CN112760391A - 口腔中幽门螺杆菌耐药性分型检测的方法和试剂盒 - Google Patents
口腔中幽门螺杆菌耐药性分型检测的方法和试剂盒 Download PDFInfo
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Abstract
由于胃活检样本内窥镜取样困难、成本高,幽门螺杆菌(Hp)生长条件苛刻难以培养,Hp耐药性的侵入性诊断方法并不理想。本发明提供一种非侵入性幽门螺杆菌耐药性检测的分子诊断方法,采用巢氏PCR技术结合测序技术对粪便样本进行快速检测与解析,利用核苷酸序列如表2及表3所示的引物联合检测23SrRNA,gyrA,PBP1,16SrRNA,porD,oorD,rpoB等7种耐药基因的52个位点的突变及rdxA耐药基因的随机突变,进而对目前临床常用7种抗生素耐药情况进行判定。本发明属于生物技术领域,快速灵敏,适合临床应用,在指导幽门螺杆菌患者的个性化诊疗根除方面有较大应用前景。
Description
技术领域
本发明属于生物技术领域,具体涉及一种检测口腔中幽门螺杆菌耐药性的诊断方法及试剂盒。
背景技术
幽门螺杆菌(H.pylori,Hp)是革兰氏阴性螺旋微需氧菌,宿主主要是人类,人体胃幽门是H.p特定的定居部位,其在全球约50-70%的人口的胃上皮中定植,在发展中国家感染率已达到90%,并且存在一定的地理差异。Hp也是目前所知的唯一一种能在人胃中生存的微生物,被认为是导致慢性胃炎,消化性溃疡,胃癌的主要诱因。
1983年,由Marshall和Warren在胃活检中发现Hp并成功培养。1989年,Krajedn从口腔唾液和牙菌斑生物膜中首次成功分离出Hp。1993年,Ferguson等从胃炎患者唾液中培养出活性Hp,从此口腔作为Hp的第二个宿主部位引起人们的关注。口腔Hp可以伴随口腔中的食物和唾液经消化系统的吞咽过程进入胃内,反之,胃内Hp可通过反酸与食物返流的方式流入到消化道后进入口腔。后来经证实,口腔Hp和胃内Hp具有较高的同源性。研究发现慢性萎缩性胃炎(CAG)患者的口腔中Hp的检出率要高于浅表性胃炎和消化性溃疡(PU)患者,胃中Hp感染和口腔Hp检出存在着一定的联系。Hp根除后的二次感染也与口腔Hp存在着一定的相关性。因此,唾液和牙菌斑可作为诊断Hp感染相关诊断的可靠非侵入性检查标本。综上所述,根除口腔中的Hp也是防止胃内Hp再感染的一项重要措施,检测口腔Hp 抗生素耐药性在临床上有非常重要的意义。
目前幽门螺杆菌的根除治疗方案主要包括质子泵抑制剂(PPI)和胃黏膜保护剂以及一种或两种抗生素,可进行三联或四联治疗,然而,在一些地区,标准三联或四联疗法根除率已低于《马斯特里赫特条约》(MaastrichtConsensus)的80%,Hp对一线抗生素的耐药率(包括多重耐药率)呈逐年上升趋势,目前抗生素耐药性已成为成功根除幽门螺杆菌的关键。用于根除幽门螺杆菌的抗生素包括克拉霉素(CAM),左氧氟沙星(LVX),阿莫西林(AMX),四环素 (TET),利福平(RIF),呋喃唑酮(FUR)和甲硝唑(MTZ)等。
目前已有单一或多种抗生素耐药性的检测方法,但是临床常用的仍为药敏实验,即侵入性获取胃活检组织,分离培养Hp,由于胃中Hp分布不均匀,患者依从性差,从而导致Hp含量少,培养成功率低。并因Hp培养条件苛刻,生长缓慢,培养周期长等因素而不适用于临床耐药性快速诊断与即时用药指导。综上,目前临床上亟需一种取样简便的可靠的非侵入性Hp 耐药性诊断方法。
本发明提出一种以口腔内唾液及牙菌斑为标本,非侵入性检测幽门螺杆菌7种抗生素耐药性的分子诊断方法及试剂盒,具体方法基于巢氏PCR技术与测序技术,其敏感性、特异性和检测准确率较高,可科学、安全、快速、准确地指导幽门螺杆菌感染患者用药。
发明内容
本发明的主要目的是提供一种准确、快速、检测流程简单的高灵敏分子诊断方法,可以同时检测目前幽门螺杆菌7种耐药基因,能够更加科学、安全、快速、准确地指导幽门螺杆菌感染患者用药。
一种检测和判定幽门螺旋杆菌耐药性的诊断方法,其特征在于,标本来源为患者口腔中唾液及牙菌斑,为非侵入性检测。
进一步,由于口腔中种幽门螺杆菌含量少,该诊断方法采用灵敏度更高的巢氏PCR技术进行检测。
进一步,该诊断方法包括对克拉霉素、左氧氟沙星、阿莫西林、四环素、呋喃唑酮、利福平、甲硝唑耐药基因的突变区域进行PCR引物设计的步骤;
其中,克拉霉素耐药基因23SrRNA突变区域的引物为F1和R1;
阿莫西林耐药基因PBP1突变区域的引物为F2和R2;
四环素耐药基因16SrRNA突变区域的引物为F3和R3;
左氧氟沙星耐药基因gyrA突变区域的引物为F4和R4;
呋喃唑酮耐药基因porD突变区域的引物为F5和R5;
呋喃唑酮耐药基因oorD突变区域的引物为F6和R6;
利福平耐药基因rpoB突变区域的引物为F7和R7;
甲硝唑耐药基因rdxA突变区域的引物为F8和R8;
该诊断方法包括对克拉霉素耐药基因进行巢氏PCR引物设计的步骤;
其中,克拉霉素耐药基因23SrRNA基因位点G1939A、T1942C的引物为F9和R9;
克拉霉素耐药基因23SrRNA基因位点G2111A、A2115G、A2116G、T2117C、G2141A、A2142G、A2142G/C/T、A2143G/C/T、A2144G、A2146G、C2147G、G2172T、T2182C、C2224A、T2243C、C2245T、C2289T的引物为F10和R10;
克拉霉素耐药基因23SrRNA基因位点C2611A、C2694A与T2717C的引物为F11和R11。
进一步,该诊断方法包括对阿莫西林耐药基因的进行巢氏PCR引物设计的步骤;
其中,阿莫西林耐药基因PBP1基因位点A320V的引物为F12和R12;
阿莫西林耐药基因PBP1基因位点F366L、A369T及V374L的引物为F13和R13;
阿莫西林耐药基因PBP1基因位点S414R与L423F的引物为F14和R14;
阿莫西林耐药基因PBP1基因位点T556S、N562Y、T593A、G595S及A599T的引物为F15和R15。
进一步,该诊断方法包括对四环素耐药基因的进行巢氏PCR引物设计的步骤;
其中,四环素耐药基因16SrRNA基因位点 AGA926-928TTC\AGC\TGC\GGA\CGA\ATC\GTA\GCA的引物为F16和R16。
进一步,该诊断方法包括对左氧氟沙星耐药基因的进行巢氏PCR引物设计的步骤;
左氧氟沙星耐药基因gyrA基因位点N87L\I\A\Y\K、D 91A\G\Y\N\H、A 88N\V及R130K 的引物为F17和R17。
进一步,该诊断方法包括对呋喃唑酮耐药基因的进行巢氏PCR引物设计的步骤;
其中,呋喃唑酮耐药基因porD基因位点C346A、C347T\G、G353A、A356G及C357T 的引物为F18和R18;
呋喃唑酮耐药基因oorD基因位点A41G、A78G、A112G、A122G、C156T及C165T的引物为F19和R19;
呋喃唑酮耐药基因oorD基因位点A335G及C349A\G的引物为F20和R20;
进一步,该诊断方法包括对利福平耐药基因的进行巢氏PCR引物设计的步骤;
其中,利福平耐药基因rpoB基因位点L525I\P、S526L、Q527K\R、D530Y\N\I\G\V\L\F、 H 540A\N\Y、I 586P\N\L、V147F、T62C、D540V\L、S545L及Q527R的引物为F21和R21。
进一步,该诊断方法包括对甲硝唑耐药基因的进行巢氏PCR引物设计的步骤;
其中,甲硝唑耐药基因rdxA随机突变位点的引物为F22~29及R22~29。
本发明还在于公开一种应用上述诊断方法的试剂盒。
本方法只需要简单的对患者口腔中唾液及牙菌斑进行全基因组提取,即可进行下游PCR 反应。利用巢氏PCR技术将这些待测片段实现扩增,结合测序技术即可获得幽门螺杆菌耐药情况。该方法设计合理、操作流程简单、检测结果快速、准确,可以满足不同临床和科研的检测需求,有利于指导临床幽门螺杆菌的根除,为幽门螺杆菌的感染治疗提供分子基础。
巢式PCR是一种变异的聚合酶链反应(PCR),使用两对PCR引物扩增完整的片段。第一对PCR引物扩增片段和普通PCR相似。第二对引物称为巢式引物,它们结合在第一次PCR产物内部,使得第二次PCR扩增片段短于第一次扩增。巢式PCR的优势在于,如果第一次扩增产生了错误片段,则第二次能在错误片段上进行引物配对并扩增的概率极低。因此,巢式PCR的扩增更加特异。
为实现以上目的,设计覆盖23SrRNA、16SrRNA、gyrA、PBP1、porD、oorD、rpoB及rdxA等基因耐药相关突变区域特异性引物共29对,包括PCR特异性引物8对,及巢氏PCR 特异性引物21对。
其中,23SrRNA、16SrRNA、gyrA、PBP1、porD、oorD、rpoB及rdxA等基因的耐药相关突变位点如表1所示,PCR特异性引物序列如表2所示,巢氏PCR特异性引物序列如表3 所示。
表1
表2
注:表2中的各基因突变区域的序列具体见表12。
表3
注:表2和表3中的F代表正向引物,R代表反向引物。
具体实施方式
下面将通过实施例对本发明的快速联合检测和判定口腔中唾液牙菌斑标本中幽门螺杆菌的克拉霉素、左氧氟沙星、阿莫西林、四环素、呋喃唑酮、利福平及甲硝唑耐药性的方法进行清楚、完整的描述。
实施例1口腔中唾液及牙菌斑标本中幽门螺旋杆菌全基因组提取过程
1.取样要求:
1)确认受试者受检前停止服用抗生素、质子泵抑制剂、铋剂四周以上,中药二周以上,且经快速脲酶试纸及尿素呼气试验(UBT)验证为阳性。
2)标本来源于口腔唾液和牙菌斑。标本定时采集,简单操作如下:基础状态下,受试者于早晨空腹(除喝水外禁食)未刷牙前,戴无菌手套静坐,并通过吞咽排空口腔唾液然后通过自然流出的方法收集1mL唾液标本,并取一支口腔拭子在面颊内壁两侧各反复刮拭6次,均收集于无菌EP管,及时用封口膜密封管口,以防交叉污染。2-8度冷藏运输,-20或-80℃冰箱内可保存1-2周。
3)多次根除治疗失败的患者,建议距上次治疗结束至少3个月。
4)确认采样的口腔内标本新鲜无污染,无食物残渣遗留。
2.口腔标品进行全基因组提取:
唾液及牙菌斑标品处理操作过程于生物安全柜内进行,超净台使用前紫外灯照30min灭菌,打开排风吹散臭氧后方可进行实验。采用改进的碘化钾法进行基因组提取,耗时约40min, 具体操作步骤如下:
取200μL唾液及牙菌斑标本于1.5ml无菌离心管,加无菌PBS 600μL,漩涡震荡混匀1min,11000×g离心5min,弃上清;沉淀加入60μL 5mol/L碘化钾溶液,漩涡震荡1min,加入110μL生理盐水和170μL DNA Extraction solution(酚:氯仿:异丙醇=25:24:1),漩涡震荡 1min,11000×g离心5min,溶液分层后取上清,加入300μL异丙醇,吹打混匀,11000×g离心5min,弃上清;加入600μL无水乙醇洗涤,11000×g离心5min,弃上清;加入300μL无水乙醇洗涤,11000×g离心5min,弃上清;沉淀于室温晾干10-15min后,加入30-50μL洗脱液ATE或者超纯水,震荡混匀,置于56℃孵育5min,期间颠倒混匀3回,一回3-5次。
实施例2 PCR扩增过程:
同时以ddH2O做阴性对照,以幽门螺杆菌标准株ATCC700392TM,ATCC43504TM为阳性对照; PCR扩增试剂可采用试剂TaKaRaTaqTMVersion 2.0(TaKaRa,Code No.R004A)(Takara),是即用型的PCR预混合溶液,只需加入引物和模板即可进行扩增简化实验的操作步骤。
1.PCR引物扩增:
采用TaKaRaTaqTMVersion 2.0(TaKaRa,Code No.R004A),冷启动法,按照表5所示组分、表5 所示程序进行扩增。将产物低温保存。
表4
表5
2.巢氏PCR引物扩增:
采用TaKaRaTaqTM Version 2.0(TaKaRa,Code No.R004A),冷启动法,按照表6所示组分、表7所示程序进行扩增。产物低温保存。
表6
试剂 | 使用量 |
DNA模板(<500ng/μl) | 1μl |
F Primer(Primer 1-8,10μM) | 3μl |
R Primer(Primer 1-8,10μM) | 3μl |
Premix Taq<sup>TM</sup> | 25μl |
Nuclease-Free Water | 18μl |
Total | 50μL |
表7
实施例3测序部分:
1.DNA琼脂糖切胶纯化
由PCR产物电泳结果切割所需DNA目的条带,纯化方式见SK8131胶回收说明书。
2.PCR测序反应
(1)取0.2ml的PCR管,编号后插于颗粒冰中,按下表加入试剂:
表8
不加轻矿物油或石蜡油,盖紧PCR管,用手指弹管混匀,稍离心。
(2)将PCR管置于BBI PCR仪上进行扩增。98℃变性2min后进行PCR循环,PCR循环参数为 96℃10s,50℃5s,60℃4min,25个循环,扩增结束后设置4℃保温。
3.醋酸钠/乙醇法纯化PCR产物
(1)将混合物离心,将扩增产物转移到1.5ml EP管中。
(2)加入25μl醋酸钠/乙醇混合液,充分振荡,置冰上10min以沉淀DNA。12 000r/min 于4℃离心30min,小心弃上清。
(3)加70%(V/V)的乙醇50μl洗涤沉淀2次。12 000r/min于4℃离心5min,小心弃上清和管壁的液珠,真空干燥沉淀10~15min。
4.电泳前测序PCR产物的处理。
(1)加入12μl的TSR于离心管中,剧烈振荡,让其充分溶解DNA沉淀,稍离心。
(2)将溶液转移至盖体分离的0.2ml PCR管中,稍离心。
(3)在PCR仪上进行热变性(95℃,2min),冰中骤冷,待上机。
5.上机操作按仪器操作说明书安装毛细管,进行毛细管位置的校正,人工手动灌胶和建立运行的测序顺序文件。仪器将自动灌胶至毛细管,1.2kV预电泳5min,按编程次序自动进样,再预电泳(1.2kV,20min),在7.5kV下电泳2h。电泳结束后仪器会自动清洗,灌胶,进下一样品,预电泳和电泳。每一个样品电泳总时间为2.5h。
实施例4测序结果比对与分析
利用软件SnapGen,参考如表1所示常见突变位点对测序得到的序列与标准株突变区域序列(ATCC700392TM,ATCC43504TM,如表12所示)进行比对,得到耐药性诊断结果,结果如表 10、11所示。
表10具体实施例结果
表11
根据表10、11得出结论:
样本3和4对应的患者均对克拉霉素,阿莫西林,呋喃唑酮及甲硝唑存在耐药风险,建议采用左氧氟沙星,四环素,氟喃唑酮及利福平中两种抗生素联合治疗;样本1对应的病人对克拉霉素,左氧氟沙星,呋喃唑酮,甲硝唑存在耐药性的风险,建议采用阿莫西林,四环素,氟喃唑酮及利福平中两种抗生素联合治疗;样本2对应的病人对克拉霉素,呋喃唑酮,甲硝唑存在耐药性的风险,建议采用阿莫西林,左氧氟沙星,四环素,氟喃唑酮及利福平中两种抗生素联合治疗。
以上对本发明创造的具体实施例进行了详细说明,但所述内容仅为本发明创造的较佳实施例,不能被认为用于限定本发明创造的实施范围。凡依本发明创造申请范围所作的均等变化与改进等,均应仍归属于本发明创造的专利涵盖范围之内。
表12
序列表
<110> 南开大学
<120> 口腔中幽门螺杆菌耐药性分型检测的方法和试剂盒
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 999
<212> DNA
<213> 幽门螺杆菌(Helicobacter pylori)
<400> 1
tcgcgataag gtgtgccaca gcgatgtggt ctcagcaaag agtccctccc gactgtttac 60
caaaaacaca gcactttgcc aactcgtaag aggaagtata aggtgtgacg cctgcccggt 120
gctcgaaggt taagaggatg cgtcagtcgc aagatgaagc gttgaattga agcccgagta 180
aacggcggcc gtaactataa cggtcctaag gtagcgaaat tccttgtcgg ttaaataccg 240
acctgcatga atggcgtaac gagatgggag ctgtctcaac cagagattca gtgaaattgt 300
agtggaggtg aaaattcctc ctacccgcgg caagacggaa agaccccgtg gacctttact 360
acaacttagc actgctaatg ggaatatcat gcgcaggata ggtgggaggc tttgaagtaa 420
gggctttggc tcttatggag ccatccttga gataccaccc ttgatgtttc tgttagctaa 480
ctggcctgtg ttatccacag gcaggacaat gcttggtggg tagtttgact ggggcggtcg 540
cctcctaaaa agtaacggag gcttgcaaag gttggctcat tgcggttgga aatcgcaagt 600
tgagtgtaat ggcacaagcc agcctgactg taagacatac aagtcaagca gagacgaaag 660
tcggtcatag tgatccggtg gttctgtgtg gaagggccat cgctcaaagg ataaaaggta 720
ccccggggat aacaggctga tctcccccaa gagctcacat cgacggggag gtttggcacc 780
tcgatgtcgg ctcatcgcat cctggggctg gagcaggtcc caagggtatg gctgttcgcc 840
atttaaagcg gtacgcgagc tgggttcaga acgtcgtgag acagttcggt ccctatctgc 900
cgtgggcgta ggaaagttga ggagagctgt ccctagtacg agaggaccgg gatggacgtg 960
tcactggtgc accagttgtt ctgccaagag catcgctgg 999
<210> 2
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<212> DNA
<213> 幽门螺杆菌(Helicobacter pylori)
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cgtgatcata gggcgcgcct taccggacgc tagagatggc ttaaagcctg tgcataggcg 60
tattttgtat gcgatgcatg aattaggcct tacttccaaa gtcgcttata aaaaaagcgc 120
taggatcgtg ggtgatgtga tcggtaaata ccacccccat ggcgataatg cggtttatga 180
tgcgctagtg agaatggcgc aagatttttc tatgcgtttg gaattagtgg atgggcaggg 240
caactttggc tctattgatg gcgataacgc tgcagcgatg cgttacactg aagccagaat 300
gactaaggcg agtgaagaaa ttttaaggga tattgataaa gacaccattg attttgtgcc 360
taattatgat gacaccttaa aagagccaga tattttacca agccgtctgc ctaacctttt 420
agtcaatggg gctaatggga tcgctgtggg gatggcgact tctatccccc ctcataggat 480
tgatgaaatc atagacgctt tagtgcatgt cttagaaaac cctaacgctg aattagatga 540
aattttggaa tttgtcaaag ggcctgattt tcccaccggt gggatcattt atggcaaggc 600
gggcattatt gaagcctata aaacggggcg agggcgcgtg aaagtgcggg ccaaagtgca 660
tgtggaaaag acaaaaaata aagaaatcat cgttttagat gaaatgcctt tccaaac 717
<210> 3
<211> 1204
<212> DNA
<213> 幽门螺杆菌(Helicobacter pylori)
<400> 3
ggatttcttc taacgagctc aaaggcgctc tcaatgaagt gccaattatc tataaccaaa 60
cttccacgca aaacatcgct ccctatgtcg tggatgaagt gttgaaacaa ttggatcaat 120
tagacgggtt aaaaactcaa ggctatacca taaagctcac gatagatttg gattaccaac 180
gcttagcgtt agagtccttg cgttttgggc atcaaaaaat cttagaaaaa attgctaaag 240
aaaagccaaa aactaacgcc tctaatgaag atgaagacaa cttgaacgcc agcatgatag 300
ttacagacac aagcaccggt aagattttag ctttagtggg ggggattgat tataaaaaaa 360
gcgctttcaa tcgcgccacg caagccaaac ggcaatttgg gagcgcgata aagccttttg 420
tgtatcagat cgcttttgat aatggctatt ccaccacttc taaaatccct gataccgcgc 480
gaaattttga aaatggcaat tatagtaaaa acagcgaaca aaaccacgca tggcacccta 540
gcaattattc tcgcaagttt ttagggcttg taaccttaca agaagctttg agccattcgt 600
taaatctagc cacgatcaat ttaagcgatc agcttggctt tgaaaaaatt tatcaatctt 660
taagcgatat ggggtttaaa aacctcccta aagacttgtc tattgtgtta ggcagctttg 720
ctatctcacc gattgaagcg gccgaaaagt attctttatt ttctaattac ggcaccatgc 780
tcaaacccat gctcattgaa agcatcactg accaacaaaa cgatgtcaaa actttcacgc 840
ccatggaaac taaaaagatc acctctaaag agcaagcttt tttaaccctt tcagtgctga 900
tgaatgcggt agaaaatggc acaggaagtt tggctcgcat taaaggttta gaaatcgccg 960
gtaaaaccgg gagttccaat aacaatattg atgcctggtt cattggcttt acccccacct 1020
tacaaagcgt gatctggttt gggagagacg ataacacgcc cattggcaaa ggagcgacag 1080
gaggcgttgt gagtgcacct gtgtattcgt acttcatgcg caatatttta gcgattgagc 1140
cttctttaaa aagaaagttt gatgtcccca aaggcttgcg taaagaaatc gtggataaaa 1200
tccc 1204
<210> 4
<211> 722
<212> DNA
<213> 幽门螺杆菌(Helicobacter pylori)
<400> 4
gcaacatggc tgatttgcga ttactagcga ttccagcttc atgcaggcga gttgcagcct 60
acaatccgaa ctgagaggtg ttttgaagat tggctccact tcacagtatt gcttctcttt 120
gtgcacccca ttgtagcacg tgtgtagccc taggcgtaag ggccatgatg acttgacgtc 180
gtccccacct tcctcctcct tacggaggca gtatccttag agttctcagc ataacctgtt 240
agcaactaag aaagggggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga 300
gctgacgaca gccgtgcagc acctgttttt caaggtctag caagctagac actccactat 360
ttctagcgga ttctctcaat gtcaagccta ggtaaggttc ttcgtgtatc ttcgaattaa 420
accacatgct ccaccgcttg tgcgggtccc cgtctattcc tttgagtttt aatcttgcga 480
ccgtactccc caggcgggat gcttaatgcg ttagctgcat tactggagag actaagccct 540
ccaacaacta gcatccatcg tttagggcgt ggactaccag ggtatctaat cctgtttgct 600
ccccacgctt tcgcgcaatc agcgtcagta atgttccagc aggtcgcctt cgcaatgagt 660
attcctcttg atctctacgg attttacccc tacaccaaga attccaccta cctctcccac 720
ac 722
<210> 5
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<212> DNA
<213> 幽门螺杆菌(Helicobacter pylori)
<400> 5
ccacgatttg ggtgtgcaag aatttgtgat catcaacgat ctagctttag ggcatgacgc 60
ttccattatc cattcttttt tggccgatta tgagtctttg aaattgctca agcaaaccga 120
aaaaattgat gatgaaaacg ctctagcggc gattcgtatc cataaggtta tgaaaccagg 180
cgatcccgtt acgactgaag tggctaagca gtttgtcaaa aaacttttct ttgatccaga 240
acgctatgat ttgaccatgg tggggcgtat gaaaatgaat cacaagttag gcttgcatgt 300
gcctgattac atcacgactt taacgcatga agatattatc accaccgtta aatacctcat 360
gaaaatcaaa aacaatcagg gcaagattga tgatagggat cacttgggca atcgtaggat 420
cagagcggta ggggaattat tggctaatga attgcattca ggcttagtga aaatgcaaaa 480
gaccattaaa gacaagctca ctaccatgag cggggctttt gattcgctca tgccccatga 540
cttggttaat tctaaaatga tcaccagcac catcatggaa tttttcatgg gcggtcagct 600
ctcgcaattt atggatcaaa ctaacccttt gagtgaggtt acgcacaaac gacgcctttc 660
agcgctcggt gaagggggat tggtgaaaga tagggtaggg tttgaagcca gagatgtgca 720
ccccacgcat tatggccgaa tttgtcccat tgagacccca gaaggtcaaa atattggtct 780
gatcaacacc ctttccactt tcacaagagt gaatgattta ggctttattg aagcccctta 840
taaaaaggtt gtggatggca aagtggtggg cgagacgatt tatttgaccg ctattcaaga 900
agacagccac atcatcgctc c 921
<210> 6
<211> 593
<212> DNA
<213> 幽门螺杆菌(Helicobacter pylori)
<400> 6
gcaagaagtc attgacgcta acatgctcgc tatccacaga gctttatgaa gaagttcact 60
aacattaagg attatacatg aaagattgga atgaatttga aatgggagcg gtgctcttcc 120
cttttgaaaa aaacgcgcaa agcgaaatgg aaaaacacaa cgatgagcgc cattacaccg 180
agcaaagtta cttcaccact tcagtggctc attggcgcgt ggctaagcct gtgcataaca 240
ataatatttg catcaattgc tttaattgtt gggtttattg cccagacgct gctattcttt 300
caagagaggg taagttaaag ggcgtggatt attctcattg taaaggctgt ggcgtgtgcg 360
tggatgtctg ccctaccaac cctaaatcgc tatggatgtt tgaagaacaa attgagcccg 420
ctaccgctct cactcaatgg ccccaaaaac aagaaaagaa aaaatcataa ggaaaaaata 480
tggcaaaaag tattgaattg caagagatag aagtgtggga tggcaacacc gctagttcta 540
acgctttaag acaggctcaa attgatgtca tcgcagccta tcctatcacc cca 593
<210> 7
<211> 763
<212> DNA
<213> 幽门螺杆菌(Helicobacter pylori)
<400> 7
cttgcctgga aatcctgtag ggagcgtgag cttagaagca attagagccg tggttttccc 60
taaaaaaacg gatgtcttgt attttgtgaa aatgccggat aagaaacatg ctttcagcgc 120
gacttataaa gagcatgtaa aaaacattaa tctttctaat aatcattttt aagattaagg 180
taaatggggc gttttttctt ttaaattgag taaaaagtgt ttagtatttt ctcattttaa 240
ttttaagttc ttactattaa agcattttag attttattaa aaaaggcttg ctacaatttt 300
agccaaattt tgattgctag ggctttagaa tacagctttt agcacaaagg agaatgaatg 360
gctaaaatga gcgctccaga tggggttacc gtttgggtga atgaagacag gtgtaagggt 420
tgtgatattt gcgtatcggt atgccctgct ggggttcttg gcatggggat tgaaaaagaa 480
agggtgcttg gaaaagtggc caaagtagcc tacccagaga gttgtatcgg ttgcgtgcaa 540
tgcgagttgc actgcccgga ttttgcgatt tatgtggctg acagaaagga tttcaaattc 600
gctaaagttt ctaaagaagc ccaagaaaga agcgaaaagg ttaaggccaa taaatacatg 660
ctcttagaag agactatttt agaagggaga ggcaactaat gcgtgagatt atttctgatg 720
ggaatgaatt agtcgctaaa gcggcgattg aagtggggtg tcg 763
<210> 8
<211> 990
<212> DNA
<213> 幽门螺杆菌(Helicobacter pylori)
<400> 8
tttgagcatg gggcagattt taagcttatt tatggtaatt gtttcgttag ggattttatt 60
gtatgctaca aaaaattcta aaaaaataaa ggaaaatcaa tgaaattttt ggatcaagaa 120
aaaagaagac aattattaaa cgagcgccat tcttgcaaga tgtttgatag ccattatgag 180
ttttctagca cagaattaga agaaatcgct gaaatcgcca ggctatcgcc aagctcttac 240
aacacgcagc catggcattt tgtgatggtt actgataagg atttaaaaaa acaaattgca 300
gcgcacagct atttcaatga agagatgatt aaaagcgctt cagcgttaat ggtggtatgc 360
tctttaagac ccagcgagtt gttaccacac ggccactaca tgcaaaatct ctatccggag 420
tcttataaag ttagagtgat cccctctttt gctcaaatgc ttggcgtgag attcaaccac 480
agcatgcaaa gattagaaag ctatatttta gagcaatgct atatcgctgt ggggcaaatt 540
tgcatgggcg tgagcttgga ttggatagtt gcattattgg aggctttgat cctttaaagg 600
tgggcgaagt tttagaagag cgtatcaata agcctaaaat cgcatgcttg atcgctttgg 660
gcaaagggtg gcagaagcga gtcaaaaatc aagaaaatca aaagttgatg cgattacttg 720
gttgtgatta aacaaaatca aaaacttttt aactataatc aaacctaaat taaagttcaa 780
ggagtggcat tttgtttaaa agaatggttt aatcgctctt ttaggggtgt tttcaagcgt 840
tcattaagcg ctaagagtct tttaagagat gatgggattt agtctctgat ttaaagggca 900
tgaaatcaga actatctgat gctcctgctt gggtttttga agacgctaaa gccccctacg 960
aagaaatggg cgtggcgtat atccctgtta 990
Claims (6)
1.一种检测和判定幽门螺旋杆菌耐药性的诊断方法,其特征在于,此方法为非侵入性诊断,该幽门螺旋杆菌的来源为口腔内唾液及牙菌斑。
2.应用如权利要求1所述的诊断方法,其特征在于,采用巢氏PCR技术结合测序技术,灵敏度、特异性与准确率较高。
3.应用如权利要求1所述的诊断方法,其特征在于,口腔内唾液及牙菌斑标本进行全基因组提取时,采用改良的碘化钾提取方法,整个提取流程操作简单、耗时短、成本低,与试剂盒法相比,得到的后续PCR结果相同,能够满足当前实验需求,适合于临床样本的快速提取。
4.应用如权利要求1所述的诊断方法,其特征在于,包括对克拉霉素、左氧氟沙星、阿莫西林、四环素、呋喃唑酮、利福平、甲硝唑耐药基因的耐药相关突变区域进行PCR引物设计的步骤;
其中,克拉霉素耐药基因23SrRNA突变区域的引物为F1和R1;
阿莫西林耐药基因PBP1突变区域的引物为F2和R2;
四环素耐药基因16SrRNA突变区域的引物为F3和R3;
左氧氟沙星耐药基因gyrA突变区域的引物为F4和R4;
呋喃唑酮耐药基因porD突变区域的引物为F5和R5;
呋喃唑酮耐药基因oorD突变区域的引物为F6和R6;
利福平耐药基因rpoB突变区域的引物为F7和R7;
甲硝唑耐药基因rdxA突变区域的引物为F8和R8。
5.应用如权利要求1所述的诊断方法,其特征在于,包括对克拉霉素耐药基因进行巢氏PCR引物设计的步骤;
其中,克拉霉素耐药基因23SrRNA基因位点G1939A、T1942C的引物为F9和R9;
克拉霉素耐药基因23SrRNA基因位点G2111A、A2115G、A2116G、T2117C、G2141A、A2142G、A2142G/C/T、A2143G/C/T、A2144G、A2146G、C2147G、G2172T、T2182C、C2224A、T2243C、C2245T、C2289T的引物为F10和R10;
克拉霉素耐药基因23SrRNA基因位点C2611A、C2694A与T2717C的引物为F1和R11;
其特征在于,包括对阿莫西林耐药基因的进行巢氏PCR引物设计的步骤;
其中,阿莫西林耐药基因PBP1基因位点A320V的引物为F12和R12;
阿莫西林耐药基因PBP1基因位点F366L、A369T及V374L的引物为F13和R13;
阿莫西林耐药基因PBP1基因位点S414R与L423F的引物为F14和R14;
阿莫西林耐药基因PBP1基因位点T556S、N562Y、T593A、G595S及A599T的引物为F15和R15;
其特征在于,包括对四环素耐药基因的进行巢氏PCR引物设计的步骤;
其中,四环素耐药基因16SrRNA基因位点AGA926-928TTC\AGC\TGC\GGA\CGA\ATC\GTA\GCA的引物为F16和R16;
其特征在于,包括对左氧氟沙星耐药基因的进行巢氏PCR引物设计的步骤;
左氧氟沙星耐药基因GYRA基因位点N87L\I\A\Y\K,D 91A\G\Y\N\H及A 88N\V、的引物为F17和R17。
其特征在于,包括对呋喃唑酮耐药基因的进行巢氏PCR引物设计的步骤;
其中,呋喃唑酮耐药基因porD基因位点C346A、C347T\G、G353A、A356G及C357T的引物为F18和R18;
呋喃唑酮耐药基因oorD基因位点A41G、A78G、A112G、A122G、C156T及C165T的引物为F19和R19;
呋喃唑酮耐药基因oorD基因位点A335G及C349A\G的引物为F20和R20;
其特征在于,包括对利福平耐药基因的进行巢氏PCR引物设计的步骤;
其中,利福平耐药基因rpoB基因位点L525I\P、S526L、Q527K\R、D530Y\N\I\G\V\L\F、H540A\N\Y、I 586P\N\L、V147F、T62C、D540V\L、S545L及Q527R的引物为F21和R21。
其特征在于,包括对甲硝唑耐药基因的进行巢氏PCR引物设计的步骤;
其中,甲硝唑耐药基因rdxA随机突变位点的引物为F22~29、R22~29。
6.应用如权利要求1-11任一所述诊断方法的试剂盒。
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