCGTCC BHQ1 3’。
本发明的具体原理是利用一对靶核酸序列的特异性引物和探针，采用实时荧光定量PCR技术，通过PCR实现靶核酸序列片断的扩增。所使用的探针为两端分别标记荧光报告基团（R）和荧光淬灭基团（Q）的寡核苷酸。在探针完整时，报告基团发出的荧光被淬灭基团吸收，在PCR扩增过程中，DNA聚合酶的5’端外切酶活性将特异结合在靶核苷酸片断上的荧光探针酶切降解，使报告基团的荧光信号可以被检测，荧光信号量的变化与扩增产物量成正比，从而可以通过荧光强弱来判断待测样本中靶核苷酸序列的存在。
 
附图说明
图1是利用引物对MPF/MPR和探针2064P检测肺炎支原体23S rRNA 2064位点A:G突变阳性样本的荧光PCR扩增图。
 
具体实施方式
1.      引物和探针的设计：通过分别对所有已知的肺炎支原体23S rDNA序列进行比较分析，选择2064位点所在区段，设计多对引物和探针，引物长度一般为20碱基左右。最优引物探针序列组合如下：
上游引物MPF： 5’ GGTGTAACCATCTCTTGA 3’
下游引物MPR： 5’ CCTGATCAATATTAAGCTACAG 3’
探针2064P：    5’  FAM CGGGGTCTCTCCGTCC BHQ1 3’。
2. 反应体系的优化：利用临床分离的耐药肺炎支原体灭活液作为待检样品，用磁珠裂解法抽提肺炎支原体核酸，分装后贮存于－80℃。
2.1 引物浓度的优化 在反应体系中其它条件相同的情况下，将肺炎支原体引物浓度分别从0.1μmol/L至1.6μmol/L作倍比连续稀释，通过对试验结果的分析比较，确定最佳引物浓度是0.2μmol/L。
2.2 探针浓度的优化 在反应体系中其它条件相同的情况下，将肺炎支原体突变检测用探针浓度分别从0.1μmol/L至0.5μmol/L作倍比连续稀释，通过对试验结果的分析比较，确定最佳探针浓度是0.2μmol/L。
利用上述引物和探针进行反应体系的建立，最后确定采用的肺炎支原体23S rRNA 2064位点A:G突变的实时荧光PCR反应体系为25μL。按照荧光定量PCR试剂盒说明进行反应液的配置。
3. 仪器检测通道的选择
选择的荧光检测通道应与探针所标记的报告荧光基团一致，具体按照仪器使用说明书进行设置。
4. PCR反应条件如下
50℃ 2min进行UNG酶反应；95℃ 1min，1 个循环；91℃ 15s，64℃ 1min，40 个循环，在64℃ 1min阶段收集荧光。
5. 检测结果分析
如果待检样本中含有发生2064位点A:G突变的肺炎支原体，则显示阳性扩增曲线，其检测灵敏度可以达到1000 copies/mL，如果待检样本中没有2064位点A:G突变的肺炎支原体，则显示阴性扩增曲线，即无扩增信号，提示上述引物和探针具有良好的特异性和灵敏度。
本发明的优点在于：
（1）  本发明提供的引物和探针的检测灵敏度可以达到1000 copies/mL，说明其灵敏度良好。
（2） 本发明提供的引物和探针对不含2064位点A:G突变的肺炎支原体的样本没有检测信号，说明其特异性强。
（3）  由于本发明是针对2064A:G突变位点，进行了程序和体系优化，避免了假阴性和假阳性结果的出现。

Claims (2)

1.一种用于检测和鉴定肺炎支原体23S rRNA 2064位点A:G突变的特异性引物序列，其特征在于所述的引物序列包括上游引物MPF序列为5’ GGTGTAACCATCTCTTGA  3’，下游引物MPR序列为5’ CCTGATCAATATTAAGCTACAG  3’。
2.一种用于检测和鉴定肺炎支原体23S rRNA 2064位点A:G突变的特异性探针序列，其特征在于所述的探针序列为 5’FAM CGGGGTCTCTCCGTCC BHQ1 3’。
CN201310550193.3A 2013-11-08 2013-11-08 肺炎支原体23S rRNA 2064位点A:G突变检测特异性引物和探针 Active CN104630328B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310550193.3A CN104630328B (zh) 2013-11-08 2013-11-08 肺炎支原体23S rRNA 2064位点A:G突变检测特异性引物和探针

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310550193.3A CN104630328B (zh) 2013-11-08 2013-11-08 肺炎支原体23S rRNA 2064位点A:G突变检测特异性引物和探针

Publications (2)

Publication Number Publication Date
CN104630328A true CN104630328A (zh) 2015-05-20
CN104630328B CN104630328B (zh) 2016-09-07

Family

ID=53209572

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310550193.3A Active CN104630328B (zh) 2013-11-08 2013-11-08 肺炎支原体23S rRNA 2064位点A:G突变检测特异性引物和探针

Country Status (1)

Country Link
CN (1) CN104630328B (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106191239A (zh) * 2016-07-11 2016-12-07 宁波基内生物技术有限公司 一种实时荧光pcr检测肺炎支原体核酸及耐药突变的引物、探针、方法及试剂盒
WO2021075655A1 (ko) * 2019-10-18 2021-04-22 고려대학교 산학협력단 마이코플라즈마 폐렴균 및 마이코플라즈마 폐렴균 돌연변이 동시 검출용 프라이머와 프로브 및 이의 용도
WO2022016153A1 (en) * 2020-07-17 2022-01-20 Gen-Probe Incorporated Detection of macrolide-resistant mycoplasma genitalium

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102002522A (zh) * 2009-11-10 2011-04-06 复旦大学附属华山医院 一种检测肺炎支原体耐药突变的方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102002522A (zh) * 2009-11-10 2011-04-06 复旦大学附属华山医院 一种检测肺炎支原体耐药突变的方法

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106191239A (zh) * 2016-07-11 2016-12-07 宁波基内生物技术有限公司 一种实时荧光pcr检测肺炎支原体核酸及耐药突变的引物、探针、方法及试剂盒
WO2021075655A1 (ko) * 2019-10-18 2021-04-22 고려대학교 산학협력단 마이코플라즈마 폐렴균 및 마이코플라즈마 폐렴균 돌연변이 동시 검출용 프라이머와 프로브 및 이의 용도
KR20210046888A (ko) * 2019-10-18 2021-04-29 고려대학교 산학협력단 마이코플라즈마 폐렴균 및 마이코플라즈마 폐렴균 돌연변이 동시 검출용 프라이머와 프로브 및 이의 용도
KR102250376B1 (ko) * 2019-10-18 2021-05-12 고려대학교 산학협력단 마이코플라즈마 폐렴균 및 마이코플라즈마 폐렴균 돌연변이 동시 검출용 프라이머와 프로브 및 이의 용도
WO2022016153A1 (en) * 2020-07-17 2022-01-20 Gen-Probe Incorporated Detection of macrolide-resistant mycoplasma genitalium

Also Published As

Publication number Publication date
CN104630328B (zh) 2016-09-07

Similar Documents

Publication Publication Date Title
CN103614489B (zh) 一种登革热病毒的恒温扩增检测试剂盒及检测方法
CN104630329A (zh) 肺炎支原体23S rRNA 2063位点A:G突变检测特异性引物和探针
CN106520977A (zh) 环介导等温扩增法检测苜蓿根腐病菌的引物及方法
Ullah et al. Detection of circulating cell-free DNA to diagnose Schistosoma japonicum infection
CN101676405A (zh) 结核分枝杆菌荧光定量pcr检测方法及其试剂盒
CN104630328A (zh) 肺炎支原体23S rRNA 2064位点A:G突变检测特异性引物和探针
CN112391483A (zh) 用于恒温扩增检测鼠疫杆菌的核酸序列、试剂盒及方法和应用
CN102952850B (zh) 用于检测结核分枝杆菌的实时荧光定量pcr方法、引物、探针及试剂盒
Wang et al. Graphene-oxide-based bioassay for the fluorometric determination of agrC gene transcription in methicillin-resistant Staphylococcus aureus that uses nicking-enzyme-assisted target recycling and a hybridization chain reaction
CN103045717A (zh) 焦磷酸测序技术检测结核分枝杆菌利福平耐药性的方法
CN105154584A (zh) 一种快速区分prrsv经典毒株和变异毒株的hrm非标记探针方法及其引物和探针
CN103215362A (zh) TaqMan-MGB探针实时荧光定量PCR技术联合检测艰难梭菌菌属基因及毒素基因的方法学建立
CN103184283A (zh) Pcr酶切分型检测军团菌试剂盒及其应用
CN109234432A (zh) 一种基于重组酶聚合酶扩增方法检测大豆猝倒病的引物、探针和试剂盒
CN102146467A (zh) 检测鼠疫耶尔森氏菌的试剂及荧光定量pcr检测鼠疫耶尔森氏菌的方法
CN103045716A (zh) 焦磷酸测序技术检测结核分枝杆菌异烟肼耐药性的方法
CN102417931B (zh) 一种白色念珠菌pcr荧光检测试剂盒及其检测方法
CN102002537A (zh) 一种辅助鉴定藜草花叶病毒的试剂及其应用
CN104946638A (zh) 一种向日葵白锈病菌和黑茎病菌的多重dpo-pcr检测试剂盒及其应用
CN102140537B (zh) 一种hbv基因分型的pcr检测方法
CN104946735A (zh) 试剂盒及确定待测dna样本预定snp位点的基因型的方法
CN105400908B (zh) 一种利用焦磷酸测序技术检测斑点叉尾鮰病毒的引物、试剂盒和检测方法
CN103509859B (zh) 山羊结核病pcr检测试剂盒
CN102787161A (zh) 一种检测结核分枝杆菌方法及其专用引物
CN102127591A (zh) 家兔须癣毛癣菌鉴定方法

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
CB02 Change of applicant information

Address after: 225300 building R19, road, Taizhou hi tech Development Zone, Zhejiang Province, China

Applicant after: JIANGSU MOLE BIOSCIENCE CO., LTD.

Address before: 225300, R19 building, No. 1, drug city road, Hailing District, Jiangsu, Taizhou

Applicant before: JIANGSU MOLE BIOSCIENCE CO., LTD.

COR Change of bibliographic data
C14 Grant of patent or utility model
GR01 Patent grant