WO2015180484A1 - 对普罗威登斯菌031，041，042，043和050特异的核苷酸及其应用 - Google Patents

对普罗威登斯菌031，041，042，043和050特异的核苷酸及其应用 Download PDF

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WO2015180484A1
WO2015180484A1 PCT/CN2015/000304 CN2015000304W WO2015180484A1 WO 2015180484 A1 WO2015180484 A1 WO 2015180484A1 CN 2015000304 W CN2015000304 W CN 2015000304W WO 2015180484 A1 WO2015180484 A1 WO 2015180484A1
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providencia
nucleotides
specific
seq
pcr
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王磊
夏香红
冯露
刘斌
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南开大学
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

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  • the present invention relates to nucleotides specific for the serotypes of Providencia O31, O41, O42, O43 and O50, in particular to the ovarian O antigen gene cluster of the O. fuligis O31, O41, O42, O43 and O50 Single gene-specific nucleotides and their applications.
  • Providencia is a common Gram-negative bacterium in the family Enterobacteriaceae and is an opportunistic pathogen. Infections caused by nosocomial infections or eating seafood can cause respiratory infections, and can also cause sepsis, urinary tract infections, and secondary meningitis. Separated from diarrhea stools, urinary tract infections, wounds, burns and bacteremia specimens.
  • Bacterial typing and identification methods mainly include traditional phenotypic methods, serological methods and molecular identification methods.
  • traditional serotyping and identification methods have certain problems. For example, serotyping requires a large amount of antiserum, while antiserum is generally incomplete and insufficient in quantity. Antisera also presents some difficulties in preparation and storage.
  • the serotyping method takes a long time, has low sensitivity, high missed detection rate, and poor accuracy, and cross-reaction often exists between antiserums produced by different O antigens. Therefore, the establishment of serum identification methods based on molecular biology techniques has become the development direction.
  • the molecular identification of Providencia has been paid more and more attention, and it has become an important basis for the identification of strains and strains of Providencia, and many new bacteria have also been produced.
  • Molecular biology detection technology has the advantages of high speed and easy to use on a large scale. It is the internationally recognized development direction of pathogen detection, but the current difficulty lies in the low molecular specificity of DNA targets.
  • the diversity of polysaccharide antigens on the surface of bacteria is caused by the genetic diversity of the gene cluster responsible for their synthesis. For a long time, people have been trying to explore the diversity of the important pathogenic factors of surface polysaccharide antigens present in Providencia and the corresponding genetic evolutionary basis.
  • ITS transcribed spacer
  • RAPD random amplified length polymorphism
  • RFLP Fragment length polymorphism
  • PCR Polymerase chain reaction
  • nucleotides complementary to the nucleotide set forth in SEQ ID NO: 1-10 for example, having a nucleotide sequence that can be complementary to a sequence amplified by an O31 serotype-specific primer;
  • the SEQ ID NO: 1-10 is as follows:
  • the present invention also provides a PCR kit comprising a PCR primer, dNTP, a buffer, and a DNA polymerase, the PCR primer being at least one of the nucleotides shown in SEQ ID NO: 1-10.
  • the kit further comprises the following reagents: 10 mM dNTP 30 ⁇ l; 10 ⁇ enzyme specific reaction buffer 50 ⁇ l; 5 U/ ⁇ l thermostable DNA polymerase 5 ⁇ l; primer mixture 10 ⁇ l; positive control 10 ⁇ l; negative control 10 ⁇ l; ddH 2 O 5ml.
  • the PCR primer described therein is preferably at least one of the nucleotides shown in SEQ ID NO: 1-10.
  • the present invention further discloses that the nucleotides of SEQ ID NO: 1-10 specific for the serotypes of Providencia O31, O41, O42, O43 and O50 are prepared for the detection of Providencia tyrosii O31. , O41, O42, O43 and O50 serotype PCR kits, gene chips or microarray applications.
  • the provenved bacteria of the present invention can be sampled in a crude extract of a culture of diarrhea stool, urinary tract infection, wound, burn and bacteremia specimen, or a crude extract of a pure culture of Providencia. .
  • the collection of the genome of the extract of Providencia is obtained by a conventional method.
  • the entire detection step includes sample pretreatment-amplification-electrophoresis detection results.
  • the reagents required for the primers and the PCR reaction system have been previously added to the amplification tube, and the user only needs to add the pre-treated sample to the amplification tube to initiate the amplification reaction, and the detection work can be completed simply and quickly.
  • the invention also provides a liquid phase detecting chip comprising a liquid phase magnetic bead and an oligonucleotide probe attached to the liquid magnetic bead, and corresponding primers corresponding to the fragment of the corresponding probe; wherein the nucleotide Preferably as SEQ ID NO: 1-10 Nucleotides shown.
  • the invention also provides a microarray comprising the nucleotides described above; wherein the nucleotides are preferably nucleotides as set forth in SEQ ID NOs: 1-10.
  • the present invention discloses nucleotides specific for the serotypes of Providencia O31, O41, O42, O43 and O50. Compared with the prior art, the present invention has the following advantages:
  • the invention provides a PCR reaction system capable of detecting Providen's bacteria and providing specific primers for serotyping detection, and the PCR method can be used for detecting clinical specimens.
  • the present invention obtains a target band by PCR reaction on each serotype-specific gene of Providencia, and compares the obtained target fragment with the known length to obtain the path of the genus Providencia. Serotype.
  • Figure 1 shows the electrophoresis results of the P1 and P2 primers of the O31 serotype wzy gene of the invention, and the screening of the other strains of the serotypes of the serotypes of the serotypes of the Wzy gene, the screening of the pzy gene P1 and P2 primers, the target band is 263 bp, and the remaining serotypes do not have any Bands. Specific strain information is shown in Table 2.
  • Fig. 2 is a graph showing the electrophoresis results of species specificity of P31 and P2 of the O31 serotype wzy gene of the present invention, wherein 6 strains of Vibrio, 1 strain of Salmonella and 1 strain of Escherichia coli were detected without any bands.
  • the specific strain information is shown in Table 2.
  • Figure 3 is a graph showing the electrophoresis results of the P41 and P4 primers of the O41 serotype wzy gene of the present invention, and the screening of the Wzy gene P3 and P4 primers, the target band is 170 bp, and the remaining serotypes are not Strips, specific strain information is shown in Table 2;
  • Figure 4 is a graph showing the results of electrophoresis of the species specificity of the O41 serotype wzy gene P3 and P4 of the present invention, 6 strains of Vibrio and 1 strain of Salmonella and 1 strain of Escherichia coli were detected, and there were no bands.
  • the specific strain information is shown in Table 2;
  • Figure 5 is a graph showing the electrophoresis results of the P4 and P6 primers of the O42 serotype wzy gene of the present invention for the detection of other serotypes of the serotypes of the genus of the genus P., and the screening of the pzy gene P5 and P6 primers, the target band is 323 bp, and the remaining serotypes are not Bands. Specific strain information is shown in Table 2.
  • Fig. 6 is a graph showing the electrophoresis results of species specificity of the O4 serotype wzy gene P5 and P6 in the present invention, wherein 6 strains of Vibrio, 1 strain of Salmonella and 1 strain of Escherichia coli were detected without any bands.
  • the specific strain information is shown in Table 2.
  • Figure 7 is a graph showing the electrophoresis results of the P49 and P8 primers of the O43 serotype wzy gene of the present invention for the detection of the other strains of the serotypes of the serotypes of the W., and the screening of the wzy gene P7 and P8 primers, the target band is 210 bp, and the remaining serotypes are not Strips, specific strain information is shown in Table 2;
  • Figure 8 is a graph showing the electrophoresis results of species specificity of the O4 serotype wzy gene P7 and P8 of the present invention, wherein six Vibrio species, one Salmonella and one strain of Escherichia coli were detected using the wzy gene P7 and P8 primers, none of which For any band, the specific strain information is shown in Table 2;
  • Figure 9 is a view showing the electrophoresis results of the P49 and P10 primers of the O50 serotype wzy gene of the present invention for the detection of other serotypes of the serotypes of the genus of the genus P., and the screening of the wzy gene P9 and P10 primers, the target band is 99 bp, and the remaining serotypes are not Strips, specific strain information is shown in Table 2;
  • Figure 10 is a graph showing the electrophoresis results of the species specificity of the O50 serotype wzy gene P9 and P10 of the present invention, wherein six Vibrio species, one Salmonella and one strain of Escherichia coli were detected using the wzy gene P9 and P10 primer primers. There are no bands, the specific strain information is shown in Table 2;
  • Figure 11 shows the electrophoresis results of the corresponding serotypes amplified by O31, O41, O42, O43 and O50 specific primers respectively.
  • the specific strain information is shown in Table 2;
  • the cultivation of the bacteria in the 37 ° C nutrient broth medium, collecting bacteria, and extracting the genome are as follows:
  • the cells were resuspended in 500 ul of 50 mM Tris-HCl (pH 8.0) and 10 ul of 0.4 M EDTA, incubated at 37 ° C for 20 minutes, and then incubated with 10 ul of 10 mg/ml lysozyme for 20 minutes. Thereafter, 3 ul of 20 mg/ml proteinase K, 15 ul of 10% SDS was added, incubated at 50 ° C for 2 hours, and then 3 ul of 10 mg/ml RNase was added and incubated at 65 ° C for 30 minutes.
  • the genomes of the standard strains of S. edodes O31, O41, O42, O43 and O50 were extracted, and the sequence of the serotype was obtained by genome-wide sequencing of each serotype genome of Providencia by Solexa pair-end sequencing technology.
  • the wzy and wzx genes are indeed serotype-specific genes, so the specific primers of this gene were selected to design specific primers. Since wzy is more specific, the wzy gene is mainly used as a target gene.
  • Primer design is a core part of the invention.
  • Design primers were designed based on the specific genes described in the literature.
  • the two genes, wzx and wzy, are relatively specific genes in the Provenus O antigen gene cluster and can be used as target genes for serotype identification.
  • the above gene was introduced into Primer Premier 5 for primer design, and the length of the primer was preferably between 18 and 24 bp, and the Tm value was between 50 and 55 °C.
  • Each gene is designed with a pair of primers with a single-purpose band.
  • primer design was performed in Genbank.
  • Genbank The designed primers could not be too similar to the sequence of other closely related bacteria, thus ensuring that the primers were amplified only at their own predetermined positions, and not with other related bacteria or The relative bacteria in the environment in which the specimen was collected did not produce a positive reaction. This is important to avoid the generation of non-specific bands and the success of experiments.
  • the designed primers are shown in Table 1.
  • Standard strains of the serotypes of Providen's O31, O41, O42, O43 and O50 and one of the other 13 serotypes of standard strains, 10 untyped proveneves, and 6 vibrio were collected.
  • the specificity of the strains, 1 strain of Salmonella and 1 strain of E. coli strain were verified.
  • the strain number and source are shown in Table 2.
  • the PCR system used for screening the primers for gene identification was 0.4 ⁇ l of 5 ⁇ M primer, 2.5 ⁇ l of 10 ⁇ enzyme-specific reaction buffer, 0.25 ⁇ l of 10 mM dNTP, 0.2 ⁇ l of 5 U/ ⁇ l of heat-resistant DNA polymerase, and 3 ⁇ l of the sample template to be tested.
  • the final ddH 2 O was made up to 25 ⁇ l. All primers gave positive results in their respective serotypes, and no PCR product bands were obtained in the other groups.
  • reaction cycle parameters on the PCR instrument in this step include DNA denaturation, renaturation, temperature and time of extension, and number of cycles, specifically:
  • One cycle of the pre-treatment process necessary for the denaturation to achieve the desired temperature is 95 ° C, 5 minutes; the denaturation temperature and time is 95 ° C, 45 seconds;
  • Refolding temperature and time is 55 ° C / 68 ° C, 1 minute;
  • the extension temperature and time are 72 ° C, 1 minute;
  • the number of cycles of denaturation, renaturation, and extension is 35 cycles;
  • the temperature and time for one cycle to stabilize the amplified product is 72 ° C, 5 minutes
  • O2 was amplified at 68 °C
  • O4 was amplified at 61 °C
  • O13 was amplified using 55-65 °C
  • O15 was amplified using 55-58 °C
  • O18 was amplified using 65 °C.
  • the work of primer screening after the basic PCR reaction was substantially completed, and the necessary length adjustment had little effect on the overall reaction conditions.
  • the primer sequences used in the present invention are all summarized in Table 1.
  • composition of the PCR kit :
  • Each kit can be used to detect 10 samples.
  • 10 ⁇ Buffer, dNTP and Taq polymerase were provided by Bao Bioengineering Co., Ltd.; the primer mixture was provided to Shanghai Yingjun Biotechnology Co., Ltd. for the self-designed sequence; positive control, negative control and ddH 2 O were prepared by our. .
  • the PCR detection method for detecting Providencia using the above PCR kit includes the following steps:
  • the environmental sample template in the above step (1) is a crude extract of a culture of tap water, river water, sea water, or the like, or a crude extract of pure culture of Providencia or pure DNA, or a positive control. Products and negative controls.
  • the extraction method of the environmental sample template in the above step (1) is:
  • reaction cycle parameters on the PCR instrument in the above step (3) include denaturation, renaturation, temperature and time of extension, and number of cycles, specifically:
  • One cycle of the pre-treatment process necessary for the denaturation to reach the desired temperature is 95 ° C, 5 minutes;
  • Denaturation temperature and time is 95 ° C, 45 seconds;
  • Refolding temperature and time is 55 ° C / 68 ° C, 1 minute;
  • the extension temperature and time are 72 ° C, 1 minute;
  • the number of cycles of denaturation, renaturation, and extension is 35 cycles;
  • the temperature and time for one cycle to stabilize the amplification product was 72 ° C for 5 minutes.
  • step (4) electrophoresis amplification product in the electrophoresis apparatus, and the specific steps of recording the result are:
  • the invention combines the components required for the PCR detection method by configuring a PCR kit capable of detecting the industrial production of Providencia, and extracts the sample to be tested while using the simple operation.
  • the sequence can be tested quickly, sensitively and conveniently.
  • the amount and concentration of each component in the kit are obtained by experiments.
  • the test equipment used to detect the provenance of the provence is simple and the detection cost is low.
  • the purpose of using positive and negative controls is to control the entire process to give an accurate judgment. If the type of O antigen of the origin of Provedenia is included, a band at the same position as the positive control can be observed from the electrophoresis results; if the O antigen type of Proudenium is not contained, it is the same as the negative control. Have this strip.
  • the amount of the reagent in the kit used in the primary detection test of the present invention is shown in Table 3 below, and the amount of the DNA template is 3 ⁇ l.
  • thermostable DNA polymerase in the present invention is a Taq enzyme.
  • the above positive control is a sample that has been determined to be each O antigen type of Providencia
  • the negative control is a sample that is not determined by the laboratory to be Notice.
  • the PCR kit is obtained by PCR amplification using a bacterial suspension of Providencia and the obtained DNA as a template. There is no difference in sensitivity and specificity, so that the extraction step of the template DNA can be omitted, and the operation method can be simplified. At the same time, compared with the conventional biochemical detection method, the sample to be tested used in the method can be directly used as a clinical sample culture solution, or can be detected by simply separating and culturing the test sample, thereby saving manpower and material resources.

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Abstract

本发明提供了对普罗威登斯菌031、041、042、043和050血清型特异的核苷酸及其应用，所述核苷酸包括1）SEQ ID NO：1-10所示的核苷酸中的至少一种；2）与SEQ ID NO：1-10所示的核苷酸互补的核苷酸中的至少一种。这些核苷酸可用于制备检测普罗威登斯菌用PCR试剂盒和基因芯片。

Description

对普罗威登斯菌O31，O41，O42，O43和O50特异的核苷酸及其应用 技术领域
本发明涉及对普罗威登斯菌O31，O41，O42，O43和O50血清型特异的核苷酸，尤其涉及对普罗威登斯菌O31，O41，O42，O43和O50血清型O抗原基因簇中单个基因特异的核苷酸及其应用。
背景技术
普罗威登斯菌(Providencia)是肠杆菌科中一种常见的革兰氏阴性菌，是一种机会致病菌。主要通过院内感染或者食用海鲜感染该菌，可以引起呼吸道感染，也可引发败血症、泌尿系感染、继发性脑膜炎等。分离于腹泻大便、尿道感染、伤口、烧伤和菌血症标本。
细菌分型与鉴定方法主要有传统的表型方法、血清学方法以及分子鉴定方法。然而，随着分子生物学的发展，传统的血清分型和鉴定方法存在着一定的问题，如血清分型这种诊断方法需要大量的抗血清，而抗血清一般种类不全，数量不足，大量的抗血清在制备和储存中也存在一些困难。另一方面血清分型方法耗时长、灵敏度低、漏检率高、准确性差，不同的O抗原产生的抗血清之间经常存在交叉反应。因此，建立基于分子生物学技术的血清鉴定方法成为发展方向。
普罗威登斯菌的分子鉴定越来越受到人们的重视，成为普罗威登斯菌菌种与株型鉴定的重要依据，不少新菌也因此产生。分子生物学检测技术具有速度快且易于大规模使用的优点，是目前国际上公认的致病菌检测的发展方向，但当前的难点在于DNA靶标分子特异性较低。细菌表面多糖抗原的多样性是由负责其合成的基因簇的遗传多样性导致的。长期以来，人们一直试图探索存在于普罗威登斯菌中的表面多糖抗原这一重要致病因子的多样性情况及相应的遗传进化基础。但该研究方向上总体进展非常缓慢，针对其多糖抗原多样性和变异规律方面的认识基本还是空白，例如：人们尚不了解普罗威登斯菌中存在多少种表面多糖抗原以及哪些表面多糖抗原对于该菌的致病性和流行性具有重要意义，负责其表面多糖抗原合成的基因簇尚未被定位(其它单位预测的部分表面多糖 抗原基因均不能与解析的多糖抗原化学结构很好对应)等。造成上述现象的主要原因是：多糖抗原合成基因簇组成复杂、基因功能较难预、糖合成基因鉴定困难等。此外，已进行部分相关研究的单位大多缺乏前期研究基础。
前期研究中，我们在深入分析细菌表面多糖抗原进化机理的基础上提出了表面抗原基因簇中的特定基因对于其编码的表面抗原具有极高的特异性理论，并以此为基础建立了从细菌表面抗原基因簇中筛选特异分子标识的技术。我们据此建立了当前国际上容量最大的致病微生物特异分子标识库(包括300余种不同细菌的特异分子标识)并建立了较为完善的细菌分子型检测系统。这些技术已被国内外包括美国农业部、美国疾病预防控制中心、法国食品安全署、荷兰食品与消费品安全机构等几十家单位广泛应用。
在本项目中，我们将在获得普罗威登斯菌全部表面多糖抗原基因簇序列信息的基础上，建立普罗威登斯菌分子血清学分型系统和快速检测方法，具有重要的科学意义和较强的应用价值。
近年来，越来越多的分子技术用于病原菌的分型、鉴定、检测及病害诊断，包括转录间隔区(ITS)序列分析、随机扩增长度多态性(RAPD)分析、核糖体DNA限制性片段长度多态性(RFLP)分析等。分子生物学方法不仅可用于普罗威登斯菌的快速血清分型筛查，稳定的鉴定结果可以弥补表型特征鉴定方法的不足。和传统检测技术相比，这些基于多聚酶链式反应(PCR)的分子检测技术，不需要经过病原菌的分离、纯培养等过程，而且具有快速、灵敏、特异性强等优点。
聚合酶链式反应技术(Polymerase chain reaction，简称PCR技术)作为微生物检测技术目前正在得到认同和推广，该技术相对于传统的方法有高通量、检测速度快、特异性强、灵敏度高等优点，只需对样品进行简单的预增菌或增菌过程，再通过离心及裂解制备细菌DNA模板，就可以在高特异性引物介导下的PCR过程中扩增目标序列，达到检测样品中是否含有待测的致病性微生物的目的。PCR的扩增过程仅需1个半小时。这对检验检疫部门和临床检验无疑极大提高了工作速度和降低了工作成本。不论从国内和国际的角度来看，快速、准确地鉴定出血清类型，为普罗威登斯菌的防控提供有效技术支持是十分重要的。
发明内容
本发明的目的在于提供了本发明涉及对普罗威登斯菌O31，O41，O42，O43和O50血 清型特异的核苷酸，其特征在于所述的核苷酸具有：
1)SEQ ID NO：1-10所示的核苷酸中的至少一种；
2)与SEQ ID NO：1-10所示的核苷酸互补的核苷酸中的至少一种(例如，具有可以与O31血清型特异引物所扩增出序列互补配对的核苷酸序列；
所述的SEQ ID NO：1-10如下：
Figure PCTCN2015000304-appb-000001
本发明还提供了一种PCR试剂盒，包括PCR引物、dNTP、缓冲液和DNA聚合酶，所述PCR引物为如SEQ ID NO：1-10所示的核苷酸中的至少一种。所述的试剂盒，还包括如下试剂：10mM dNTP 30μl；10×酶特异性反应缓冲液50μl；5U/μl耐热DNA聚合酶5μl；引物混合物10μl；阳性对照品10μl；阴性对照品10μl；ddH2O 5ml。其中所述的PCR引物优选为所述如SEQ ID NO：1-10所示的核苷酸中的至少一种。
本发明进一步公开了对普罗威登斯菌O31，O41，O42，O43和O50血清型特异的SEQ ID NO：1-10核苷酸在制备用于检测普罗威登斯菌普罗威登斯菌O31，O41，O42，O43和O50血清型的PCR试剂盒、基因芯片或微阵列方面的应用。本发明所述普罗威登斯菌可以取样于腹泻大便、尿道感染、伤口、烧伤和菌血症标本的培养物的粗提液，或是普罗威登斯菌的纯培养物的粗提液等。
收集普罗威登斯菌提取基因组是采用常规方法制备获得。
针对普罗威登斯菌的PCR试剂盒，整个检测步骤包括样品预处理-扩增-电泳检测结果。引物和PCR反应体系所需要的试剂已预先加入扩增管中，使用者只需将预处理后的样本加入扩增管启动扩增反应即可，简单快速的完成检测工作。
本发明还提供一种液相检测芯片，包括液相磁珠和连接在液相磁珠上的寡核苷酸探针，以及相应探针所在片段所对应的相应引物；其中所述核苷酸优选为如SEQ ID NO：1-10 所示的核苷酸。
本发明还提供一种微列阵，其包括上述的核苷酸；其中所述核苷酸优选为如SEQ ID NO：1-10所示的核苷酸。
本发明公开的对普罗威登斯菌O31，O41，O42，O43和O50血清型特异的核苷酸与现有技术相比，本发明具有如下优点：
(1)实用性强
本发明建立的一种PCR反应体系，可检测普罗威登斯菌，提供血清分型检测所用到的特异引物，利用该PCR方法可以对临床标本进行检测。
(2)准确性高
本发明通过对普罗威登斯菌的各血清型特异的基因的PCR反应，每个样品得到一条目的条带，将得到目的片段与已知长度相比较，就可以得到普罗威登斯菌所属的血清型。
(3)检测成本相对较低
可以推广应用于食品卫生监督、环境监测、尚品监测检验检疫等领域，并为其他不同致病菌检测组合提供技术模式。
以上所述，仅是本发明的操作和实施方法而已，并非对本发明作任何形式上的限制，凡是依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。
附图说明：
图1表示本发明O31血清型wzy基因P1和P2引物检测普罗威登斯菌其他血清型标准菌株电泳结果图，wzy基因P1和P2引物的筛选，目的条带为263bp，其余的血清型没有任何条带。具体菌株信息见表2
图2表示本发明O31血清型wzy基因P1和P2引物种特异性的鉴定电泳结果图，其中检测了6株弧菌以及1株沙门菌、1株大肠杆菌，均无任何条带。具体菌株信息见表2。
图3表示本发明O41血清型wzy基因P3和P4引物检测普罗威登斯菌其他血清型标准菌株电泳结果图，wzy基因P3和P4引物的筛选，目的条带为170bp，其余的血清型没有任何条带，具体菌株信息见表2；
图4表示本发明O41血清型wzy基因P3和P4引物种特异性的鉴定电泳结果图，其 中检测了6株弧菌以及1株沙门菌、1株大肠杆菌，均无任何条带，具体菌株信息见表2；
图5表示本发明O42血清型wzy基因P5和P6引物检测普罗威登斯菌其他血清型标准菌株电泳结果图，wzy基因P5和P6引物的筛选，目的条带为323bp，其余的血清型没有任何条带。具体菌株信息见表2
图6表示本发明O42血清型wzy基因P5和P6引物种特异性的鉴定电泳结果图，其中检测了6株弧菌以及1株沙门菌、1株大肠杆菌，均无任何条带。具体菌株信息见表2。
图7表示本发明O43血清型wzy基因P7和P8引物检测普罗威登斯菌其他血清型标准菌株电泳结果图，wzy基因P7和P8引物的筛选，目的条带为210bp，其余的血清型没有任何条带，具体菌株信息见表2；
图8表示本发明O43血清型wzy基因P7和P8引物种特异性的鉴定电泳结果图，其中用wzy基因P7和P8引物检测了6株弧菌以及1株沙门菌、1株大肠杆菌，均无任何条带，具体菌株信息见表2；
图9表示本发明O50血清型wzy基因P9和P10引物检测普罗威登斯菌其他血清型标准菌株电泳结果图，wzy基因P9和P10引物的筛选，目的条带为99bp，其余的血清型没有任何条带，具体菌株信息见表2；
图10表示本发明O50血清型wzy基因P9和P10引物种特异性的鉴定电泳结果图，其中用wzy基因P9和P10引物引物检测了6株弧菌以及1株沙门菌、1株大肠杆菌，均无任何条带，具体菌株信息见表2；
图11表示分别用O31，O41，O42，O43和O50特异引物扩增对应血清型的电泳结果图，具体菌株信息见表2；
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件。其中普罗威登斯菌来源于波兰罗兹大学。
实施例1：基因组的提取
37℃营养肉汤培养基培养普罗威登斯菌，收集细菌，提取基因组具体步骤如下：
用500ul 50mM Tris-HCl(pH8.0)和10ul 0.4M EDTA重悬细胞，37℃温育20分钟，然后加入10ul 10mg/ml的溶菌酶继续保温20分钟。之后加入3ul 20mg/ml的蛋白酶K、15ul 10％SDS，50℃温育2小时，再加入3ul 10mg/ml的RNase，65℃温育30分钟。加等体积酚抽提混合物，取上清液，再用等体积的酚∶氯仿∶异戊醇(25∶24∶1)溶液抽提两次，取上清液，再用等体积的乙醚抽提以除去残余的酚。上清液用2倍体积乙醇沉淀DNA，用玻璃丝卷出DNA并用70％乙醇洗DNA，最后将DNA重悬于30ul TE中。基因组DNA通过0.4％的琼脂糖凝胶电泳检测。
实施例2：序列破译
提取普罗威登斯菌O31，O41，O42，O43和O50血清型标准菌株的基因组，通过Solexa pair-end测序技术对普罗威登斯菌各个血清型基因组进行全基因组测序获得该血清型的序列，运用Blast及PSI-Blast进行序列比对，采用TMHMM 2.0program进行跨膜结构预测，运用ClustalW program进行序列对齐及筛选保守和特定基因片段，最终获得普罗威登斯菌各个血清型的O抗原基因簇序列及破译结果。
实施例3：引物设计
根据基因簇破译情况，我们发现wzy和wzx基因确实是血清型特异的基因，所以选取该基因特异区段设计特异引物。由于wzy更加特异，所以主要以wzy基因为靶基因。
引物设计是该发明的核心部分。设计引物根据文献中叙述的特异基因来设计。wzx和wzy这两个基因是普罗威登斯菌O抗原基因簇中比较特异的基因，可以作为血清型鉴定的靶基因。将上述基因导入Primer Premier 5进行引物设计，引物的长度最好在18～24bp之间，Tm值在50～55℃。每个基因设计一对引物，有单一目的条带。
引物设计之后在Genbank中进行BLAST，设计的引物不能与其它近缘细菌的序列相似性过高，这样就能确保该引物只在自己的预定位置进行扩增，而不与其它的近缘菌或者采集标本的环境中的近缘菌不产生阳性反应。这一点对于避免非特异条带的产生和实验的成败十分重要。
设计出的引物如表1所示。
表1用于PCR的引物序列
Figure PCTCN2015000304-appb-000002
实施例4：特异引物的筛选
收集了普罗威登斯菌O31，O41，O42，O43和O50血清型的标准菌株及其他13种血清型的标准菌株各一株，10株未分型的普罗威登斯菌，6株弧菌属菌株，1株沙门菌属菌株以及1株大肠杆菌菌株验证引物的特异性，菌株编号和来源见表2.
表2用于特异性检测的菌株
Figure PCTCN2015000304-appb-000003
Figure PCTCN2015000304-appb-000004
基因鉴定引物筛选用到的PCR体系为5μM引物0.4μl、10×酶特异性反应缓冲液2.5μl、10mM dNTP 0.25μl、5U/μl耐热DNA聚合酶0.2μl及3μl的待测样品模板到0.2ml的薄壁PCR管中，最后ddH2O补足到25μl。所有引物都在各自所对应的血清型中得到阳性结果，在其他组中没有得到任何PCR产物带。
这个步骤中的PCR仪上的反应循环参数包括DNA的变性、复性、延伸的温度和时间、循环次数，具体为：
前期为使变性能够达到所需的温度而必需的前期处理过程的一个循环为95℃，5分钟；变性温度和时间为95℃，45秒；
复性温度和时间为55℃/68℃，1分钟；
延伸温度和时间为72℃，1分钟；
变性、复性、延伸的循环次数为35个循环；
为稳定扩增产物而进行一个循环的温度和时间为72℃，5分钟
其中，O2使用68℃扩增，O4使用61℃扩增，O13使用55-65℃扩增，O15使用55-58℃扩增均可，O18使用65℃扩增。
上述步骤在电泳设备中电泳扩增产物，记录结果的具体步骤为：
①取2～5μl扩增产物与6×溴酚蓝上样缓冲液以1∶1的体积比混合；
②将混合液上样于1.0％的琼脂糖凝胶上；
③将琼脂糖凝胶电泳120v稳压电泳约20分钟，用DL2000Marker进行对照；
④观察并记录结果。
通过基本PCR反应之后引物筛选的工作基本结束，必要的长度调整对整体反应条件影响不大，本发明中用到的引物序列全部总结在表1中。
表1用于PCR的引物序列
Figure PCTCN2015000304-appb-000005
实施例6：PCR检测试剂盒的制备及应用
1、PCR试剂盒的组成：
Figure PCTCN2015000304-appb-000006
Figure PCTCN2015000304-appb-000007
每个试剂盒可用于检测10个样品。
其中10×Buffer、dNTP、Taq聚合酶由宝生物工程有限公司提供；引物混合物为自行设计的序列提供给上海英骏生物技术公司合成；阳性对照品、阴性对照品和ddH2O由我们自行制备。
2、仪器设备
其中10×Buffer、dNTP、Taq聚合酶由上海生工提供；引物混合物为自行设计的序列提供给上海英骏生物技术公司合成；阳性对照品、阴性对照品和ddH2O由我们自行制备。实验的设备PCR仪(又名DNA热循环扩增仪)、电泳设备(包括电泳仪及电泳槽)、凝胶成像仪、-20℃冰箱、高速离心机、微量移液器和0.2ml PCR薄壁管。
3、PCR试剂盒的使用具体实例
使用上述的PCR试剂盒检测普罗威登斯菌的PCR检测方法包括如下步骤：
(1)提取待测环境样品模板；
(2)在PCR薄壁管中加入、dNTP、10×Buffer、Taq聚合酶、引物、待测样品模板和ddH2O混匀；
(3)将薄壁PCR管中混匀的混合物在PCR仪上扩增；
(4)在电泳设备中电泳扩增产物，记录结果；
(5)分析并进行结果判断。
上述步骤(1)中的环境样品模板为自来水、河流水、海水等的培养物的粗提液，或是普罗威登斯菌的纯培养物的粗提液或是纯DNA，或是阳性对照品和阴性对照品。
上述步骤(1)中的环境样品模板的提取方法为：
①取1.5ml培养物，在12000rpm条件下离心1分钟，去掉上清液；
②取500μl的ddH2O重悬沉淀，在8000rpm条件下离心5分钟，去掉上清液，烘干；
③取100μl ddH2O重悬沉淀，在100℃沸水中水浴10分钟；
④再置于冰上10分钟后，在12000rpm条件下离心2分钟；
⑤取3μl中层上清作为PCR模板
上述步骤(3)中的PCR仪上的反应循环参数包括DNA的变性、复性、延伸的温度和时间、循环次数，具体为：
前期为使变性能够达到所需的温度而必需的前期处理过程的一个循环为95℃，5分钟；
变性温度和时间为95℃，45秒；
复性温度和时间为55℃/68℃，1分钟；
延伸温度和时间为72℃，1分钟；
变性、复性、延伸的循环次数为35个循环；
为稳定扩增产物而进行一个循环的温度和时间为72℃，5分钟。
上述步骤(4)在电泳设备中电泳扩增产物，记录结果的具体步骤为：
①取2～5μl扩增产物与6×溴酚蓝上样缓冲液以5∶1的体积比混合；
②将混合液上样于1.0％的琼脂糖凝胶上；
③将琼脂糖凝胶电泳120v稳压电泳约20分钟，用DL2000Marker进行对照；
④观察并记录结果。
本发明通过配置一种可检测普罗威登斯菌的可产业化生产的PCR试剂盒，将PCR检测方法需要使用的组分组合在一起，使用时，提取待测样品，同时经过较为简单的操作层序就可以进行快速、灵敏、简便的检测，试剂盒中各组分的用量和浓度均为试验所得，用该试剂盒检测普罗威登斯菌所使用的试验设备简单，检测成本低。
使用阳性和阴性对照品的目的是用于质控整个操作过程，以便得出准确的判断。若含有普罗威登斯菌目的O抗原类型，则从电泳结果中可以观察到与阳性对照品相同位置的条带；若不含有普罗威登斯菌目的O抗原类型，则与阴性对照品一样不具有这一条带。本发明一次检测试验所使用的试剂盒中的试剂量见下表3所示，DNA模板量为3μl
表3一次检测试验所使用的试剂盒中的试剂量
成分 浓度 加样量(μl)
ddH2O   18.65
10×PCR缓冲液 10× 2.5
10mM dNTP 10mM 0.25
引物混合物 5mM 0.4
Taq酶 5U/μl 0.2
总体积   25
本发明中的耐热DNA聚合酶为Taq酶。
上述的阳性对照品为已确定是普罗威登斯菌各个O抗原类型的样品，阴性对照品则为经实验室确定不是普罗威登斯菌的样品。
本PCR试剂盒若用普罗威登斯菌的菌悬液进行PCR扩增，与经提取得到的DNA作为模板所得结果一直。敏感度和特异性无差别，这样，可省去模板DNA的提取步骤，使操作方法得以简化。同时，相比常规生化检测方法而言，本方法所采用的待测样品可以直接是临床样品培养液，或者对检测样品进行简单分离培养就可以进行检测，因而节省了人力物力。
4、待测样品的提供
收集了普罗威登斯菌O31，O41，O42，O43和O50血清型标准菌株，6株弧菌属其他菌株，1株沙门菌属菌株以及1株大肠杆菌菌株验证引物的特异性，菌株编号和来源见表2。

Claims (8)

  1. 对普罗威登斯菌O31，O41，O42，O43和O50血清型特异的核苷酸，其特征在于所述的核苷酸具有：
    1)SEQ ID NO：1-10所示的核苷酸中的至少一种；
    2)与SEQ ID NO：1-10所示的核苷酸互补的核苷酸中的至少一种。
  2. 一种PCR试剂盒，包括PCR引物、dNTP、缓冲液和DNA聚合酶，所述PCR引物为如SEQ ID NO：1-10所示的核苷酸中的至少一种。
  3. 权利要求2所述的试剂盒，还包括如下试剂：10mM dNTP 30μl；10×酶特异性反应缓冲液50μl；5U/μl耐热DNA聚合酶5μl；引物混合物10μl；阳性对照品10μl；阴性对照品10μl；ddH2O 1ml。
  4. 权利要求1所述的SEQ ID NO：1-10特异核苷酸在制备用于检测普罗威登斯菌PCR试剂盒方面的应用。
  5. 权利要求1所述的SEQ ID NO：1-10特异核苷酸在制备用于检测普罗威登斯菌的基因芯片方面的应用。
  6. 权利要求1所述的SEQ ID NO：1-10特异核苷酸在制备用于检测普罗威登斯菌微阵列方面的应用。
  7. 权利要求4-6所述的应用，其中所述的检测普罗威登斯菌指的是检测引起呼吸道感染、败血症、泌尿系感染、继发性脑膜炎的细菌。
  8. 权利要求4-6所述的应用，其中所述的普罗威登斯菌指的是取样于腹泻大便、尿道感染、伤口、烧伤和菌血症标本培养物的粗提液或是普罗威登斯菌的纯培养物的粗提液。
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