一组人肺腺癌和人结直肠癌诊断用LncRNA生物标志物
本发明涉及生物技术领域，特别是涉及一组人肺腺癌和人结直肠癌诊断用LncRNA生物标志物、相关试剂盒以及他们的用途。本发明提供一组人肺腺癌和/或人结直肠癌诊断用LncRNA生物标志物，所述LncRNA生物标志物包括：uc001gzl.3、ENST、uc004bbl.1、ENST和NR_034174。本发明所提供的人肺腺癌和/或人结直肠癌诊断用LncRNA生物标志物，可用来区分人早期肺腺癌和人结直肠癌，其区分早期肺腺癌和配对正常肺组织的AUC可达0.978，灵敏度和特异性分别为92%和98%。而对于结直肠癌样本的诊断其AUC亦可达到0.963，灵敏度和特异性分别为94.4%和88.9%。
专利类型：
申请（专利）号：
申请日期：
公开(公告)日：
公开(公告)号：
主分类号：
C12Q1/68,C12Q1/00,C,C12,C12Q,C12Q1
C12Q1/68,C12Q1/00,C12N15/11,C12N15/00,C,C12,C12Q,C12N,C12Q1,C12N15,C12Q1/68,C12Q1/00,C12N15/11,C12N15/00
申请（专利权）人：
中国科学院上海微系统与信息技术研究所
发明（设计）人：
毛红菊,王萍,卢韶华,白亚楠,张宏莲,金庆辉,赵建龙
主申请人地址：
200050 上海市长宁区长宁路865号
专利代理机构：
上海光华专利事务所 31219
国别省市代码：
一组人肺腺癌和/或人结直肠癌诊断用LncRNA生物标志物，所述LncRNA生物标志物包括：uc001gzl.3、ENST、uc004bbl.1、ENST和NR_034174。
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&&8:00-11:30,13:00-17:00(工作日)Nanometer detecting probe chip without amplifying genom DNA and detection method - Patent - Europe PMC
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Nanometer detecting probe chip without amplifying genom DNA and detection method
(PAT - CN)
HONGJU MAO
JIANLONG ZHAO
CHUNPING JIA
QINGHUI JIN
Patent: 
Unexamined APPLIC. open to Public inspection - China
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Classifier:Patent CN1651579A - 一种基因芯片内参照、制备方法及其应用 - Google PatentsCN1651579 AApplicationCN Aug 10, 2005Dec 3, 2004Dec 3, 2004.8, CN 1651579 A, CN 1651579A, CN , CN-A-1651579, CN1651579 A, CN1651579A, CN, CN.8, , , , , , , ,
(3) , 一种基因芯片内参照、制备方法及其应用
CN 1651579 A南京迪安医学检测中心有限公司丙肝测序分型试剂盒及其检测方法International ClassificationC06PublicationC10Request of examination as to substanceC14GrantedRotatePatent CN1635150A - Medicine and bacterium resistant detection chip, method for preparation and ... - Google PatentsCN1635150 AApplicationCN Jul 6, 2005Oct 21, 2004Oct 21, 2004.3, CN 1635150 A, CN 1635150A, CN , CN-A-1635150, CN1635150 A, CN1635150A, CN, CN.3, , , , , , , , , ,
(4) , Medicine and bacterium resistant detection chip, method for preparation and application thereof
CN 1635150 A
The invention relates to a detection chip for familiar resistant organisms and its preparation and application methods belonging to microorganism detection field. The invention employs DAN chip method including fixing the synthesized oligonucleotide probes for clinical detection of familiar resistant organisms on the slide surface to form a dot-plot, hybridizing the pending sample DNA with the chip to obtain a great deal of gene sequence information relative to the bacteria identification and drug tolerance, identifying the kinds of the familiar pathogenicbacterias existing in the clinical sample, thus to implement the identification of the clinical familiar pathogenicbacterias and detection of the main drug resistant spectrum. The invention is simple, convenient, quick, sensitive and specific, and can obviously shorten the disease diagnosis time.
1.一种耐药细菌检测芯片，在一张经修饰的载玻片上，固定有一系列寡核苷酸点阵探针，其特征在于：(1)所说的探针是针对不同细菌23S rRNA基因特异序列及其主要耐药基因包括mecA基因、PBP2B基因、vanA基因、vanB基因、SHV基因和CTX-M基因的特异序列或突变位点设计的；(2)每一探针的熔解温度(Tm)相近；(3)突变位点尽可能位于探针的中间； A detecting chip resistant bacteria, on a modified glass slide, fixed series of oligonucleotides probes lattice, characterized in that: (1) said probe for different bacterial 23S rRNA gene-specific sequence and its major resistance genes, including mecA gene, PBP2B genes, vanA gene, vanB gene, gene-specific sequence SHV and CTX-M genes (2) Each probe melting temperature ( Tm) (3) possible mutation sites in the
2.按权利要求1所述的耐药细菌检测芯片，其特征在于：所说的探针主要针对23SrRNA基因，mecA基因，PBP2B基因，vanA基因，vanB基因，SHV基因，CTX-M基因。 2. resistant bacteria according to claim 1, wherein the detecting chip, wherein: said probe targeted 23SrRNA gene, mecA gene, PBP2B gene, vanA gene, vanB gene, SHV gene, CTX-M gene.
3.按权利要求1所述的耐药细菌检测芯片，其特征在于：所说的针对不同细菌23S基因特异序列设计的探针如下表所示：编号
NH2-poly(T)16-GACCGATAGTGAACCAGTACCG2
NH2-poly(T)16-CGGAGTTACAAAGGACGACAT3
葡萄球菌通用探针
NH2-poly(T)16-GTAGGACACTCTATACGGAGAC4
肠球菌通用探针
NH2-poly(T)16-GAGGTAGACGCAGAGAACTGAA5
NH2-poly(T)16-CGAAATGCTR(A/G)ACAACACCTAG6
NH2-poly(T)16-CGAAATGTGGAAGACACCTA7
肺炎链球菌1
NH2-poly(T)16-AGAAGAATGATTTGGGAAGATC8
肺炎链球菌2
NH2-poly(T)16-GTAGGACTGCAATGTGGACTC9
大肠埃希菌1
NH2-poly(T)16-CCAGAGCCTGAATCAGTGTGT10
大肠埃希菌2
NH2-poly(T)16-CCAGAGCCTGAATCAGTATGTG11
铜绿假单胞菌
NH2-poly(T)16-GCTTCATTGATTTTAGCGGAAC12
变形杆菌属1
NH2-poly(T)16-AGCAGTGTCAGGAGAACGGTCT13
变形杆菌属2
NH2-poly(T)16-AGCCCCGTATCTGAAGATGCT14
洋葱伯克霍尔德菌
NH2-poly(T)16-GTATTGTTAGCCGAACGCTCT15
嗜麦芽窄食单胞菌
NH2-poly(T)16-CCCTGTATCTGAAAGGGCCA16
阴沟肠杆菌
NH2-poly(T)16-CCCGTACACGAAAATGCAC17
鲍曼不动杆菌
NH2-poly(T)16-ACGAAAGGGCACACATAATGAT18
脆弱拟杆菌
NH2-poly(T)16-ACGGCATGTGTGGGGTT19
阴沟肠杆菌2
NH2-poly(T)16-AAAGTCGCACGGTACAGG20
阴沟肠杆菌3
NH2-poly(T)16-CACGAAAGCACACAGGCT21
普通变形杆菌
NH2-poly(T)16-CAATAGCAGCATCAGGAGAA22
摩氏摩根菌
NH2-poly(T)16-TCTGAAAGCACTGGTGTTGT23
弗劳地柠檬酸杆菌
NH2-poly(T)16-AAAAGTGCATGTGTTGTGAAC24
洛菲不动杆菌
NH2-poly(T)16-AGGGCTTATATGATGATGTCG25
产气肠杆菌
NH2-poly(T)16-AATGCACAGGTTGTGAACTC26
肺炎克雷伯菌4
NH2-poly(T)16-AATGCACAGGCTGTGAACTC27
伤寒沙门菌
NH2-poly(T)16-AAAGCGCATATGCTGTGA28
其他沙门菌属
NH2-poly(T)16-AAAGCGCATGTGCTGTGA29
肺炎克雷伯菌3
NH2-poly(T)16-CCCGTACACCAAAATGCA30
NH2-poly(T)16-CGGCTGAGAGGCAGTCAG31
消化链球菌
NH2-poly(T)16-CAGAATAGAGACACTTTAAGAAGAG32
NH2-poly(T)16-AAAGATATAGCGTTTTAGCAGAAT33
NH2-poly(T)16-ACGGAGTTACAAAAGAAAGTTATAA 3. resistant bacteria according to claim 1 wherein the detecting chip, wherein: said different bacterial 23S gene-specific probe sequences designed as shown in the following table: No. 1 universal probe probe sequence NH2-poly ( T) 16-GACCGATAGTGAACCAGTACCG2 aureus NH2-poly (T) 16-CGGAGTTACAAAGGACGACAT3 aureus universal probe NH2-poly (T) 16-GTAGGACACTCTATACGGAGAC4 enterococci universal probe NH2-poly (T) 16-GAGGTAGACGCAGAGAACTGAA5 faecalis NH2- poly (T) 16-CGAAATGCTR (A / G) ACAACACCTAG6 feces Enterococcus NH2-poly (T) 16-CGAAATGTGGAAGACACCTA7 Streptococcus pneumoniae 1 NH2-poly (T) 16-AGAAGAATGATTTGGGAAGATC8 Streptococcus pneumoniae 2 NH2-poly (T) 16- GTAGGACTGCAATGTGGACTC9 coli 1 NH2-poly (T) 16-CCAGAGCCTGAATCAGTGTGT10 coli 2 NH2-poly (T) 16-CCAGAGCCTGAATCAGTATGTG11 aeruginosa NH2-poly (T) 16-GCTTCATTGATTTTAGCGGAAC12 Proteus 1 NH2-poly (T) 16-AGCAGTGTCAGGAGAACGGTCT13 Proteus 2 NH2-poly (T) 16-AGCCCCGTATCTGAAGATGCT14 Burkholderia cepacia NH2-poly (T) 16-GTATTGTTAGCCGAACGCTCT15 maltophilia Aeromonas NH2-poly (T) 16- CCCTGTATCTGAAAGGGCCA16 cloacae NH2-poly (T) 16-CCCGTACACGAAAATGCAC17 Acinetobacter baumannii NH2-poly (T) 16-ACGAAAGGGCACACATAATGAT18 B.fragilis NH2-poly (T) 16-ACGGCATGTGTGGGGTT19 cloacae 2 NH2-poly (T) 16 -AAAGTCGCACGGTACAGG20 cloacae 3 NH2-poly (T) 16-CACGAAAGCACACAGGCT21 Proteus NH2-poly (T) 16-CAATAGCAGCATCAGGAGAA22 Morganella morganii NH2-poly (T) 16-TCTGAAAGCACTGGTGTTGT23 Citrobacter NH2-poly ( T) 16-AAAAGTGCATGTGTTGTGAAC24 Loffi Acinetobacter NH2-poly (T) 16-AGGGCTTATATGATGATGTCG25 aerogenes NH2-poly (T) 16-AATGCACAGGTTGTGAACTC26 Klebsiella pneumoniae 4 NH2-poly (T) 16-AATGCACAGGCTGTGAACTC27 Salmonella typhi NH2-poly (T) 16-AAAGCGCATATGCTGTGA28 other Salmonella NH2-poly (T) 16-AAAGCGCATGTGCTGTGA29 Klebsiella pneumoniae 3 NH2-poly (T) 16-CCCGTACACCAAAATGCA30 Mycobacterium NH2-poly (T) 16-CGGCTGAGAGGCAGTCAG31 digest Streptococcus NH2-poly (T) 16-CAGAATAGAGACACTTTAAGAAGAG32 Peptococcus NH2-poly (T) 16-AAAGATATAGCGTTTTAGCAGAAT33 Listeria NH2-poly (T) 16-ACGGAGTTACAAAAGAAAGTTATAA
4.按权利要求1所述的耐药细菌检测芯片，其特征在于：所述的针对不同耐药基因特异序列或突变位点设计的探针如下表所示：编号
序列(5'＞3')34
NH2-poly(T)16-GCGCTATAGATTGAAAGGATC35
NH2-poly(T)16-GCGCGTTCAGGCTCATC36
NH2-poly(T)16-GCGGGCATCGCCGTTC37
NH2-poly(T)16-CTCAGGCTTACGGTTCATTC38
NH2-poly(T)16-AGCTGGTGACATGGATGAAA39
CTX-M-13组
NH2-poly(T)16-ACCGCCATTCCCGGCGA40
SHV-1(1-12)
NH2-poly(T)16-GGAGCTGGCGAGCG41
SHV-1(1-11)
NH2-poly(T)16-CTGGCGAGCGGGGT42
NH2-poly(T)16-CTGGCGAACGGGGT43
NH2-poly(T)16-GGAGCTAGCAAGCGGG44
SHV通用探针
NH2-poly(T)16-TTATCGCCGATAAGACCG探针长度相近为20mer。 4. resistant bacteria according to claim 1 wherein the detecting chip, wherein: said resistance genes for different specific sequences or mutations designed probes shown in the following table: Number probe sequences (5 '& 3 ') 34 mecA NH2-poly (T) 16-GCGCTATAGATTGAAAGGATC35 vanAN NH2-poly (T) 16-GCGCGTTCAGGCTCATC36 vanBN NH2-poly (T) 16-GCGGGCATCGCCGTTC37 pbp2bm NH2-poly (T) 16-CTCAGGCTTACGGTTCATTC38 CTX-M-1 group NH2-poly (T) 16-AGCTGGTGACATGGATGAAA39 CTX-M-13 group NH2-poly (T) 16-ACCGCCATTCCCGGCGA40 SHV-1 (1-12) NH2-poly (T) 16-GGAGCTGGCGAGCG41 SHV-1 (1-11) NH2 -poly (T) 16-CTGGCGAGCGGGGT42 SHV-11 NH2-poly (T) 16-CTGGCGAACGGGGT43 SHV-12 NH2-poly (T) 16-GGAGCTAGCAAGCGGG44 SHV universal probe NH2-poly (T) 16-TTATCGCCGATAAGACCG probe length similar to 20mer.
5.按权利要求1或2或3或4所述的耐药细菌检测芯片的制备方法，其特征在于包括如下步骤：(1)根据所设计的寡核苷酸探针进行探针的合成；(2)用去离子水将合成的探针稀释，并与点样溶液(spotting solution)等体积混合，使终浓度为75pmol/μl；(3)载玻片表面使用醛基修饰；(4)利用Cartesian公司的微阵列芯片制作系统将探针点阵在经醛基修饰的载玻片表面；(5)置于相对湿度70％，室温条件下经48～72小时进行固定；(6)室温下将载玻片浸入0.2％SDS中振荡数分钟，再浸入双蒸水中振荡数分钟，再浸入100℃双蒸水30秒，晾干，备用。 5. The method as claimed in preparation of resistant bacteria detecting chip 1 or 2 or 3 or 4, characterized by comprising the steps of: (1) synthesis of the probe according to the design of ol (2) Synthesis of deionized water and diluted probes, and the volume of the solution is mixed with the spotting (spotting solution), so that the final concentration of 75 pmol/μl; (3) the use of aldehyde-modified (4) utilizing Cartesian's microarray production system through the probe in a dot aldehyde m (5) placed in a relative humidity of 70%, dried at room temperature for 48 to 72 (6) at room temperature The slides were dipped at 0.2% SDS with shaking for several minutes, and then immersed in double distilled water shaking for several minutes, then immersed in 100 ℃ double distilled water for 30 seconds to dry and set aside.
6.按权利要求5所述的耐药细菌检测芯片的制备方法，其特征在于所说的寡核苷酸探针的5'末端需附加一定长度16mer的连接臂(Poly T)，该连接臂的5'末端需进行氨基修饰。 6. Preparation of resistant bacteria detection chip according to claim 5, wherein, wherein the 5 'end of said oligonucleotide probe for additional connection arm length 16mer of (Poly T), the connection arm The 5 'end of the need for an amino modified.
7.按权利要求1所述的耐药细菌检测芯片的应用方法，其特征在于包括以下步骤：(1)标本处理：取1～1.5ml临床标本1,500rpm离心5分钟，吸取上清，弃沉淀；12,000rpm离心5分钟，弃上清，加入1.5ml ddH2O重悬沉淀；12,000rpm离心5分钟，弃上清，再加入1.5ml ddH2O重悬沉淀；12,000rpm离心5分钟，弃上清，加入100μl STET、2μl溶葡萄球菌酶(1mg/ml)振荡混匀，经37℃10分钟，95℃10分钟处理；5,000rpm离心1分钟，留取上清作为扩增模板，获得待测标本的DNA，置4℃保存备用；(2)荧光标记：获得待测标本的DNA后，进行荧光标记处理：取制备好的标本DNA溶液2μl与23μl PCR扩增体系混匀，所述扩增体系包括2.5μl 10×PCR反应缓冲液5nmol dATP，5nmol dTTP，5nmol dGTP，5nmol dCTP，各15pmol的上游及下游引物，其中一条引物有Cy3或Cy5标记，1.25U DNA聚合酶，通过以下热循环过程制备荧光标记的标本DNA目的片段：在95℃保温5min，然后以95℃30S，55℃30S，72℃30S循环40次，最后72℃保温5min；(3)芯片杂交；(4)检测信号分析，最后用General Scanning公司的芯片信号分析系统Scanarray3000扫描并分析结果； 7. The application of the chip resistant bacteria detection method as claimed in claim 1, characterized by comprising the steps of: (1) the sample processing: Take 1 ~ 1.5ml clinical specimens 1,500rpm rpm for 5 minutes, the supernatant, disc 12,000rpm centrifuged for 5 min, the supernatant was discarded, adding 1.5ml ddH2O res 12,000rpm centrifuged for 5 min, the supernatant was discarded, then add 1.5ml ddH2O res 12,000rpm centrifuged for 5 min, the supernatant was discarded, adding 100μl STET, 2μl lysostaphin (1mg / ml) oscillation mixing, by 37 ℃ 10 分钟, 95 ℃ 10 分钟 5,000rpm centrifuged for 1 min, the supernatant specimens as an amplification template, DNA obtained test specimen, at 4 ℃ (2) fluorescence labeling: DNA test specimens obtained after fluorescent labeling process: take DNA samples prepared solution 2μl and 23μl PCR amplification system mix, the amplification system includes 2.5μl 10 × PCR reaction buffer 5nmol dATP, 5nmol dTTP, 5nmol dGTP, 5nmol dCTP, 15pmol of each upstream and downstream primers, wherein the primers have a Cy3 or Cy5 labeled, 1.25U DNA polymerase, prepared by the following thermal cycling fluorescently labeled the sample DNA fragment: incubation at 95 ℃ 5min, then to 95 ℃ 30S, 55 ℃ 30S, 72 ℃ 30S 40 cycles, and finally 72 ℃ incubated 5 (3) (4) detecting a signal analysis, and finally with General Scanning the company's chip signal analysis system Scanarray3000 scan and
8.按权利要求7所述的耐药细菌检测芯片的应用方法，其特征在于所说的芯片杂交是：取含荧光标记标本DNA目的片段样品2μl，与13μl Easy Hyb杂交液混匀，98℃变性7min，冰浴后取15μl滴于芯片表面，覆盖盖玻片，置于40℃湿盒中杂交60min，然后在洗液中室温避光漂洗10min，晾干。 8. The application of the chip resistant bacteria detection method as claimed in claim 7, wherein said microarray is: take samples containing fluorescence-labeled DNA fragment sample 2μl, 13μl Easy Hyb hybridization solution with mixing, 98 ℃ degeneration 7min, the ice bath after taking 15μl drops on the chip surface, covered with coverslips, placed 40 ℃ wet box hybridization 60min, then at room temperature in the dark rinse wash 10min, dry.
9.按权利要求7所述的耐药细菌检测芯片的应用方法，其特征在于所说的上游和下游引物如下： 9. The method of application of the resistant bacteria detection chip as claimed in claim 7, wherein said upstream and downstream primers are as follows:
表中所列各对引物均采用Cy3或Cy5标记其中一条引物的5'端，除23s rRNA基因引物采用Cy5标记外，其余引物均采用Cy3标记。 Each pair of primers listed in the table are made of Cy3 or Cy5 labeled 5 'end of one primer where, in addition to 23s rRNA gene using Cy5-labeled primer, the rest are used Cy3-labeled primer.
耐药菌检测芯片及其制备方法和应用方法 Resistant detection chip and its preparation method and application methods
技术领域 FIELD
本发明属微生物检验领域，涉及耐药菌快速检测的方法，特别是一种常见耐药菌检测芯片及其制备方法和应用方法。 The invention belongs to the field of microbiology testing, rapid detection method involves resistant, particularly resistant to detect a common chip and its preparation method and application methods.
背景技术 BACKGROUND
近年发展起来的DNA芯片检测技术，利用或借助微电子芯片技术的集约化和平行处理原理，可以快速、高效地获取或处理大量的生命信息(包括基因的识别、基因突变检测、基因表达谱检测和对外来分子的识别等)，在生物学研究如，生命科学、医学诊断、新药筛选和司法鉴定等研究中发挥了重要作用。 In recent years, developed a DNA microarray technology, or by the use of parallel processing intensive and principles of microelectronic chip technology that can quickly and efficiently process large amounts of life or get information (including identifying genes, gene mutations, gene expression profiling to detect and recognition of foreign molecules, etc.), such as life sciences, medical diagnostics, drug screening and forensic research has played an important role in biological research.
当前，感染性疾病仍然是威胁人类健康和生命的重要疾病，据2003年世界卫生组织将健康报告，2002年全球62.25亿人口中，感染性疾病占死亡原因的第二位(26.2％)，仅次于心血管病(29.2％)，而在致残或劳动力丧失的原因中则高居榜首(30％)。 Currently, infectious diseases are still a threat to human health and life of important diseases, according to the 2003 World Health Organization report on the health, in 2002 the world's population of 6.225 billion, infectious diseases accounted for the second cause of death (26.2%), only secondary to cardiovascular disease (29.2%), while the labor force in the cause of disability or the loss of the top of the list (30%). 感染性疾病治愈率低、病死率高的主要原因有以下两方面：一是病原诊断不及时，未能早期正确进行抗感染治疗，二是细菌耐药性严重，治疗困难。 Infectious disease cure rate is low, mainly due to high mortality in the following two aspects: First, the pathogen is not timely diagnosis, failure to correct anti-infection treatment early, two serious bacterial resistance is difficult to treat. 临床医疗工作中，在未能明确病原前，常予以经验治疗，然而即使是基于循证医学拟定的经验治疗方案亦有较高的主观性和盲目性。 Clinical work, failed to clear the pathogen before, often to be the experience of treatment, however, is based on the experience, even if evidence-based medicine regimens also drawn high subjectivity and blindness. 在欧洲一个大系列临床调查结果发现对感染者的经验治疗方案属不恰当者占25％，属此种情况者由于抗感染治疗方案不正确导致42％～60％的病死率，而治疗方案正确者病死率仅17.7％。 A large series of clinical findings of the European experience in the discovery of treatment programs for those infected belong inappropriate accounted for 25% of those who belong to this case because of anti-infection treatment regimens lead to incorrect 42% to 60% mortality, and treatment options right mortality was only 17.7%. 不恰当的治疗往往表现为滥用广谱抗菌药，其结果除疗效差外，尚导致耐药菌的明显增多，以致治疗更为困难，医疗费用亦大幅上升。 Inappropriate treatment is often manifested in the abuse of broad-spectrum antibiotics, which results in addition to poor efficacy, but still result in a significant increase in drug-resistant, so that treatment is more difficult, health care costs have risen sharply.
造成临床上抗感染治疗不恰当的根本原因在于目前病原诊断水平低、费时间。 Resulting in anti-infective therapy clinically inappropriate fundamental reason is that the current low levels of pathogen diagnosis, time-consuming. 以细菌而言，细菌检出及细菌药敏测定需4～5日，至少3日，血培养常需5～7日。 In bacteria, the bacteria detection and antimicrobial susceptibility determination takes 4 to 5 days, at least 3 days, blood cultures usually take 5 to 7 days. 感染病原菌耐药，又缺乏病原和药敏的资料，不能及时正确针对性抗感染治疗，是导致耐药菌感染治疗失败的主因。 Pathogens resistant pathogens and lack of sensitivity of the information can not be properly targeted anti-infection treatment, leading to drug-resistant infections are the main cause of treatment failure. 近年来，日益增多的耐药菌感染出现在细菌中，多重耐药的非发酵菌、肠杆菌科细菌和葡萄球菌感染逐年增多，以假单胞菌、不动杆菌等非发酵菌为例，已经自20世纪90年代的占革兰阴性杆菌的23％～25％至目前的约35％，且为重症监护室感染的第1、2位病原菌，常呈多重耐药，其所致肺部感染、血流感染的病死率可达20％～40％。 In recent years, an increasing number of drug-resistant infections occur in bacteria, multi-drug resistant non-fermenting bacteria, Enterobacteriaceae and Staphylococcus aureus infections has increased every year, with Pseudomonas, Acinetobacter and other non-fermenting bacteria, for example, has been since 23% to 25% to about 35% of the current Gram-negative bacilli in the 1990s, and for the intensive care unit of the 1st and 2nd place pathogen infection, often multi-drug resistance, which caused lung infections, bloodstream infections fatality rate can reach 20% to 40%. 此均给治疗带来了很大的困难。 This was brought to the treatment very difficult.
临床上重要的耐药菌在革兰阳性菌中是甲氧西林耐药葡萄球菌、万古霉素耐药肠球菌和青霉素不敏感肺炎链球菌，而产β-内酰胺酶是革兰阴性杆菌耐药的重要机制，其中最重要的机制之一是产超广谱β-内酰胺酶(extended spectrumβ-lactamases，ESBLs)。 Clinically important drug-resistant methicillin-resistant Staphylococcus aureus in Gram-positive bacteria, Streptococcus pneumoniae is not sensitive to vancomycin-resistant enterococci and penicillin, while β- lactamase producing Gram-negative bacilli resistant One of the most important mechanism in which an important mechanism for the drug, is producing extended-spectrum β- lactamase (extended spectrumβ-lactamases, ESBLs).
研究报道，耐甲氧西林葡萄球菌的主要耐药机制是由于葡萄球菌胞膜上的青霉素结合蛋白(penicillin binding proteins，PBPs)发生改变。 Studies have reported that the main resistance mechanism of methicillin-resistant Staphylococcus aureus is due to penicillin-binding protein on the cell membrane (penicillin binding proteins, PBPs) change. 耐甲氧西林葡萄球菌产生的青霉素结合蛋白称为PBP2a或PBP2'。 Methicillin-resistant Staphylococcus aureus penicillin-binding protein produced called PBP2a or PBP2 '. 由于PBP2a与β-内酰胺类抗生素的亲和力极低且能替代其它PBPs的功能，使得耐甲氧西林葡萄球菌能够在高浓度β-内酰胺类抗生素存在的环境中生存。 Because affinity PBP2a and β- lactam antibiotics is very low and can replace other PBPs features that make methicillin-resistant Staphylococcus aureus can survive in high concentrations within β- lactam antibiotics occurring environment. 编码PBPs的结构基因为mecA，该基因位于耐甲氧西林葡萄球菌的环状染色体上，受mec1和mecR1基因调控。 Structural gene encoding PBPs of mecA, the gene is located on methicillin-resistant Staphylococcus ring chromosome, gene regulation by mec1 and mecR1.
耐万古霉素肠球菌的耐药机制主要是由于肠球菌携带了vanA、vanB、vanC、vanD、vanE等耐药基因后，细胞壁的生物合成途径发生了改变，使其合成的肽聚糖前体与万古霉素等糖肽类抗生素亲和力下降，从而导致肠球菌对万古霉素耐药。 Vancomycin-resistant enterococci resistant mechanism is mainly due to enterococci carrying resistance genes vanA, vanB, vanC, vanD, vanE Once, cell wall biosynthesis pathway changed to synthetic peptidoglycan body before vancomycin and other glycopeptides affinity decreased, resulting in vancomycin-resistant enterococci. 目前研究较多的是vanA、vanB、vanC。 More current research is vanA, vanB, vanC. 携带vanA的肠球菌对万古霉素及替考拉宁均高度耐药，而携带vanB的肠球菌对万古霉素呈不同程度的耐药，但对替考拉宁敏感，携带vanC的肠球菌对万古霉素低度耐药，对替考拉宁敏感，通常以前两者较为多见，最具临床意义。 Carrying vanA enterococci to vancomycin and teicoplanin are highly resistant enterococci and carry vanB vancomycin showed varying degrees of resistance, but sensitive to teicoplanin, carry vanC of enterococci low-level resistance to vancomycin, teicoplanin sensitive, usually before the two more common, most clinical significance.
青霉素不敏感肺炎链球菌的耐药机制主要是由于编码PBPs的基因pbp1a，pbp2x和pbp2b发生变异，它们的编码产物与青霉素的亲和力下降，从而导致肺炎链球菌对青霉素耐药。 Not sensitive to penicillin-resistant Streptococcus pneumoniae mechanism is mainly due to the gene encoding PBPs of pbp1a, pbp2x and pbp2b mutate their encoded products and affinity of penicillin decreased, resulting in penicillin-resistant Streptococcus pneumoniae. 该过程的发生，是一个上述3个基因逐步变异而导致耐药程度逐步提高，且存在多条变异途径的渐进过程，其中pbp2b的变异是高度耐药所必需的。 The process takes place, the gene is one of said three phase variation caused gradually increase the degree of drug resistance and multiple pathways exist gradual process variation, wherein the variation is highly resistant pbp2b necessary.
ESBLs主要有TEM型、SHV型和CTX-M型、OXA型和其他型。 There are TEM-type ESBLs, SHV and CTX-M-type model, OXA-type and other types. TEM、SHV型是在广谱酶TEM-1、2、SHV-1的基础上发生1～5个氨基酸产生点突变而来，由于氨基酸的改变引起底物谱的扩大从而导致细菌对第三代头孢菌素和单环β-内酰胺类抗生素的耐药。 TEM, SHV-type extended spectrum is the enzyme TEM-1,2, 1 ~ 5 amino acids occur SHV-1 based on the generated from point mutations, caused to expand due to changes in the amino acid substrate spectrum causing bacteria to the Third Generation cephalosporins and single ring β- lactam antibiotics. SHV、TEM家族的组内同源性较高，与普通的广谱酶序列仅相差一至几个氨基酸，其中TEM家族在国内较少报道，CTX-M按基因序列同源性可分为4～5个组，组间同源性较低，均为ESBLs，是国内肠杆菌科细菌最常见的型别。 SHV, TEM homology within the family group is high, broad-spectrum enzyme ordinary sequence difference of only one to several amino acids, including TEM family in the country reported less, CTX-M by gene sequence homology can be divided into 4 ~ 5 groups, low homology between the two groups, both ESBLs, Enterobacteriaceae is the most common type.
传统的病原学诊断及其耐药性检测主要依赖于形态学、培养等方法，其成本相对较低，方法也不复杂。 The traditional etiological diagnosis and drug resistance detection method depends on the morphology, cultivation, relatively low cost, the method is not complicated. 但最大的缺点在于耗时长，近年来虽然出现了快速培养的技术，但大大提高了成本，也难于在短时间内同时获取细菌鉴定与药敏试验数据，仍然不能满足临床需要。 But the biggest drawback is time-consuming, although in recent years there has been rapid culture technology, but greatly increases the cost, while also difficult to obtain bacterial identification and susceptibility testing of data in a short time, it can not meet the clinical needs. 常用的耐药性检测方法有纸片扩散法、肉汤稀释法、琼脂稀释法、E-试验法等，这些方法均须在获得纯培养的细菌后才能进行，因此，耐药性检测在病原学检测基础上至少还需要增加一天的时间以获取结果。 Commonly used methods of resistance testing disk diffusion method, broth dilution, agar dilution method, E- test method, etc. These methods must be carried out in obtaining pure cultures of bacteria after, so resistance in pathogen detection Studies based on the detection of at least one additional time needed to get results. 鉴于此，耐药菌的快速诊断成为临床微生物学的一个重要发展方向。 In view of this, the rapid diagnosis of drug-resistant bacteria has become an important direction of development of clinical microbiology.
分子生物学检测技术是以病原菌的核酸为研究对象，通过鉴定的病原和耐药基因的核酸分子来进行病原学诊断。 Molecular biology is a nucleic acid detection techniques for the study of pathogenic bacteria, pathogens and nucleic acid molecules through identification of resistance genes for etiological diagnosis. 如核酸杂交技术，靶基因的扩增技术等，但是这些技术共同的缺点在于能够检测的靶位点较少，实用性较差，有些技术需要进行RNA操作，技术复杂，操作要求高，在临床上难以推广应用。 The nucleic acid hybridization, amplification of the target gene technology, but a common drawback of these techniques is that fewer target sites that can be detected, poor practicality, some techniques require RNA operation, technical complexity, operational requirements, in clinical the difficult application.
发明内容 SUMMARY
本发明的目的是克服上述已有技术的不足，提供一种常见耐药菌检测芯片及其制备方法。 Object of the present invention to overcome the deficiencies of the prior art, there is provided a common resistant detection chip and its preparation method. 本发明的进一步目的是提供耐药菌检测芯片的应用方法，以实现对临床常见致病细菌鉴定以及主要耐药性测定的敏感、特异、快速的检测。 A further object of the present invention is to provide a detection chip resistant application method, in order to achieve the sensitivity of the identification of pathogenic bacteria of clinical drug resistance and is mainly determined, specific and rapid detection.
本发明利用DNA芯片的方法，将已经合成的用于检测常见耐药菌的寡核苷酸探针固定于载玻片表面形成点阵，通过将待测样本的DNA片断与芯片杂交，即可获取大量与细菌鉴定以及耐药性相关的基因序列信息，从而同期实现临床常见致病菌的鉴定以及主要耐药谱的检测，为感染病的治疗提供参考。 The present invention is a method using a DNA chip, which has been synthesized oligonucleotides probes for detecting drug-resistant common fixed lattice is formed on the surface of the slide, by the DNA fragment hybridized with the chip of the test sample, can be get a lot of gene sequence information and identification of bacteria and drug-related, and thus achieve earlier detection and identification of pathogens in clinical major resistance patterns, and provide reference for the treatment of disease infection.
本发明通过下述技术方案实现：一种耐药细菌检测芯片，在一张经修饰的载玻片上，固定有一系列寡核苷酸点阵探针，其特征在于： The present invention is realized by the following technical scheme: a detecting chip resistant bacteria, on a modified glass slide, fixed lattice series of oligonucleotides probes, wherein:
(1)所说的探针是针对不同细菌23S rRNA基因特异序列及其主要耐药基因包括mecA基因、PBP2B基因、vanA基因、vanB基因、SHV基因和CTX-M基因的特异序列或突变位点而设计的；(2)每一探针的熔解温度(Tm)相近；(3)突变位点尽可能位于探针的中间；所说的探针主要针对23S rRNA基因，mecA基因，PBP2B基因，vanA基因，vanB基因，SHV基因，CTX-M基因。 (1) said the probe is different bacterial 23S rRNA gene-specific sequence and its major resistance genes, including mecA gene, PBP2B genes, vanA gene, vanB gene, gene-specific sequence SHV and CTX-M genes or
(2) of each probe melting temperature (Tm) (3) possible mutation sites in the said probes targeted 23S rRNA gene, mecA gene, PBP2B gene, vanA gene, vanB gene, SHV gene, CTX-M genes.
针对不同细菌23S基因各自特异序列设计的探针如表1所示表1.菌种检测探针序列编号
NH2-poly(T)16-GACCGATAGTGAACCAGTACCG2
NH2-poly(T)16-CGGAGTTACAAAGGACGACAT3
葡萄球菌通用探针
NH2-poly(T)16-GTAGGACACTCTATACGGAGAC4
肠球菌通用探针
NH2-poly(T)16-GAGGTAGACGCAGAGAACTGAA5
NH2-poly(T)16-CGAAATGCTR(A/G)ACAACACCTAG6
NH2-poly(T)16-CGAAATGTGGAAGACACCTA7
肺炎链球菌1
NH2-poly(T)16-AGAAGAATGATTTGGGAAGATC8
肺炎链球菌2
NH2-poly(T)16-GTAGGACTGCAATGTGGACTC9
大肠埃希菌1
NH2-poly(T)16-CCAGAGCCTGAATCAGTGTGT10
大肠埃希菌2
NH2-poly(T)16-CCAGAGCCTGAATCAGTATGTG11
铜绿假单胞菌
NH2-poly(T)16-GCTTCATTGATTTTAGCGGAAC12
变形杆菌属1
NH2-poly(T)16-AGCAGTGTCAGGAGAACGGTCT13
变形杆菌属2
NH2-poly(T)16-AGCCCCGTATCTGAAGATGCT14
洋葱伯克霍尔德菌
NH2-poly(T)16-GTATTGTTAGCCGAACGCTCT15
嗜麦芽窄食单胞菌
NH2-poly(T)16-CCCTGTATCTGAAAGGGCCA For each of the different bacterial 23S gene probes specific sequence designed as Table 1. The number of bacteria detection probe sequence probe sequence a universal probe NH2-poly (T) as shown in 1 16-GACCGATAGTGAACCAGTACCG2 aureus NH2-poly ( T) 16-CGGAGTTACAAAGGACGACAT3 aureus universal probe NH2-poly (T) 16-GTAGGACACTCTATACGGAGAC4 Enterococcus universal probe NH2-poly (T) 16-GAGGTAGACGCAGAGAACTGAA5 faecalis NH2-poly (T) 16-CGAAATGCTR (A / G) ACAACACCTAG6 feces Enterococcus NH2-poly (T) 16-CGAAATGTGGAAGACACCTA7 Streptococcus pneumoniae 1 NH2-poly (T) 16-AGAAGAATGATTTGGGAAGATC8 Streptococcus pneumoniae 2 NH2-poly (T) 16-GTAGGACTGCAATGTGGACTC9 coli 1 NH2-poly (T) 16-CCAGAGCCTGAATCAGTGTGT10 coli 2 NH2-poly (T) 16-CCAGAGCCTGAATCAGTATGTG11 aeruginosa NH2-poly (T) 16-GCTTCATTGATTTTAGCGGAAC12 Proteus 1 NH2-poly (T) 16-AGCAGTGTCAGGAGAACGGTCT13 Proteus 2 NH2- poly (T) 16-AGCCCCGTATCTGAAGATGCT14 Burkholderia cepacia NH2-poly (T) 16-GTATTGTTAGCCGAACGCTCT15 maltophilia Aeromonas NH2-poly (T) 16-CCCTGTATCTGAAAGGGCCA
阴沟肠杆菌
NH2-poly(T)16-CCCGTACACGAAAATGCAC17
鲍曼不动杆菌
NH2-poly(T)16-ACGAAAGGGCACACATAATGAT18
脆弱拟杆菌
NH2-poly(T)16-ACGGCATGTGTGGGGTT19
阴沟肠杆菌2
NH2-poly(T)16-AAAGTCGCACGGTACAGG20
阴沟肠杆菌3
NH2-poly(T)16-CACGAAAGCACACAGGCT21
普通变形杆菌
NH2-poly(T)16-CAATAGCAGCATCAGGAGAA22
摩氏摩根菌
NH2-poly(T)16-TCTGAAAGCACTGGTGTTGT23
弗劳地柠檬酸杆菌
NH2-poly(T)16-AAAAGTGCATGTGTTGTGAAC24
洛菲不动杆菌
NH2-poly(T)16-AGGGCTTATATGATGATGTCG25
产气肠杆菌
NH2-poly(T)16-AATGCACAGGTTGTGAACTC26
肺炎克雷伯菌4
NH2-poly(T)16-AATGCACAGGCTGTGAACTC27
伤寒沙门菌
NH2-poly(T)16-AAAGCGCATATGCTGTGA28
其他沙门菌属
NH2-poly(T)16-AAAGCGCATGTGCTGTGA29
肺炎克雷伯菌3
NH2-poly(T)16-CCCGTACACCAAAATGCA30
NH2-poly(T)16-CGGCTGAGAGGCAGTCAG31
消化链球菌
NH2-poly(T)16-CAGAATAGAGACACTTTAAGAAGAG32
NH2-poly(T)16-AAAGATATAGCGTTTTAGCAGAAT33
NH2-poly(T)16-ACGGAGTTACAAAAGAAAGTTATAA针对不同耐药基因特异序列或突变位点而设计的探针如下述表2所示：表2.耐药基因检测探针编号
序列(5'＞3')34
NH2-poly(T)16-GCGCTATAGATTGAAAGGATC35
NH2-poly(T)16-GCGCGTTCAGGCTCATC36
NH2-poly(T)16-GCGGGCATCGCCGTTC 16 E. cloacae NH2-poly (T) 16-CCCGTACACGAAAATGCAC17 Acinetobacter baumannii NH2-poly (T) 16-ACGAAAGGGCACACATAATGAT18 B.fragilis NH2-poly (T) 16-ACGGCATGTGTGGGGTT19 cloacae 2 NH2-poly (T) 16 -AAAGTCGCACGGTACAGG20 cloacae 3 NH2-poly (T) 16-CACGAAAGCACACAGGCT21 Proteus NH2-poly (T) 16-CAATAGCAGCATCAGGAGAA22 Morganella morganii NH2-poly (T) 16-TCTGAAAGCACTGGTGTTGT23 Citrobacter NH2-poly ( T) 16-AAAAGTGCATGTGTTGTGAAC24 Loffi Acinetobacter NH2-poly (T) 16-AGGGCTTATATGATGATGTCG25 aerogenes NH2-poly (T) 16-AATGCACAGGTTGTGAACTC26 Klebsiella pneumoniae 4 NH2-poly (T) 16-AATGCACAGGCTGTGAACTC27 Salmonella typhi NH2-poly (T) 16-AAAGCGCATATGCTGTGA28 other Salmonella NH2-poly (T) 16-AAAGCGCATGTGCTGTGA29 Klebsiella pneumoniae 3 NH2-poly (T) 16-CCCGTACACCAAAATGCA30 Mycobacterium NH2-poly (T) 16-CGGCTGAGAGGCAGTCAG31 digest Streptococcus probe NH2-poly (T) 16-CAGAATAGAGACACTTTAAGAAGAG32 Peptococcus NH2-poly (T) 16-AAAGATATAGCGTTTTAGCAGAAT33 Listeria NH2-poly (T) 16-ACGGAGTTACAAAAGAAAGTTATAA for different specific sequences or mutations resistance genes and design As shown in the following Table 2: Table 2. Number resistance gene probe sequence detection probe (5 '& 3') 34 mecA NH2-poly (T) 16-GCGCTATAGATTGAAAGGATC35 vanAN NH2-poly (T) 16-GCGCGTTCAGGCTCATC36 vanBN NH2 -poly (T) 16-GCGGGCATCGCCGTTC
NH2-poly(T)16-CTCAGGCTTACGGTTCATTC38
NH2-poly(T)16-AGCTGGTGACATGGATGAAA39
CTX-M-13组
NH2-poly(T)16-ACCGCCATTCCCGGCGA40
SHV-1(1-12)
NH2-poly(T)16-GGAGCTGGCGAGCG41
SHV-1(1-11)
NH2-poly(T)16-CTGGCGAGCGGGGT42
NH2-poly(T)16-CTGGCGAACGGGGT43
NH2-poly(T)16-GGAGCTAGCAAGCGGG44
SHV通用探针
NH2-poly(T)16-TTATCGCCGATAAGACCG探针长度相近为20mer。 37 pbp2bm NH2-poly (T) 16-CTCAGGCTTACGGTTCATTC38 CTX-M-1 group NH2-poly (T) 16-AGCTGGTGACATGGATGAAA39 CTX-M-13 group NH2-poly (T) 16-ACCGCCATTCCCGGCGA40 SHV-1 (1-12) NH2 -poly (T) 16-GGAGCTGGCGAGCG41 SHV-1 (1-11) NH2-poly (T) 16-CTGGCGAGCGGGGT42 SHV-11 NH2-poly (T) 16-CTGGCGAACGGGGT43 SHV-12 NH2-poly (T) 16-GGAGCTAGCAAGCGGG44 SHV universal probe NH2-poly (T) 16-TTATCGCCGATAAGACCG probe length similar to 20mer.
上述耐药细菌检测芯片的制作方法包括如下步骤：(1)根据所设计的寡核苷酸探针进行探针的合成；(2)用去离子水将合成的探针稀释，并与点样溶液(spotting solution)等体积混合，使终浓度为75pmol/μl；(3)载玻片表面使用醛基修饰；(4)利用Cartesian公司的微阵列芯片制作系统将探针点阵在经醛基修饰的载玻片表面；(5)置于相对湿度70％，室温条件下经48～72小时进行固定；(6)室温下将载玻片浸入0.2％SDS中振荡数分钟，再浸入双蒸水中振荡数分钟，再浸入100℃双蒸水30秒，晾干，备用；所说的寡核苷酸探针的5'末端需附加一定长度16mer的连接臂(Poly T)，该连接臂的5'末端需进行氨基修饰；上述耐药细菌检测芯片的应用方法包括以下步骤：(一)标本处理：(1)取1～1.5ml临床标本1,500rpm离心5分钟，吸取上清，弃沉淀；(2)12,000rpm离心5分钟，弃上清，加入1.5ml ddH2O重悬沉淀； Detecting the above-described method of manufacturing the chip resistant bacteria comprising the steps of: (1) in accordance with the designed oligonucleotide pro (2) was diluted with deionized water to probe synthesis, and with the point-like solution (spotting solution) mixed in equal volumes, a final concentration of 75 pmol/μl; (3) the use of aldehyde-modified (4) the use of Cartesian's microarray probes lattice in the production system via an aldehyde group m (5) were placed in a relative humidity of 70%, dried at room temperature for 48 to 72 (6) at room temperature Slides were immersed in 0.2% SDS with shaking for several minutes, and then immersed in double distilled water oscillation minutes, then immersed in 100 ℃ double distilled water for 30 seconds and 5 'end of said oligonucleotide probe for additional length connecting arms (Poly T) 16mer and the tether 5 'end need to
application method of the resistant bacteria detection chip comprising the steps :( a) the sample processing: (1) Take 1 ~ 1.5ml clinical specimens 1,500rpm rpm for 5 minutes, the supernatant, dis (2) 12,000rpm centrifuged for 5 minutes, discard supernatant, resuspend pellet in 1.5ml ddH2O
(3)12,000rpm离心5分钟，弃上清，再加入1.5ml ddH2O重悬沉淀；(4)12,000rpm离心5分钟，弃上清，加入100μl STET、2μl溶葡萄球菌酶(1mg/ml)振荡混匀，经37℃10分钟，95℃10分钟处理；(5)5,000rpm离心1分钟，留取上清作为扩增模板，获得待测标本的DNA，置4℃保存备用。 (3) 12,000rpm centrifuged for 5 minutes, the supernatant discarded, and then resuspended precipitate was added 1.5ml ddH2O; (4) 12,000rpm centrifuged for 5 minutes, discard the supernatant, add 100μl STET, 2μl lysostaphin (1mg / ml) oscillation Mix by 37 ℃ 10 分钟, 95 ℃ 10 分钟 (5) 5,000rpm centrifuged for 1 min, the supernatant was used as template for amplification specimens tested were obtained specimens DNA, at 4 ℃ spare.
(二)荧光标记：获得待测标本的DNA后，进行荧光标记处理：(1)取制备好的标本DNA溶液2μl与23μl PCR扩增体系混匀(扩增体系包括2.5μl 10×PCR反应缓冲液5nmol dATP，5nmol dTTP，5nmol dGTP，5nmol dCTP，各15pmol的上游及下游引物，其中一条引物有Cy3或Cy5标记，1.25U DNA聚合酶)，通过以下热循环过程制备荧光标记的标本DNA目的片段：95℃
重复40个循环72℃
5min(2)芯片杂交(3)检测信号分析，最后用General Scanning公司的芯片信号分析系统Scanarray3000扫描并分析结果；所说的芯片杂交是：取含荧光标记标本DNA目的片段样品2μl，与13μl Easy Hyb杂交液(罗氏公司)混匀，98℃变性7min，冰浴后取15μl滴于芯片表面，覆盖盖玻片，置于40℃湿盒中杂交60min，然后在洗液I中室温避光漂洗10min，晾干。 (B) a fluorescent label: After obtaining the DNA samples tested, fluorescence labeling process: (1) Take the prepared sample DNA solution 2μl and 23μl PCR amplification system mix (amplification system comprising 2.5μl 10 × PCR reaction buffer liquid 5nmol dATP, 5nmol dTTP, 5nmol dGTP, 5nmol dCTP, each 15pmol upstream and downstream primers, one primer has Cy3 or Cy5 labeled, 1.25U DNA polymerase), sample DNA fragment prepared by fluorescent markers following thermal cycling : 95 ℃ 5min95 ℃ 30S | 55 ℃ 30S | repeated 40 cycles 72 ℃ 30S | 72 ℃ 5min (2) microarray hybridization (3) analysis of the detection signal, and finally with General Scanning Corporation chip signal analysis system Scanarray3000 scanning and a said microarray hybridization is: take DNA samples containing fluorescently labeled fragment samples 2μl, and 13μl Easy Hyb hybridization solution (Roche) mix, 98 ℃ denaturation 7min, take an ice bath after 15μl drops on the chip surface, covered with a coverslip placed 40 ℃ wet box hybridization 60min, then at room temperature in the dark rinse wash I 10min, dry.
所说的上游和下游引物如表3所示： Said upstream and downstream primers as shown in Table 3:
表3 23s RNA基因引物序列及耐药基因引物序列 Table 3 23s RNA gene sequences and primer sequences resistance gene primers
上表中所列各对引物均采用Cy3或Cy5标记其中一条引物的5'端，除23s rRNA基因引物采用Cy5标记外，其余引物均采用Cy3标记。 Listed in the table for each pair of primers are used Cy3 or Cy5-labeled primer wherein a 5 'end, in addition to 23s rRNA gene using Cy5-labeled primer, the rest are used Cy3-labeled primer.
本发明具有如下优点：(1)本发明的耐药细菌检测芯片可将临床常见耐药菌的鉴定与耐药谱测定同步完成，检测过程简便、快速；(2)本发明的耐药细菌检测芯片具有检测灵敏、特异，可将临床标本中存在的常见致病细菌鉴定到菌种；(3)本发明的耐药细菌检测芯片所用探针设计方法简单，易行，可根据实际需要扩展检测细菌的范围；(4)采用2种不同的荧光分别标记鉴定引物和耐药基因引物，可在后续分析中采用两种激发光进行扫描，在实现细菌鉴定与耐药谱测定同步检测的同时，将两类杂交信息分别进行分析，提高分析的特异性。 The present invention has the following advantages: (1) detection chip resistant bacteria present invention can be identified with common clinical drug resistant spectroscopy synchronization is complete, the detection process is simple, resistant bacteria detection (2) of the present invention chip has a detection sensitivity, specificity, clinical specimens can be present in pathogenic bacteria ident (3) drug-resistant bacterial detection chip used in the present invention probe design method is simple, easy, scalable detection according to actual nee (4) using two different fluorescent labeled primer and resistance genes identified primers, may be employed in the subsequent analyzes two types of excitation light scanning, at the same time to achieve bacterial identification and drug spectroscopy synchronous detection, The two types of information separately hybridization analysis, to improve the specificity of the analysis.
附图说明 Brief Description
图1是本发明包括43种探针的耐药细菌检测芯片用于检测实际临床标本的结果，显示，所检测标本中有携带mecA基因的金葡菌，即甲氧西林耐药金葡菌，表明本发明将菌种鉴定与耐药基因检测同步进行，一次检测获得病原学诊断的完整数据的特点，具有非常高的实用意义。 Figure 1 is a present invention includes detecting 43 kinds of bacteria resistant chip probes for detecting actual clinical specimens results show, the detected samples have carried mecA gene of S. aureus, i.e., methicillin resistant Staphylococcus aureus, show that the invention will strain identification and detection of resistance genes simultaneously, one test to obtain the characteristics of the complete data of etiological diagnosis, with a very high practical significance.
其中，左图为菌种鉴定扫描图，是635nm扫描结果，最左侧一排纵向探针为通用探针，其阳性提示存在细菌扩增产物，探针3为葡萄球菌通用探针，其阳性表示存在葡萄球菌属扩增产物，探针2为金葡菌探针，其阳性表示存在金葡菌扩增产物，结果显示所检测的标本含有金葡菌；右图为耐药基因检测结果扫描图，所示探针为mecA基因探针，其阳性表示所检测标本中含有mecA基因。 Among them, left for strain identification scan, the scan results is 635nm, the leftmost vertical row probe for universal probe, the positive amplification product indicates the presence of bacteria, staphylococcus universal probe for probe 3, the positive indicates the presence of Staphylococcus amplification products, a probe 2 probe for S. aureus, which indicates the presence of S. aureus positive amplification products showed that the detected sample containing S right is resistance gene detection scan results Figure probe shows mecA gene probe, the positive representation of the detected sample containing mecA gene.
图2为芯片探针的分布示意图。 Figure 2 is a schematic diagram of the distribution of the chip probe.
图3为芯片点样的示意图，矩阵即如图分布. Figure 3 is a schematic view of microarray spotting, i.e., as shown in the matrix distribution.
具体实施方式 DETAILED DESCRIPTION
探针设计和芯片制作根据不同细菌已知的23S rRNA基因序列以及耐药基因序列，设计长度相近的探针，约20mer。 Example 1 probe design and implementation of chip production, depending on the bacterial 23S rRNA gene sequences of known gene sequences and drug design similar to the length of the probe, about 20mer. 如表1和表2所示。 As shown in Table 1 and Table 2.
将合成的寡核苷酸探针溶于离子水中，并与点样溶液(spotting solution)等体积混合，使终浓度为75pmol/μl；利用Cartesian公司的微阵列芯片制作系统将探针点阵在经醛基修饰的载玻片表面；置于相对湿度70％，室温条件下经48～72小时进行固定；然后，室温下将载玻片浸入0.2％SDS中振荡数分钟，再浸入双蒸水中振荡数分钟，再浸入100℃双蒸水30秒，晾干，备用。 The oligonucleotide probes synthesized dissolved deionized water, and mixed with a solution of spotting (spotting solution) equal volume, so that a final concentration of 75 pmol/μl; use Cartesian's microarray production system in the probe lattice by aldehyde m disposed relative humidity of 70%, dried at room temperature for 48 to 72 then, slides were dipped at room temperature for 0.2% SDS with shaking for several minutes, and then immersed in double distilled water oscillation minutes, then immersed in 100 ℃ double distilled water for 30 seconds to dry and set aside.
标本DNA目的片段的荧光标记设计两对引物，并将每对引物中一条的5'端标记Cy3或Cy5，利用PCR扩增产生荧光标记的标本DNA目的片段，其中模板为细菌基因组DNA，23s RNA基因引物序列及耐药基因引物序列见表3。 Fluorescence Example 2 sample DNA fragment implemented tag design two pairs of primers, and the primer 21 of the 5 'end tag of each pair of Cy3 or Cy5, by PCR amplification of the sample DNA fragment to produce a fluorescent label, wherein the template for the bacterial genomic DNA , 23s RNA gene sequences of the primers and the resistance gene primer sequences are shown in Table 3. 扩增体系如下：标本DNA溶液2μl与23μl PCR扩增体系混匀(扩增体系包括2.5μl 10×PCR反应缓冲液5nmol dATP，5nmoldTTP，5nmol dGTP，5nmol dCTP，各15pmol的上游及下游引物，)。 Amplification system as follows: the sample DNA solution 2μl and 23μl PCR amplification system mix (amplification system comprising 2.5μl 10 × PCR reaction buffer 5nmol dATP, 5nmoldTTP, 5nmol dGTP, 5nmol dCTP, 15pmol each upstream and downstream primer) . 此体系在95℃保温5min，然后以95℃30S，55℃30S，72℃30S循环40次，最后72℃保温5min。 This was incubated at 95 ℃ 5min, then to 95 ℃ 30S, 55 ℃ 30S, 72 ℃ 30S 40 cycles, and finally 72 ℃ incubated 5min.
标记的目的片段与芯片杂交取含荧光标记标本DNA目的片段样品2μl，与13μl Easy Hyb杂交液(罗氏公司)混匀，99℃变性5min，冰浴后取15μl滴于芯片表面，覆盖盖玻片，置于40℃湿盒中杂交60min，然后在洗液I(1×SSC，0.1％SDS)中室温避光漂洗10min，晾干。 Fragment and microarray hybridization Example 3 A mixture of labeled DNA fragment fluorescently labeled specimens sample 2μl, and 13μl Easy Hyb hybridization solution (Roche) mix, 99 ℃ denaturation 5min, after taking an ice bath 15μl drops on the chip surface, cover coverslip, placed in a humidified chamber 40 ℃ hybridization 60min, then wash I (1 × SSC, 0.1% SDS) in the dark at room temperature rinsing 10min, dry.
信号检测和分析杂交后DNA芯片用General Scanning公司的芯片信号分析系统Scanarray3000进行扫描分析，采用635nm和532nm波长同时扫描芯片，记录扫描图像。 Example 4 signal detection and analysis of DNA microarray hybridization chip company with General Scanning Signal Analysis System Scanarray3000 scan analysis, using 635nm and 532nm wavelengths simultaneously scan the chip to record the scanned image. 以635nm波长激发的杂交信号判读菌种鉴定结果，以532nm波长激发的杂交信号判读耐药结果。 The signal interpretation of hybrid species identification results 635nm wavelength excitation, 532nm wavelength to excite the hybridization signal interpretation resistance results. 然后与相应临床标本的常规检测结果进行对比。 Then the corresponding test results with conventional clinical specimens were compared.
临床血标本检测50份血标本按前述方法进行标本处理、制备模板DNA、目的片段的荧光标记，并采用本发明制备的芯片进行杂交以及信号检测与分析。 Example 5 implementation of clinical blood samples to detect 50 blood samples as previously described specimens were processed to prepare template DNA, fluorescence-labeled fragment, and the present invention is prepared using chip hybridization and signal detection and analysis. 结果显示，芯片检测结果与临床常规检测结果的符合率为100％。 The results show that the chip test results and clinical routine test results was 100%. 用本发明耐药细菌检测芯片检测有耐药细菌存在的临床标本敏感性可达90％以上，特异性可达100％。 Resistant bacteria detected by microarray sensitivity of the present invention there is resistance to the presence of bacteria in clinical samples of up to 90% and a specificity of 100%. 证实本发明耐药细菌检测芯片，从临床标本处理、PCR扩增(标本DNA目的片段的荧光标记)，到获得检测结果仅需4小时的时间，可大大缩短疾病的诊断时间，为感染性疾病患者的救治赢得宝贵时间。 Confirmed that the present invention is resistant bacteria detection chip, from clinical specimens treated, PCR amplification (fluorescence-labeled sample DNA target fragment), to obtain the detection result only 4 hours, can greatly shorten the time of disease diagnosis, for infectious diseases the patient's treatment to win precious time.
本发明还可用于尿、胸水、腹水、脑脊液标本检测。 The present invention can also be used in urine, pleural effusion, ascites, cerebrospinal fluid specimens.
表4是检测结果与临床微生物实验室常规检测结果对比(血标本芯片与常规检测方法比较)。 Table 4 shows the test results (comparison of blood samples chips and conventional detection methods) and routine clinical microbiology laboratory test results compared.
表4细菌名称
常规方法检
芯片方法检出
符合份数出例数
例数革兰阴性菌
36甲氧西林敏感金黄色葡萄球菌
3甲氧西林耐药金黄色葡萄球菌
2甲氧西林敏感凝固酶阴性葡萄球菌
20甲氧西林耐药凝固酶阴性葡萄球菌
7青霉素不敏感肺炎链球菌
2革兰阴性菌
14大肠埃希菌
4肺炎克雷伯菌
3铜绿假单胞菌
2洋葱伯克霍尔德菌
3甲型副伤寒杆菌
1摩根摩根菌
50 Table 4. Name the conventional method of bacterial detection method detection chip shares a number of cases in line with the number of cases of gram-negative bacteria 363 636 methicillin-sensitive Staphylococcus aureus 333 methicillin-resistant Staphylococcus aureus 222 A methicillin-sensitive coagulase-negative staphylococci 202,020 methicillin-resistant coagulase-negative staphylococci 111 feces enterococci 777 penicillin non-susceptible Streptococcus pneumoniae Listeria 110 222 14 Gram-negative bacteria 1414 E. coli and Klebsiella pneumoniae 444 333 222 Pseudomonas aeruginosa Burkholderia cepacia 33 paratyphoid bacillus bacteria Morgan Morgan 111 111 Total 50 5050
SEQUENCE LISTING&110&复旦大学附属华山医院；中国科学院上海微系统与信息技术研究所&120&耐药菌检测芯片及其制备方法和应用方法&130&&33&170&PatentIn version 3.3&210&1&211&38&212&DNA&213&人工序列&220& SEQUENCE LISTING & 110 & Huashan H Shanghai Institute of Microsystem and Information Technology Institute & 120 & resistant detection chip and its preparation method and application methods & 130 &
& 160 & 33 & 170 & PatentIn version 3.3 & 210 & 1 & 211 & 38 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&1tttttttttt ttttttgacc gatagtgaac cagtaccg
38&210&2&211&37&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 1tttttttttt ttttttgacc gatagtgaac cagtaccg 38 & 210 & 2 & 211 & 37 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&2tttttttttt ttttttcgga gttacaaagg acgacat
37&210&3&211&38&212&DNA&213&人工序列 & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 2tttttttttt ttttttcgga gttacaaagg acgacat 37 & 210 & 3 & 211 & 38 & 212 & DNA & 213 & artificial sequence
&220& & Lt; 220 &
&221&modified_base&222&(1)..(1)&400&3tttttttttt ttttttgtag gacactctat acggagac
38&210&4&211&38&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 3tttttttttt ttttttgtag gacactctat acggagac 38 & 210 & 4 & 211 & 38 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&4tttttttttt ttttttgagg tagacgcaga gaactgaa
38&210&5&211&39&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 4tttttttttt ttttttgagg tagacgcaga gaactgaa 38 & 210 & 5 & 211 & 39 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&5tttttttttt ttttttcgaa atgctragac aacacctag
39&210&6&211&36&212&DNA&213&人工序列 & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 5tttttttttt ttttttcgaa atgctragac aacacctag 39 & 210 & 6 & 211 & 36 & 212 & DNA & 213 & artificial sequence
&220& & Lt; 220 &
&221&modified_base&222&(1)..(1)&400&6tttttttttt ttttttcgaa atgtggaaga caccta
36&210&7&211&30&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 6tttttttttt ttttttcgaa atgtggaaga caccta 36 & 210 & 7 & 211 & 30 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&7ttttttttag aagaatgatt tgggaagatc
30&210&8&211&37&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 7ttttttttag aagaatgatt tgggaagatc 30 & 210 & 8 & 211 & 37 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&8tttttttttt ttttttgtag gactgcaatg tggactc
37&210&9&211&37&212&DNA&213&人工序列&400&9tttttttttt ttttttccag agcctgaatc agtgtgt
37 & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 8tttttttttt ttttttgtag gactgcaatg tggactc 37 & 210 & 9 & 211 & 37 & 212 & DNA & 213 & artificial sequence & 400 & 9tttttttttt ttttttccag agcctgaatc agtgtgt 37
&210&10&211&38&212&DNA&213&人工序列&220& & Lt; 210 & 10 & 211 & 38 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&10tttttttttt ttttttccag agcctgaatc agtatgtg
38&210&11&211&38&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 10tttttttttt ttttttccag agcctgaatc agtatgtg 38 & 210 & 11 & 211 & 38 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&11tttttttttt ttttttgctt cattgatttt agcggaac
38&210&12&211&38&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 11tttttttttt ttttttgctt cattgatttt agcggaac 38 & 210 & 12 & 211 & 38 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&12tttttttttt ttttttagca gtgtcaggag aacggtct
38&210&13 & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 12tttttttttt ttttttagca gtgtcaggag aacggtct 38 & 210 & 13
&211&37&212&DNA&213&人工序列&220& & Lt; 211 & 37 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&13tttttttttt ttttttagcc ccgtatctga agatgct
37&210&14&211&37&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 13tttttttttt ttttttagcc ccgtatctga agatgct 37 & 210 & 14 & 211 & 37 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&14tttttttttt ttttttgtat tgttagccga acgctct
37&210&15&211&36&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 14tttttttttt ttttttgtat tgttagccga acgctct 37 & 210 & 15 & 211 & 36 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&15tttttttttt ttttttccct gtatctgaaa gggcca
36&210&16&211&35&212&DNA & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 15tttttttttt ttttttccct gtatctgaaa gggcca 36 & 210 & 16 & 211 & 35 & 212 & DNA
&213&人工序列&220& & Lt; 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&16tttttttttt ttttttcccg tacacgaaaa tgcac
35&210&17&211&38&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 16tttttttttt ttttttcccg tacacgaaaa tgcac 35 & 210 & 17 & 211 & 38 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&17tttttttttt ttttttacga aagggcacac ataatgat
38&210&18&211&33&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 17tttttttttt ttttttacga aagggcacac ataatgat 38 & 210 & 18 & 211 & 33 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&18tttttttttt ttttttacgg catgtgtggg gtt
33&210&19&211&34&212&DNA&213&人工序列 & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 18tttttttttt ttttttacgg catgtgtggg gtt 33 & 210 & 19 & 211 & 34 & 212 & DNA & 213 & artificial sequence
&220& & Lt; 220 &
&221&modified_base&222&(1)..(1)&400&19tttttttttt ttttttaaag tcgcacggta cagg
34&210&20&211&34&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 19tttttttttt ttttttaaag tcgcacggta cagg 34 & 210 & 20 & 211 & 34 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&20tttttttttt ttttttcacg aaagcacaca ggct
34&210&21&211&36&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 20tttttttttt ttttttcacg aaagcacaca ggct 34 & 210 & 21 & 211 & 36 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&21tttttttttt ttttttcaat agcagcatca ggagaa
36&210&22&211&36&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 21tttttttttt ttttttcaat agcagcatca ggagaa 36 & 210 & 22 & 211 & 36 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&22tttttttttt tttttttctg aaagcactgg tgttgt
36&210&23&211&37&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 22tttttttttt tttttttctg aaagcactgg tgttgt 36 & 210 & 23 & 211 & 37 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&23tttttttttt ttttttaaaa gtgcatgtgt tgtgaac
37&210&24&211&37&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 23tttttttttt ttttttaaaa gtgcatgtgt tgtgaac 37 & 210 & 24 & 211 & 37 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&24tttttttttt ttttttaggg cttatatgat gatgtcg
37&210&25&211&36&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 24tttttttttt ttttttaggg cttatatgat gatgtcg 37 & 210 & 25 & 211 & 36 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1) & Lt; 221 & modified_base & 222 & (1) .. (1)
&400&25tttttttttt ttttttaatg cacaggttgt gaactc
36&210&26&211&36&212&DNA&213&人工序列&220& & Lt; 400 & 25tttttttttt ttttttaatg cacaggttgt gaactc 36 & 210 & 26 & 211 & 36 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&26tttttttttt ttttttaatg cacaggctgt gaactc
36&210&27&211&34&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 26tttttttttt ttttttaatg cacaggctgt gaactc 36 & 210 & 27 & 211 & 34 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&27tttttttttt ttttttaaag cgcatatgct gtga
34&210&28&211&34&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 27tttttttttt ttttttaaag cgcatatgct gtga 34 & 210 & 28 & 211 & 34 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&28 & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 28
tttttttttt ttttttaaag cgcatgtgct gtga
34&210&29&211&33&212&DNA&213&人工序列&220& tttttttttt ttttttaaag cgcatgtgct gtga 34 & 210 & 29 & 211 & 33 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&29tttttttttt ttttttcccg tacaccaaaa tgc
33&210&30&211&26&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 29tttttttttt ttttttcccg tacaccaaaa tgc 33 & 210 & 30 & 211 & 26 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&30ttttttttcg gctgagaggc agtcag
26&210&31&211&41&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 30ttttttttcg gctgagaggc agtcag 26 & 210 & 31 & 211 & 41 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&31tttttttttt ttttttcaga atagagacac tttaagaaga g
41 & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 31tttttttttt ttttttcaga atagagacac tttaagaaga g 41
&210&32&211&40&212&DNA&213&人工序列&220& & Lt; 210 & 32 & 211 & 40 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&32tttttttttt ttttttaaag atatagcgtt ttagcagaat
40&210&33&211&41&212&DNA&213&人工序列&220& & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 32tttttttttt ttttttaaag atatagcgtt ttagcagaat 40 & 210 & 33 & 211 & 41 & 212 & DNA & 213 & artificial sequence & 220 &
&221&modified_base&222&(1)..(1)&400&33tttttttttt ttttttacgg agttacaaaa gaaagttata a
41 & Lt; 221 & modified_base & 222 & (1) .. (1) & 400 & 33tttttttttt ttttttacgg agttacaaaa gaaagttata a 41 上海生物芯片有限公司Beta-lactamase SHV gene variation detecting chip and application thereof浙江大学检测芯片的制备方法及利用该芯片检测病原体的方法武汉大学快速检测尿液中粪肠球菌的荧光定量pcr试剂盒 *中国检验检疫科学研究院Composition and method for detecting drug resistance of staphylococcus aureus *中国检验检疫科学研究院Composition and method for detecting drug resistance of staphylococcus aureus *复旦大学附属华山医院Visualized pathogen detection chip as well as preparation method and application thereof *上海中优医药高科技有限公司Method for detecting baumanii carrying qacE *1-sull geneCapitalbio CorporationMethods, microarray, and kits for detection of drug resistance genes in gram-negative bacteria *Capitalbio CorporationMethods, microarray, and kits for detection of drug resistance genes in gram-negative bacteriaInternational ClassificationC06PublicationC10Entry into substantive examinationC14Grant of patent or utility modelEXPYTermination of patent right or utility modelRotate