CN104419703A - Method for quickly detecting common pathogenic bacteria with high flux - Google Patents

Abstract

The invention provides a method for quickly detecting common pathogenic bacteria with high flux. The method comprises: performing PCR (polymerase chain reaction) amplification on a sample by adopting a universal primer pair specific to multiple pathogenic bacteria iconic DNA sequences, to obtain an iconic DNA sequence of the pathogenic bacteria; making the iconic DNA sequence of the pathogenic bacteria in contact with specific capture probes specific to the pathogenic bacteria iconic DNA sequences respectively to obtain a cross product with different detectable markers; and detecting signals of the detectable markers to determine the presence or quantity of the pathogenic bacteria in the sample. The invention also provides a kit for detecting the pathogenic bacteria. The method and the kit can be used for accurately and quickly detecting the pathogenic bacteria with high flux and have the advantages of easiness in operation, strong specificity, high sensitivity and the like, so the method and the kit have a wide application prospect.

Description

A kind of method of high-throughput rapid detection common pathogen

Technical field

The present invention relates to clinical detection, food inspection, public health and bacteriology field.Specifically, the present invention relates to the novel method of a kind of high-throughput, rapid detection pathogenic bacterium (preferred clinical common pathogenic bacteria).

Background technology

Bacterial infection disease (such as urinary system infection, as urinary tract infections) serious threat human health and sickness rate improve day by day.Common pathogenic bacteria can cause various diseases, such as: the diarrhoea, the septicemia that are caused by intestinal bacteria (E.coli); The suppurative infection, food poisoning, the pneumonia that are caused by streptococcus aureus (Staphyloccocus aureus); The upper respiratory tract caused by Klebsiella Pneumoniae (K.peneumoniae), intestinal tract infections; The urinary tract infections caused by enterococcus faecalis (E.faecalis), endocarditis, cholecystitis, meningitis and wound infection etc.; The nosocomial infection caused by enterobacter cloacae (Enterobacter cloacae), urinary tract infection, respiratory tract infection and septicemia etc.; The nosocomial infection, postoperative wound infection, suppurative otitis media etc. that are caused by Pseudomonas aeruginosa (P.Aeruginosa); The urinary system infection caused by proteus vulgaris (P.vulgaris), traumatic infection, respiratory tract infection, food poisoning and septicemia etc.; The pulmonary infection caused by Serratia marcescans (Serratiamarcescens), meningitis, endocarditis, urinary tract infections, burn after septicemia etc.

Directed toward bacteria infectious diseases uses Broad spectrum antibiotics in a large number, although can suppress most of pathogenic bacterium, Resistant strain and dissociant can be caused constantly to occur, life-time service also can cause multiple side effect, increases the complicacy of patient burden and clinical treatment.Further, some disease may be caused jointly by various pathogens, and the cause of disease is complicated.Therefore, detect specific pathogenic bacterium and suppress measured pathogenic bacterium targetedly, being conducive to rational therapy bacterial infection disease.

But, there is length consuming time, detection sensitivity by shortcomings such as culture condition restrictions in the traditional pathogenic microbes detect method based on cultivating, more and more can not meet clinical needs, thus exploitation method that is quick, responsive, that detect pathogenic bacteria is exactly the target that people pursue always.The biochip technology of development in recent years has the features such as high-throughput, integrated, microminiaturized and automatization, be used widely in the detection field such as gene locus difference and analyzing gene expression level, and in the context of detection of clinical bacteria and resistance, then the reasonable employment that can be clinical antibiotics provides scientific basis.

At present, the clinical and upper Bacteria Identification main method of food test quarantine has: traditional bacteria distribution culture & identification method, PCR, Southern Blot hybridization (i.e. southern blotting technique method), ELISA equimolecular biology and immunological method.But traditional detection method complex operation, time and effort consuming, generally need 3-5 days, although ELISA method is highly sensitive, there is false positive in vulnerable to pollution; Adopt multiple PCR technique, although quick, the accurately and easily diagnostic purpose to single bacterium can be reached, but when pathogenic bacteria is not bright, need to adopt multiple different primers to test, this is dissatisfactory concerning the diversity of the complicacy of pathogenic bacteria and PCR program.

Along with reaching its maturity of biochip technology, efficient, quick, accurate, sensitive biochip technology, is applied to increasing field, wherein just comprises clinical bacteria quick diagnosis field.Liquid-phase chip technology is the molecular diagnostic techniques platform of new generation recently developed, this platform can guarantee information quality, relatively high-throughout detection can be provided again, its high-throughput, high precision and short feature consuming time are that the detection of clinical common bacteria provides thinking, effectively can prevent the abuse of antibiotic medicine.

Current state-of-the-art technology is Tem-PCR (the target enrichedmultiplex PCR of scientist doctor Han Jian invention, target sequence enrichment multiplex PCR) R. concomitans of technology and liquid-phase chip detection technique, this technology can carry out extremely sensitive and special amplification and detection to multiple target sequence in primary first-order equation, its cardinal principle is, for each target sequence to be amplified, the Auele Specific Primer of design two pairs of nidos: F0, R0; Fi, Ri.The sequence label that Fi and Ri primer is wherein identified by general super primer (Super Primer) with an energy.The concentration of the target sequence Auele Specific Primer of nido is extremely low, for enrich target sequence in the first few circulation of PCR.Only have the concentration of super primer to be enough to carry out exponential amplification, and only have reverse super primer to carry out the mark of vitamin H.Finally by liquid-phase chip detection platform output signal, reach testing goal.This technology have compared with regular-PCR technology compatible good, high specificity, efficiency superelevation, can the advantage such as sxemiquantitative, but this technology exists and requires the shortcomings such as high, PCR long reaction time, susceptibility are strong not to required DNA profiling.

Bacterium rRNA is divided into 3 kinds by settling ratio, is respectively 5S, 16S and 23S rRNA.16S rDNA is the corresponding DNA sequence dna of 16S rRNA that bacterial chromosome is encoded, and be present in all bacterial chromosomal genes, its internal structure comprises conserved regions and variable region.16S rDNA is the gene of coding prokaryotic organism small subunit ribosome rRNA (16S rRNA), and its length is about 1500pb, is in systematic bacteriology research the most frequently used, the most useful " molecular clock ".

In ribosome-RNA(rRNA) (rRNA) characteristic sequence, think after deliberation 16S rRNA and similar rRNA gene order the most suitable as biosystem development index, its Main Basis is: they for cell common; Its function homology and the most ancient; Not only containing conserved sequence but also containing variable sequence; Molecular size is applicable to operation; Its sequence variation and evolutionary distance adapt.The life concern tree of drawing according to 16S rRNA sequence, tellurian all cells biology is divided into 3 territories (domain) by Ka Er Butterworth (Carl Woese), i.e. Archimycetes (Archaea, also claim Archaebacteria), bacterium (Bacteria also claims Eubacteria) and eukaryote (Eukarya).16S rRNA sequential analysis have also been obtained extensive approval as the Main Basis of microorganism classification system, day by day perfect along with microorganism rrna database, and this technology becomes a powerful of division bacteria and qualification.

About the database information of 16S rDNA and analysis software much abundant compared with the gene of other taxonomic identification, as RDP (http://rdp.cme.msu.edu/html/), ARB (http://www.ard-home.de/), ORS (http://soul.mikro.biologie.tu-muenchen.de/ORS/), OPD (http://www.com.msu.eduOPD/) and PRIMROSE, CLUSTAL-X, PRIMER PREMIER, DNA START etc., each analysis software has his own strong points because of the algorithm difference adopted.

But, the method simultaneously detecting various pathogens in sample is not yet provided in this area.Still in the urgent need to developing the deficiency that can overcome in current pathogenic microbes detect in this area, and the method for energy high-throughput, rapid detection various pathogens.

Summary of the invention

Main purpose of the present invention is a kind of method providing high-throughput, rapid detection common pathogen, and the method has the advantages such as high specificity, susceptibility is high, detection time is short, cross reaction is few.Another object of the present invention is the test kit being provided for the inventive method, and the application in clinical common bacteria detects of method of the present invention and test kit.

In a first aspect of the present invention, provide a kind of method obtaining the significant DNA sequence dna of pathogenic bacterium in sample, described pathogenic bacterium be selected from lower group of bacterium one or more: intestinal bacteria (E.coli), Klebsiella Pneumoniae (K.peneumoniae, i.e. KPN), enterococcus faecalis (E.faecalis, i.e. EF), enterobacter cloacae (Enterobacter cloacae, i.e. ECL), Pseudomonas aeruginosa (P.Aeruginosa, i.e. PAE), proteus vulgaris (P.vulgaris, i.e. PV), Serratia marcescans (Serratia marcescens, i.e. SM), streptococcus aureus (Staphyloccocus aureus, i.e. SA), described method comprises:

I () obtains for the general upstream primer of sequence A CGCGAAGAA CCTTACC shown in SEQ ID NO:10 and the general downstream primer for sequence TGTTGGGTTAAGTCCCGCAACGA shown in sequence TGTTGGGTTAAGTCCCGCAACGAG shown in SEQ ID NO:11 or SEQ ID NO:12;

(ii) described general upstream primer and general downstream primer is adopted to carry out pcr amplification to described sample, thus obtain the significant DNA of described pathogenic bacterium, wherein, described significant DNA sequence dna is arranged in the 16S rDNA of described pathogenic bacterium, and comprises one or more pathogenic bacterium described specific sequence exclusive separately.

In some preferences, what obtained by above-described PCR amplification method is the significant DNA sequence dna of each bacterium shown in SEQ ID NO:21-28 respectively.

In some preferences, the sequence of described general downstream primer is for sequence TGTTGGGTTAAGTCCCGCAACGA shown in SEQ ID NO:12.

In some preferences, the sequence of described general upstream primer is as shown in SEQ ID NO:1 or SEQ ID NO:3; The sequence of described general downstream primer is as shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

In other preferences, described pathogenic bacterium are be selected from least two kinds in group bacterium.

In some embodiments, described general upstream primer is 15 ~ 17 on all four sequences of continuous nucleotide sequence with sequence shown in SEQ ID NO:10; Described general downstream primer is and SEQ ID NO:11 or or the sequence of 15 ~ 23 continuous nucleotide sequence complete complementaries of SEQ ID NO:12.

In some preferences, described general downstream primer for sequence TGTTGGGTTAAGTCCCGCAACGA shown in SEQ ID NO:12 or the sequence of 15 ~ 23 continuous nucleotide sequence complete complementaries.

In other embodiments, described general upstream primer is the sequence of 15 ~ 17 continuous nucleotide sequence complete complementaries with sequence shown in SEQ ID NO:10; Described general downstream primer is 15 ~ 23 on all four sequences of continuous nucleotide sequence with SEQ ID NO:11 or SEQ ID NO:12.

In some preferences, described general downstream primer is 15 ~ 23 on all four sequences of continuous nucleotide sequence with sequence TGTTGGGTTAAGTCCCGCAACGA shown in SEQ ID NO:12.

In some preferences, the significant DNA sequence dna of described pathogenic bacterium is as follows respectively: colibacillary significant DNA sequence dna a) shown in SEQ ID NO:21; B) the significant DNA sequence dna of the Klebsiella Pneumoniae shown in SEQ ID NO:22; C) the significant DNA sequence dna of the enterococcus faecalis shown in SEQ ID NO:23; D) the significant DNA sequence dna of the enterobacter cloacae shown in SEQ ID NO:24; E) the significant DNA sequence dna of the Pseudomonas aeruginosa shown in SEQ ID NO:25; F) the significant DNA sequence dna of the proteus vulgaris shown in SEQ ID NO:26; G) the significant DNA sequence dna of the Serratia marcescans shown in SEQ ID NO:27; H) the significant DNA sequence dna of the streptococcus aureus shown in SEQID NO:28.

In a second aspect of the present invention, provide a kind of method detecting pathogenic bacterium in sample, described pathogenic bacterium be selected from lower group one or more: intestinal bacteria (E.coli), Klebsiella Pneumoniae (K.peneumoniae, i.e. KPN), enterococcus faecalis (E.faecalis, i.e. EF), enterobacter cloacae (Enterobactercloacae, i.e. ECL), Pseudomonas aeruginosa (P.Aeruginosa, i.e. PAE), proteus vulgaris (P.vulgaris, i.e. PV), Serratia marcescans (Serratia marcescens, i.e. SM), streptococcus aureus (Staphyloccocus aureus, i.e. SA), described method comprises:

(A) method of the present invention is adopted to obtain the significant DNA sequence dna of pathogenic bacterium described in sample;

(B) provide respectively for the corresponding specificity capture probe of the significant DNA sequence dna of described pathogenic bacterium;

(C) under hybridization conditions, the significant DNA sequence dna of described pathogenic bacterium is contacted with described specificity capture probe, and before described contact, when or afterwards with the mixture that significant DNA sequence dna and the described specificity capture probe of specificity capture probe described in different detectable label substance markers or described pathogenic bacterium are formed, obtain the hybrid product with different detectable;

(D) signal of the detectable be connected on described hybrid product is detected, to determine existence or the quantity of various pathogenic bacterium to be measured in sample.

In other preferences, described method is used for detecting in sample at least two kinds that are selected from group bacterium simultaneously.

In some embodiments, described method is used for clinical sample detection, food test, food processing process detects or makeup detect, and is preferred for clinical sample and detects.

In further embodiments, described detectable is selected from: coding microball or other carrier (as fluorescent marker, luminescent protein etc.).

In further embodiments, after described method is also included in step (A), be separated and/or purifying described in described pathogenic bacterium significant DNA sequence dna and/or the significant DNA sequence dna of described pathogenic bacterium is connected with detectable.In certain embodiments, described detectable is vitamin H, antibiotin, antibody, labeled cell or luminescent protein.

In some embodiments, described sample source is in biological tissue, body fluid, blood, urine or food.

In further embodiments, described sample is DNA sample or bacterium liquid.

In further embodiments, can comprise in described bacterium group in pathogenic bacterium in described sample one or more.

In other embodiments, it is characterized in that, described specificity capture probe is selected from:

A) for the specificity capture probe of the significant DNA sequence dna of intestinal bacteria shown in SEQ ID NO:21; B) for the specificity capture probe of the significant DNA sequence dna of Klebsiella Pneumoniae shown in SEQ ID NO:22; C) for the specificity capture probe of the significant DNA sequence dna of enterococcus faecalis shown in SEQ ID NO:23; D) for the specificity capture probe of the significant DNA sequence dna of enterobacter cloacae shown in SEQ ID NO:24; E) for the specificity capture probe of the significant DNA sequence dna of Pseudomonas aeruginosa shown in SEQ ID NO:25; F) for the specificity capture probe of the significant DNA sequence dna of proteus vulgaris shown in SEQ ID NO:26; G) for the specificity capture probe of the significant DNA sequence dna of Serratia marcescans shown in SEQ ID NO:27; And h) for the specificity capture probe of the streptococcus aureus shown in SEQ ID NO:28.

In other preferences, the G/C of described specificity capture probe is 45 ~ 60%, preferably 50 ~ 55%.

In further embodiments, the length of described specificity capture probe is 20-25bp, more preferably 22bp.

In further embodiments, the Tm value of described specificity capture probe is 40-60 DEG C, preferably 52 DEG C.

In further embodiments, the 5' of described specificity capture probe end will be modified or closed so that with coding microball coupling.

In further embodiments, described specificity capture probe is selected from lower group:

A) the colibacillary specificity capture probe shown in SEQ ID NO:13;

B) the specificity capture probe of the Klebsiella Pneumoniae shown in SEQ ID NO:14;

C) the specificity capture probe of the enterococcus faecalis shown in SEQ ID NO:15;

D) the specificity capture probe of the enterobacter cloacae shown in SEQ ID NO:16;

E) the specificity capture probe of the Pseudomonas aeruginosa shown in SEQ ID NO:17;

F) the specificity capture probe of the proteus vulgaris shown in SEQ ID NO:18;

H) the specificity capture probe of the Serratia marcescans shown in SEQ ID NO:19; With

I) the specificity capture probe of the streptococcus aureus shown in SEQ ID NO:20.

In other embodiments, the described detectable in step (C) is coding microball, and described coding microball adopts the marker coding that can produce different signal.

In certain embodiments, described marker is selected from: the combination of the marker of different colours or its different ratios.In further embodiments, described marker is selected from: the fluorescent marker of different colours, or the combination of two or more fluorescent marker of different ratios.

In certain embodiments, described coding microball and capture probe coupling.In certain embodiments, described coupling is undertaken by EDC method.In further embodiments, described coupling is undertaken by NHS coupling agent.In further embodiments, described microballoon is polystyrene microsphere, and according to its surface with the difference of molecule residues, can in conjunction with different probe molecules.

In other embodiments, the described detection in step (D) adopts one or more systems or the instrument that are selected from lower group: liquid-phase chip detection system, flow cytometer or solid phase chip.

In a third aspect of the present invention, provide a kind of test kit for detecting pathogenic bacterium and/or treatment pathogenic infection, described pathogenic bacterium be selected from lower group one or more: intestinal bacteria (E.coli), Klebsiella Pneumoniae (K.peneumoniae, i.e. KPN), enterococcus faecalis (E.faecalis, i.e. EF), enterobacter cloacae (Enterobacter cloacae, i.e. ECL), Pseudomonas aeruginosa (P.Aeruginosa, i.e. PAE), proteus vulgaris (P.vulgaris, i.e. PV), Serratia marcescans (Serratia marcescens, i.e. SM), streptococcus aureus (Staphyloccocus aureus, i.e. SA), described test kit comprises:

(i') for sequence A CGCGAAGAA CCTTACC shown in SEQ ID NO:10 general upstream primer and for sequence TGTTGGGTTAAGTCCCGCAACGAG shown in SEQ ID NO:11 or or SEQ ID NO:12 shown in the general downstream primer of sequence TGTTGGGTTAAGTCCCGCAACGA, described general upstream primer and general downstream primer is wherein adopted to carry out pcr amplification to described sample, the significant DNA of described pathogenic bacterium can be obtained, wherein, described significant DNA sequence dna is arranged in the 16SrDNA of described pathogenic bacterium, and comprise one or more pathogenic bacterium described specific sequence exclusive separately,

(ii') for the connector of the corresponding specificity capture probe of the significant DNA sequence dna of described pathogenic bacterium, various detectable or each species specificity capture probe and detectable;

(iii') one or more container and/or packing material;

(iv') optional, pcr amplification reagent;

(v') optional, for connecting the reagent of described specificity capture probe and detectable;

(vi') optional, the reagent of detection signal and/or instrument;

(vii') optional, working instructions;

(viii') optional, pathogenic bacterium therapeutical agent.

In some preferences, described test kit is used for detecting in sample at least two kinds of bacterium being selected from group simultaneously.

In a fourth aspect of the present invention, provide the purposes that following substances detects pathogenic bacterium in preparation and/or treats in the test kit of pathogenic infection:

(i) for sequence A CGCGAAGAA CCTTACC shown in SEQ ID NO:10 general upstream primer and for sequence TGTTGGGTTAAGTCCCGCAACGAG shown in SEQ ID NO:11 or or SEQ ID NO:12 shown in the general downstream primer of sequence TGTTGGGTTAAGTCCCGCAACGA, described general upstream primer and general downstream primer is wherein adopted to carry out pcr amplification to described sample, the significant DNA of described pathogenic bacterium can be obtained, wherein, described significant DNA sequence dna is arranged in the 16SrDNA of described pathogenic bacterium, and comprise one or more pathogenic bacterium described specific sequence exclusive separately,

(ii) for the connector of the corresponding specificity capture probe of the significant DNA sequence dna of described pathogenic bacterium, various detectable or each species specificity capture probe and detectable;

(iii) one or more container and/or packing material;

(iv) optional, pcr amplification reagent;

V () is optional, for connecting the reagent of described specificity capture probe and detectable;

(vi) optional, the reagent of detection signal and/or instrument;

(vii) optional, working instructions;

(viii) optional, pathogenic bacterium therapeutical agent.

In certain embodiments, described detectable is microballoon.

In other side of the present invention, additionally provide aforesaid method of the present invention and the purposes of test kit in detection pathogenic bacterium and/or treatment pathogenic infection.

Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.

Accompanying drawing explanation

Below in conjunction with accompanying drawing, the invention will be further described, and wherein these displays are only in order to illustrate embodiment of the present invention, instead of in order to limit to scope of the present invention.

Fig. 1: for the universal primer of 16S rDNA gene and the design diagram of specificity capture probe of bacterium A-bacterium F.

Fig. 2: adopt universal primer to the gel electrophoresis figure of (SEQ ID NO:1, SEQ ID NO:2) pcr amplification different strains.

Fig. 3: adopt universal primer to the gel electrophoresis figure of (SEQ ID NO:3, SEQ ID NO:4) pcr amplification different strains.

Fig. 4: adopt universal primer to the gel electrophoresis figure of (SEQ ID NO:3, SEQ ID NO:5) pcr amplification different strains.

Fig. 5: adopt universal primer to the gel electrophoresis figure of (SEQ ID NO:3, SEQ ID NO:6) pcr amplification different strains.

Fig. 6: adopt universal primer to the gel electrophoresis figure of (SEQ ID NO:3, SEQ ID NO:7) pcr amplification different strains.

Fig. 7: adopt universal primer to the gel electrophoresis figure of (SEQ ID NO:8, SEQ ID NO:9) pcr amplification different strains.

Embodiment

The present invention is mainly in order to overcome the deficiency in current pathogenic bacterium (especially clinical common pathogenic bacteria) detection, a kind of method of high-throughput rapid detection pathogenic bacterium is provided, adopt a pair universal primer and the design of specificity capture probe, realize by liquid-phase chip the method that signal exports, can simultaneously, rapid detection various pathogens.The method is efficient, sensitive, high specificity, cross reaction are few, and overcoming the PCR existed in multiplex PCR and fluorescence detecting system, to react design of primers loaded down with trivial details and produce the shortcomings such as cross reaction.

The novel pathogenic bacterium Fast Detection Technique being core with 16S rDNA round pcr of the present inventor's exploitation, only a pair universal primer need be adopted just can to increase to the 16S rRNA characteristic information of multiple different bacterium, the PCR primer of the 16S rDNA information for comprising multiplicity of acquisition.The PCR method of this routine, can complete amplified reaction within one hour.The PCR primer obtained, adopts the coding microball with Auele Specific Primer coupling, with liquid-phase chip detection system (as Luminex200), can realize multiplicity and high-throughout detection target.

obtain the method for the significant DNA sequence dna of pathogenic bacterium

A kind of method obtaining the significant DNA sequence dna of pathogenic bacterium in sample is provided in the present invention, described pathogenic bacterium be selected from lower group one or more: intestinal bacteria (E.coli), Klebsiella Pneumoniae (K.peneumoniae, i.e. KPN), enterococcus faecalis (E.faecalis, i.e. EF), enterobacter cloacae (Enterobactercloacae, i.e. ECL), Pseudomonas aeruginosa (P.Aeruginosa, i.e. PAE), proteus vulgaris (P.vulgaris, i.e. PV), Serratia marcescans (Serratia marcescens, i.e. SM), streptococcus aureus (Staphyloccocus aureus, i.e. SA), described method comprises:

I () obtains for the general upstream primer of sequence A CGCGAAGAACCTTACC shown in SEQ ID NO:10 and the general downstream primer for sequence TGTTGGGTTAAGTCCCGCAACGA shown in sequence TGTTGGGTTAAGTCCCGCAACGAG shown in SEQ ID NO:11 or SEQ ID NO:12;

(ii) described general upstream primer and general downstream primer is adopted to carry out pcr amplification to described sample, thus obtain the significant DNA of described pathogenic bacterium, wherein, described significant DNA sequence dna is arranged in the 16S rDNA of described pathogenic bacterium, and comprises one or more pathogenic bacterium described specific sequence exclusive separately.

As used herein, term " significant DNA sequence dna " refers to and is arranged in various pathogenic bacterium 16S rDNA and the particular sequence that can be obtained amplification by universal primer of the present invention, the two ends of described sequence comprise the total conserved sequence of each bacterium, and have the exclusive specific sequence of various pathogenic bacterium at this interior sequences.In certain embodiments of the present invention, the significant DNA sequence dna of described pathogenic bacterium is as follows respectively: colibacillary significant DNA sequence dna a) shown in SEQ ID NO:21; B) the significant DNA sequence dna of the Klebsiella Pneumoniae shown in SEQ ID NO:22; C) the significant DNA sequence dna of the enterococcus faecalis shown in SEQ ID NO:23; D) the significant DNA sequence dna of the enterobacter cloacae shown in SEQ IDNO:24; E) the significant DNA sequence dna of the Pseudomonas aeruginosa shown in SEQ ID NO:25; F) the significant DNA sequence dna of the proteus vulgaris shown in SEQ ID NO:26; G) the significant DNA sequence dna of the Serratia marcescans shown in SEQ ID NO:27; And the significant DNA sequence dna of streptococcus aureus h) shown in SEQ ID NO:28.

As used herein, term " specific sequence " or " variable region " are used interchangeably, and all refer to and are positioned at one section of exclusive region of various pathogenic bacterium 16S rDNA gene internal to be measured, various pathogenic bacterium to be measured.Existence and the particular location of this specific sequence can be determined by the 16S rDNA sequence of the various pathogenic bacterium to be measured of analysis and comparison.Usually, these specific sequence two ends have the total conserved sequence of various pathogenic bacterium.Described specific sequence can be used for the kind distinguishing pathogenic bacterium.

Sequence alignment, reference (multi-analyte suspension assay quantitatively determining human change of serum C EA, AFP, NSE and tPSA, fine strain of millet Hui Yi etc., J Mol Diagn Ther, in July, 2010, Vo1.2, No.4 can be adopted, Wang Jiali etc., High-Throughput, Sensitive, and Accurate Multiplex PCR-Microsphere FlowCytometry System for Large-Scale Comprehensive Detection of Respiratory discloses .10.1128/JCM.02501-06.2007 Viruses (with the drug-fast Golden yellow staphylococcus of new technical platform Luminex rapid detection tool Staphcillin) on May 30th, 2007 in advance, 45 (8): 2626.DOI:J.Clin.Microbiol.James R.Prudent etc.), this area routine techniquess such as experimental verification are determined the specific sequence of the pathogenic bacterium each to be measured being applicable to the inventive method or are comprised the significant DNA sequence dna of this sequence.Be well known in the art about the database information of 16S rDNA and analysis software, such as RDP (http://rdp.cme.msu.edu/html/), ARB (http://www.ard-home.de/), ORS (http://soul.mikro.biologie.tu-muenchen.de/ORS/), OPD (http://www.com.msu.eduOPD/) and PRIMROSE, CLUSTAL-X, PRIMER PREMIER, DNA START etc., each analysis software has his own strong points because of the algorithm difference adopted, and these databases and software all can be used in the present invention.

As used herein, term " conserved sequence " or " consensus sequence " are used interchangeably, and all refer to the concensus sequence in the significant DNA sequence dna of the 16S rRNA gene (16S rDNA) of various pathogenic bacterium.The universal primer of the present invention designed for " conserved sequence " can be used for amplifying the significant DNA sequence dna of various pathogenic bacterium to be measured.In order to realize the object of universal amplification, this conserved sequence is positioned at the two ends of variable region usually.

As used herein, term " universal primer to " refers to for the conservative region in the significant DNA sequence dna of pathogenic bacterium, a pair universal primer that can be used for amplifying the significant DNA sequence dna of pathogenic bacterium of the specific sequence comprising various pathogenic bacterium.General upstream primer (or claiming " forward universal primer ") is usually for the upstream of significant DNA sequence dna; General upstream primer (or claiming " reverse universal primer ") is usually for the downstream of significant DNA sequence dna.

The right design of universal primer can be carried out according to design of primers rule conventional in this area, adopt known primer-design software and/or by the service providing the company of design of primers to provide special.General upstream primer of the present invention and general downstream primer are respectively for sequence A CGCGAAGAACCTTACC shown in SEQ ID NO:10 with for sequence TGTTGGGTTAAGTCCCGCAACGA shown in sequence TGTTGGGTTAAGTCCCGCAACGAG shown in SEQ ID NO:11 or SEQ ID NO:12.Such as in one embodiment of the invention, can adopt the general upstream primer of SEQ ID NO:1 or 3 and SEQ ID NO:2,4,5, the general downstream primer of 6 or 7 is combined to form universal primer pair.

In the method for the invention, PCR method as known in the art and normal condition, employing universal primer pair of the present invention can be adopted, pcr amplification is carried out to sample.Also can carry out pcr amplification to the bacterial classification of known type, and amplified production is checked order, and order-checking information is used for follow-up specificity capture probe design.

pathogenic microbes detect method

Provide a kind of method detecting pathogenic bacterium in the present invention, described method comprises:

(A) method as above is adopted to obtain the significant DNA sequence dna of pathogenic bacterium described in sample;

(B) provide respectively for the corresponding specificity capture probe of the significant DNA sequence dna of described pathogenic bacterium (one or more, preferably at least two kinds);

(C) under hybridization conditions, the significant DNA sequence dna of described pathogenic bacterium is contacted with described specificity capture probe, and before described contact, when or afterwards with the mixture that significant DNA sequence dna and the described specificity capture probe of specificity capture probe described in different detectable label substance markers or described pathogenic bacterium are formed, obtain the hybrid product with different detectable;

(D) signal of the detectable be connected on described hybrid product is detected, to determine existence or the quantity of various pathogenic bacterium to be measured in sample.

As used herein, term " specificity capture probe " or " capture probe " are used interchangeably, all refer to for the specific sequence design of certain pathogenic bacterium that may exist in the significant DNA sequence dna of 16S rDNA, thus specificity can catch the probe of the 16S rDNA amplified production of this kind of pathogenic bacterium.As required, capture probe of the present invention can with or without marker (as radioactivity, vitamin H, fluorescent marker etc.).Can according to probe design Design with Rule probe conventional in this area, and synthetic probe.

In certain embodiments of the present invention, the sequence capture probe of SEQ ID NO:13 ~ 20 respectively specifically for and the significant DNA sequence dna of intestinal bacteria, Klebsiella Pneumoniae, enterococcus faecalis, enterobacter cloacae, Pseudomonas aeruginosa, proteus vulgaris, Serratia marcescans and streptococcus aureus can be caught respectively, be more specifically the specific sequence in significant DNA sequence dna.

Available various detectable is connected with capture probe, to make with specific mark in the sequence of being caught by specific capture probe, thus is mapped with the sequence of specified germ by specific detectable label, is convenient to distinguish different bacteriums.Described detectable includes but not limited to: coding microball, carrier (such as fluorescent marker, luminescent protein) etc., optimized encoding microballoon.The method of linkage flag thing and capture probe is well known in the art, those of ordinary skill in the art can modify capture probe or marker or transform according to selected marker, can be interconnected to obtain both (such as can see Xu Yanjun, Xiamen University's master thesis in 2008: the documents such as the preparation of carboxyl functionality polystyrene microsphere in liquid-phase chip, sign and the application in biological detection thereof and the reference quoted thereof).

As used herein, term " coding microball " refer to there is different detectable area and divide feature (such as size and/or color), can with capture probe coupling and the various microballoons distinguished of the equipment that can be detected.The material of microballoon can be: polystyrene, as MultiAnalyte microballoon or SeroMap microballoon etc.The diameter of microballoon can be 20nm-200 μm, preferably 5 ~ 10 μm, more preferably 5.6 μm.Microballoon can dye distinct colors (as fluorescence color) with the dyestuff of different ratio (preferred fluorescence dye), thus obtain can reach several, ten several, the different coding microball of tens of kinds, even over one hundred kind.Coding microball is obtained by commercially available approach, such as purchased from triumphant outstanding biological, BIO-RAD or as required designed, designed and prepare coding microball (such as can with reference to Chen Wei, the principle and characteristics progress of liquid-phase chip technology, Chengdu Medical College's Pathological Staff Room, Chengdu hospital journal, 2008 (3): 225-231, the document is included in herein as a reference in full with it).

Capture probe of the present invention can be passed through to modify or transform to have and is suitable for and the joint of coding microball coupling or group, such as, can connect AMB for being connected with coding microball further at the 5' end of capture probe.The coupling of capture probe and coding microball, method as known in the art can be adopted to carry out (such as can with reference to Han Weining etc., the applied research progress of liquid-phase chip technology in detection of nucleic acids, modern preventive medicine, 2008 (10): 1911-1915, the document is included in herein as a reference in full with it).Joint such as on applicable capture probe or group are connected with the corresponding construction on coding microball or under the condition of reacting, mixing and hatching capture probe and coding microball.

Under proper condition, employing universal primer is made to hybridize from different capture probes-coding microball conjugate the PCR primer obtained that increases, to make PCR primer with the detectable signal from coding microball.The normal condition that hybridization conditions can refer in the working specification (standard operating procedure of such as luminex coupling reagent kit is carried out) or this area that manufacturer provides is carried out.

Method as known in the art can be adopted to detect the signal of the coding microball be connected in described PCR primer, to measure existence or the quantity of pathogenic bacterium.Such as can adopt fluorescent microsphere detection system, flow cytometer or solid phase chip etc.When the specific coding microballoon signal with the capture probe coupling of specific pathogenic bacterium being detected, show in the sample be detected with this kind of specific pathogenic bacterium.If required, by the quantity of pathogenic bacterium in the strong and weak working sample of coding microball signal.

the application of test kit and reagent

Additionally provide a kind of test kit for detecting pathogenic bacterium in the present invention, it comprises:

(i') for the general upstream primer of sequence A CGCGAAGAA CCTTACC shown in SEQ ID NO:10 and the general downstream primer for sequence TGTTGGGTTAAGTCCCGCAACGA shown in sequence TGTTGGGTTAAGTCCCGCAACGAG or SEQID NO:12 shown in SEQ ID NO:11, described general upstream primer and general downstream primer is wherein adopted to carry out pcr amplification to described sample, the significant DNA of described pathogenic bacterium can be obtained, wherein, described significant DNA sequence dna is arranged in the 16S rDNA of described pathogenic bacterium, and comprise one or more pathogenic bacterium described specific sequence exclusive separately,

(ii') for the connector of the corresponding specificity capture probe of the significant DNA sequence dna of described pathogenic bacterium, various detectable or each species specificity capture probe and detectable;

(iii') one or more container and/or packing material;

(iv') optional, pcr amplification reagent;

(v') optional, for connecting the reagent of described specificity capture probe and detectable;

(vi') optional, the reagent of detection signal and/or instrument;

(vii') optional, working instructions;

(viii') optional, pathogenic bacterium therapeutical agent.

The purposes adopting method of the present invention or test kit to detect pathogenic bacterium and/or treat in pathogenic infection is further provided in the present invention.

advantage of the present invention

Utilize technology of the present invention, can at short notice (in 4 hours, as 2-3 hour) various clinical common pathogen and composition thereof is carried out accurately, rapid detection; Now (such as siemens walkaway96 microbial identification system is detected by Other Instruments with traditional detection method (microbial culture, biochemical identification, serotype etc.) and hospital, this system principle or the principle of traditional detection method) compare, the characteristics and innovation of new technology is mainly reflected in: directly can carry out quick multiplexed PCR amplification with bacterium liquid or measuring samples as pcr template, high-flux parallel detection can be carried out to multiple common pathogen simultaneously, once experiment can draw whole result, fast easy and simple to handle, high specificity, highly sensitive.

sequence table explanation

Corresponding relation in sequence table of the present invention between sequence number and concrete sequence is as shown in table 1 below:

Table 1. sequence table explanation

Embodiment

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.Those skilled in the art can make suitable amendment, variation to the present invention, and these amendments and variation are all within the scope of the present invention.

The experimental technique of unreceipted actual conditions in the following example, the ordinary method in this area can be adopted, such as with reference to " Molecular Cloning: A Laboratory guide " (third edition, New York, CSH Press, New York:Cold Spring Harbor Laboratory Press, 1989) or the condition of advising according to supplier.The sequence measurement of DNA is the method for this area routine, also can provide test by commercial company.

Unless otherwise indicated, otherwise per-cent and number calculate by weight.Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.

materials and methods

1. major experimental instrument

The major experimental instrument of this experiment is PCR instrument (BIO-RAD S1000Thermal cycler), fluorescent microsphere detection system (luminex liquichip200), QIAxcel Advanced system (Kai Jie company QIAGEN).

2. experimental strain

Table 2. embodiment experimental strain used describes

3. major experimental reagent

Intestinal bacteria, enterobacter cloacae, Klebsiella Pneumoniae, enterococcus faecalis, proteus vulgaris, Serratia marcescans, Pseudomonas aeruginosa and streptococcus aureus all adopt LB substratum 37 DEG C of overnight incubation.

LB substratum: yeast extract 0.5%, Tryptones 1%, NaCl1%;

HotStar Taq Plus standard mix reagent box (HotStar Taq Plus Master mix kit (1000), cat.No.203645, Germany's just root biology (QIAGEN)), EDC (Sigma (Sigma)), coupling microballoon (COOH beads#171-5060** Bole company (BIO-RAD)), sheath fluid (cat.NO922902, Germany just root is biological), S-PE (cat.NO922721 Germany just root is biological).

Primer and probe are synthesized by Sangon Biotech (Shanghai) Co., Ltd..

4. universal primer and capture probe design

The design of universal primer and biotin labeling, according to the conservative region of significant DNA sequence dna in the 16S rDNA of pathogenic bacterium, design a pair universal primer, position and the sequence of this primer are as follows:

General upstream primer 16S-for is for sequences Design shown in SEQ ID NO:10; General downstream primer 16S-rev is for sequences Design shown in sequence shown in SEQ ID NO:11 or SEQ ID NO:12:

Table 3. universal primer of the present invention

SEQ ID NO：	Universal primer	Sequence	5 is terminal modified
				1	General upstream primer 1	5’-ACGCGAAGAACCTTACC-3’
2	General downstream primer 2	5’-TGCGGGACTTAACCCAAC-3’	BIO
				3	General upstream primer 3	5’-GCGAAGAACCTTACC-3’
4	General downstream primer 4	5’-TGCGGGACTTAACCC-3’	BIO
				5	General downstream primer 5	5’-TCGTTGCGGGACTTAACCCAACA-3’	BIO
6	General downstream primer 6	5’-GTTGCGGGACTTAACCCAACA-3’	BIO
				7	General downstream primer 7	5’-CTCGTTGCGGGACTTAACCCAA-3’	BIO

Another design can be increased the pair of primers (with reference to the application of Zhou Huang, 16S rRNA sequence analysis in medical microbial qualification, Beijing Institute of Microbiology and Epidemiology, biotechnology communication, Vol.10, NO4,1999) of the most long segment of 16S rDNA:

Upstream primer 16S-for designs for the 8-28bp place of the 16S rDNA of each bacterium; Downstream primer 16S-rev designs for the 1460-1510 place of the 16S rDNA of each bacterium:

Table 4. total length 16S rDNA universal primer

SEQ ID NO：	Universal primer	Sequence	5 is terminal modified
				8	The general upstream primer 8 of total length 16	5’-AGAGTTTGATCCTGGCTCAG-3’
9	The general downstream primer 9 of total length 16	5’-ACGGTTACCTTGTTACGACTT-3’	BIO

Each No. Genebank, bacterium 16S rDNA is as follows: enterobacter cloacae: AJ251469.1; Klebsiella Pneumoniae: Y17657.1; Enterococcus faecalis: KC699230.1; Proteus vulgaris: DQ205432.1; Serratia marcescans: FM163485.2; Pseudomonas aeruginosa: FJ194518.1; Streptococcus aureus: DQ269498.1.

SEQ ID NO:1-7 expanding fragment length is 130-140bp, SEQ ID NO:8-9 expanding fragment length be about 1500bp, the downstream primer designed marks at 5 ' end vitamin H (Biotin), for luminex instrument Luminex-streaming fluorescence technique (also known as liquid chip, liquid-phase chip)) signal detection.

The sequence after PCR is obtained by DNA sequencing, then in conjunction with database Taxonomy in NCBI, obtain the 16S rDNA of each bacterial strain, nucleic acid comparison software DNAMAN is utilized to compare, obtain the significant sequence of each bacterial strain in universal primer interval, be the specific probe of design for sequence (be design capture probe for sequence).Design a capture probe to every kind of pathogenic bacterium, Tm value is about 52 DEG C, and closes in 5 ' end phosphorylation, for the coupling of probe and microballoon.Design concept as shown in Figure 1.The concrete sequence of capture probe is as shown in table 5:

Table 5. is for the probe sequence designed by each bacterial strain

AMB (Probes completes modification by synthetic primer) is that chemical substance aminolink (Aminolinker) C12/5' Amino End Group is modified, for linking probe and magnetic bead.

5. universal primer PCR reaction and condition

In test, the reaction system of PCR comprises (50 μ l): Hotstar enzyme 25 μ L, water 22 μ L, and universal primer (10 μm of ol/L) each 4 μ L, (bacteria concentration in template is for being greater than 1 × 10 for template 10 μ L ⁵cFU/ml).

PCR program is: 95 DEG C, 5 minutes; 94 DEG C, 40 seconds; 55 DEG C, 40 seconds; 72 DEG C, 1 minute; 34 circulations; 72 DEG C, 2 minutes; 16 DEG C, 5 minutes; Stop.

The PCR primer obtained is checked order by prompt base (Shanghai) trade Co., Ltd in the English Weihe River.

6. the coupling of probe, microballoon

(concentration of microballoon is 1.25 × 10 to vortex microballoon ⁷individual/mL) 5min, 100 μ L microballoons are gone in coupling tube, lucifuge, wash microballoon with 0.1M MES.Centrifugal microballoon 12000rpm, 3min, abandon supernatant, then suspend with 50 μ L0.1M MES.The probe reagent of 5 microlitre 100pmol/L is joined in each corresponding microballoon (i.e. the microballoon of different coding) suspension.Adding 2.5 μ L10mg/mL EDC immediately comprises in the mixing tube of probe and microballoon to each, vortex 10 seconds, incubated at room temperature mixed solution 30 minutes.

The interpolation of second time EDC and hatching (step is the same).

Secondary hatching adds the EDC solution (10mg/mL) of 2.5 μ L0.1M MES dissolvings in the mixing tube of probe-microballoon, and vortex 10 seconds, hatches 30 minutes, place in room temperature dark place.

Then, with 0.02% polysorbas20 washing microballoon, add 1mL0.02% polysorbas20 in the coupling tube of the microballoon of each coupling, vortex 10 seconds, with 12000rpm centrifugal 3 minutes, abandon supernatant.Add 1mL0.1%SDS cleaning, vortex 10 seconds, with 12000rpm centrifugal 3 minutes.Eddy diffusion, with 250 μ L1 × TE vortex 5 seconds.4 DEG C of preservations.

7. fluorescent microsphere systems axiol-ogy

Step 1: universal primer PCR amplification (with described in the 5th part)

Step 2: the reaction of universal primer PCR amplified production and probe-microballoon conjugate

A. the addition of related reagent in reaction system:

Microballoon mixed system (microballoon concentration: be greater than 5 × 10 ³individual/ml): 500 μ L-50 × N=M

50 represent extension rate; N represents the volume (μ L) of various probe-microspheres solution; M represents the volume (mol/L) of 1 × TE.

The preparation of 1 × TE (10mM Tris-HCl, pH=8.0,0.1mM EDTA pH=8.0):

1M Tris-HCl pH＝8.0，1mL；0.5M EDTA pH＝8.0，0.2mL；

100mL is settled to ultrapure water.

Table 6. reaction system forms

Composition	Volume μ L/r × n
		Microballoon mixed system	10
Detect damping fluid	35
		PCR primer	5

" r " is writing a Chinese character in simplified form of " reaction (reaction) ";

" n " is writing a Chinese character in simplified form of " quantity (number) ";

" volume μ L/r × n " represents the reagent volume needed for single reaction system.

Detect the formula of damping fluid (1.8L): 606.6mL ultrapure water, the 5M TMAC of 1080mL, 20% sarcosyl (Sarkosyl) of 9mL, the 0.5M EDTA (pH=8.0) of the 1M Tris-HCl (pH=8.0) of 90mL, 14.4mL.

Crossover process:

By microballoon diluent (microballoon mixed system+detection damping fluid) vortex 15 seconds, join in hybrid pipe or 96 orifice plates after mixing, every hole 45 μ L, joined PCR primer in respective aperture, and every hole 5 μ L, after mixing, hatches 10 minutes by orifice plate in 52 DEG C; After hatching, every hole adds 10 μ L S-PE, hatches 5 minutes for 52 DEG C; After hatching, every hole adds the ddH of the prior preheating of 120 microlitre ₂o, stops crossover process, to be measured.

Adopt fluorescent microsphere detection system (luminex liquichip200), by red, green two bundle laser detect respectively microballoon coding and reporter fluorescence to carry out quantitative and semi-quantitative.

it is general that embodiment 1. adopts shown in the general upstream primer shown in SEQ ID NO:1 and SEQ ID NO:2 downstream primer carries out pcr amplification to clinical common pathogenic bacteria

Cultivate the clinical common pathogenic bacteria that will detect and (be common in urine, also show in other sample): intestinal bacteria (E.coli), Klebsiella Pneumoniae (K.peneumoniae, i.e. KPN), enterococcus faecalis (E.faecalis, i.e. EF), enterobacter cloacae (Enterobacter cloacae, i.e. ECL), Pseudomonas aeruginosa (P.Aeruginosa, i.e. PAE), proteus vulgaris (P.vulgaris, i.e. PV), Serratia marcescans (Serratia marcescens, i.e. SM), streptococcus aureus (Staphyloccocus aureus, i.e. SA).

Bacterial concentration is adjusted to and is greater than 10 ⁵cFU/ml, directly carries out pcr amplification.

Pcr amplification result as shown in Figure 2.

Figure 2 shows that bacterial classification PCR gel electrophoresis figure, from left to right intestinal bacteria (E.coli) successively, Klebsiella Pneumoniae (K.peneumoniae, i.e. KPN), enterococcus faecalis (E.faecalis, i.e. EF), enterobacter cloacae (Enterobacter cloacae, i.e. ECL), Pseudomonas aeruginosa (P.Aeruginosa, i.e. PAE), proteus vulgaris (P.vulgaris, i.e. PV), Serratia marcescans (Serratia marcescens, i.e. SM), streptococcus aureus (Staphyloccocus aureus, i.e. SA), the mixed bacterium sample of above 8 kinds of bacterium, blank and molecular weight marker (Marker100-2000bp).

This result shows: designed primer can highly sensitive, the corresponding bacterial strain of specific amplification and bacterial strain mixed solution.

it is general that embodiment 2. adopts shown in the general upstream primer shown in SEQ ID NO:3 and SEQ ID NO:4 downstream primer carries out pcr amplification to clinical common pathogenic bacteria

The amplification method of bacterium liquid is the same (embodiment 1), and pcr amplification result as shown in Figure 3.

Figure 3 shows that bacterial classification PCR gel electrophoresis figure, from left to right intestinal bacteria (E.coli) successively, Klebsiella Pneumoniae (K.peneumoniae, i.e. KPN), enterococcus faecalis (E.faecalis, i.e. EF), enterobacter cloacae (Enterobacter cloacae, i.e. ECL), Pseudomonas aeruginosa (P.Aeruginosa, i.e. PAE), proteus vulgaris (P.vulgaris, i.e. PV), Serratia marcescans (Serratia marcescens, i.e. SM), streptococcus aureus (Staphyloccocus aureus, i.e. SA), the mixed bacterium sample of above 8 kinds of bacterium, blank and molecular weight marker (Marker100-2000bp).

This result shows: designed primer also can effectively increase acquisition PCR primer, can be used as universal primer.

it is general that embodiment 3. adopts shown in the general upstream primer shown in SEQ ID NO:3 and SEQ ID NO:5 downstream primer carries out pcr amplification to clinical common pathogenic bacteria

The amplification method of bacterium liquid is the same (embodiment 1), and pcr amplification result as shown in Figure 4.

Figure 4 shows that bacterial classification PCR gel electrophoresis figure, from left to right intestinal bacteria (E.coli) successively, Klebsiella Pneumoniae (K.peneumoniae, i.e. KPN), enterococcus faecalis (E.faecalis, i.e. EF), enterobacter cloacae (Enterobacter cloacae, i.e. ECL), Pseudomonas aeruginosa (P.Aeruginosa, i.e. PAE), proteus vulgaris (P.vulgaris, i.e. PV), Serratia marcescans (Serratia marcescens, i.e. SM), streptococcus aureus (Staphyloccocus aureus, i.e. SA), the mixed bacterium sample of above 8 kinds of bacterium, blank and molecular weight marker (Marker100-2000bp).

This result shows: designed primer also as universal primer, can obtain the PCR primer of corresponding PCR primer and mixed bacterium.

it is general that embodiment 4. adopts shown in the general upstream primer shown in SEQ ID NO:3 and SEQ ID NO:6 downstream primer carries out pcr amplification to clinical common pathogenic bacteria

The amplification method of bacterium liquid is the same (embodiment 1), and pcr amplification result as shown in Figure 5.

Figure 5 shows that bacterial classification PCR gel electrophoresis figure, from left to right intestinal bacteria (E.coli) successively, Klebsiella Pneumoniae (K.peneumoniae, i.e. KPN), enterococcus faecalis (E.faecalis, i.e. EF), enterobacter cloacae (Enterobacter cloacae, i.e. ECL), Pseudomonas aeruginosa (P.Aeruginosa, i.e. PAE), proteus vulgaris (P.vulgaris, i.e. PV), Serratia marcescans (Serratia marcescens, i.e. SM), streptococcus aureus (Staphyloccocus aureus, i.e. SA), the mixed bacterium sample of above 8 kinds of bacterium, blank and molecular weight marker (Marker100-2000bp).

This result shows: designed primer also as universal primer, can obtain the PCR primer of corresponding PCR primer and mixed bacterium.

it is general that embodiment 5. adopts shown in the general upstream primer shown in SEQ ID NO:3 and SEQ ID NO:7 downstream primer carries out pcr amplification to clinical common pathogenic bacteria

The amplification method of bacterium liquid is the same (embodiment 1), and pcr amplification result as shown in Figure 6.

Figure 6 shows that bacterial classification PCR gel electrophoresis figure, from left to right intestinal bacteria (E.coli) successively, Klebsiella Pneumoniae (K.peneumoniae, i.e. KPN), enterococcus faecalis (E.faecalis, i.e. EF), enterobacter cloacae (Enterobacter cloacae, i.e. ECL), Pseudomonas aeruginosa (P.Aeruginosa, i.e. PAE), proteus vulgaris (P.vulgaris, i.e. PV), Serratia marcescans (Serratia marcescens, i.e. SM), streptococcus aureus (Staphyloccocus aureus, i.e. SA), the mixed bacterium sample of above 8 kinds of bacterium, blank and molecular weight marker (Marker100-2000bp).

This result shows: designed primer also as universal primer, can obtain the PCR primer of corresponding PCR primer and mixed bacterium.

embodiment 6. adopts the downstream primer pair shown in the upstream primer shown in SEQ ID NO:8 and SEQ ID NO:9 clinical common pathogenic bacteria carries out pcr amplification (amplification length is greatly about 1500bp)

The amplification method of bacterium liquid is the same (embodiment 1), and pcr amplification result as shown in Figure 7.

Figure 7 shows that bacterial classification PCR gel electrophoresis figure, from left to right intestinal bacteria (E.coli) successively, Klebsiella Pneumoniae (K.peneumoniae, i.e. KPN), enterococcus faecalis (E.faecalis, i.e. EF), enterobacter cloacae (Enterobacter cloacae, i.e. ECL), Pseudomonas aeruginosa (P.Aeruginosa, i.e. PAE), proteus vulgaris (P.vulgaris, i.e. PV), Serratia marcescans (Serratia marcescens, i.e. SM), streptococcus aureus (Staphyloccocus aureus, i.e. SA), the mixed bacterium sample of above 8 kinds of bacterium, blank and molecular weight marker (Marker100-2000bp).

This result shows: designed total length 16S rDNA universal primer increase single bacterium time, can target product be obtained, but when pcr amplification is carried out to mixed bacteria liquid, must fall short product.In other words, this primer pair effectively cannot increase to mixed bacteria liquid.And the sample that will detect in actual applications normally mixed bacteria liquid (as: urine, body fluid, blood, food etc., wherein comprised microorganism is incessantly a kind of), therefore this total length 16S rDNA universal primer is to not applying to this experiment and most of clinical setting as universal primer.On the contrary, the of the present invention specific universal primer adopted in embodiment 1-5 is to effectively increasing to mixed bacteria liquid.

embodiment 7. adopts Luminex systems axiol-ogy clinical common pathogenic bacteria

The microballoon of the PCR primer of gained in the embodiment 1 comprising single bacterial strain or hybrid bacterial strain sample and coupling probe is reacted, then adopts Luminex system to detect.The PCR primer of other non-targeted bacterial strain is also joined detection system, the specificity (i.e. cross reaction situation) of detection system simultaneously.

To join probe with microballoon lotus root to be combined with PCR primer, realize signal with liquid-phase chip and export, represent by MFI value, detected result is as shown in table 7 below:

Table 7: adopt Luminex systems axiol-ogy clinical common pathogenic bacteria

Data type: mean value, n=14

Positive findings judgment basis: the fluorescence intensity median (MPI) obtained is greater than 250, or add the negative findings standard deviation of 5 times for the mean value of negative findings when numerical value is about 250 time, 2 kinds of result of determination all use, when all in threshold value, should be judged to the positive.

With in form about the data instance of ECL bacterium, calculate standard deviation the positive value.

Negative findings mean value=(102+46+46+78+59+14+103+103+85+81+59+26)/12=66.83

Positive findings threshold value=66.83+5 × 28.80=210.86, namely when obtaining result and being greater than 210.86, can think positive findings.

Reagent of the present invention and method is adopted to be the positive to the result that enterobacter cloacae detects.

Other result can according to said method be analogized.

First be the test of single positive sample be namely that monoxenic PCR primer detects to sample, to determine feasibility that detection probes designs, sensitivity and specificity.Can be drawn by the numbering 1-8 of table 7: probe design is feasible, highly sensitive, and high specificity.

Numbering 9-12 is the laboratory do not included in probe design other bacterial classification existing, and employing native system does not detect the bacterium beyond system design, shows present system high specificity, cross reaction does not occur.

Can be reached a conclusion by this experiment: system of the present invention can as the strong specificity effective system detecting clinical common pathogenic bacteria.

embodiment 8. adopts Luminex systems axiol-ogy clinical sample

Adopt 382 routine middle segment urine sample samples to detect, the detected result of its liquid-phase chip Luminex200 and siemens walkaway96 microbial identification system is as shown in table 8:

Table 8: adopt Luminex systems axiol-ogy clinical sample

Note: in blood agar detected result, "+" represents: the flat-plate bacterial colony number of 5 μ L urines is greater than 20;

In PCR detected result, "+" represents: with blank comparison, band concentration is greater than blank;

In Siemens System detected result, "+" represents: with hospital's gold standard (with reference to " national Clinical Laboratory working specification " third edition, Department of Medical Administration of Ministry of Health of the People's Republic of China) decision-making system instrument display result, bacterium is dense is greater than 1 × 10 ⁵cFU/mL;

In liquid-phase chip system detection results, "+" represents: MFI (fluorescence intensity median) is greater than 250.

Qualification brief introduction and the process of siemens walkaway96 microbial identification system are summarized as follows: siemens's identification systems are based on microorganism in-depth theory, by microbial information coding techniques, this system is analyzed various physiological condition, biochemical indicator, metabolic reaction according to microorganism, and result is converted into the data that software can identify, carry out cluster analysis, compare with known reference strains database, finally unknown bacterium is identified.Experimental identification process: urine on blood agar 37 DEG C cultivate 24 hours, further experiment is judged whether by technician, observe flat board do not grow bacterium colony or to grow the report that less bacterium colony or the more flat board of miscellaneous bacteria abandon negative, picking is judged as the microbiological contamination bacterium colony on the flat board of microbiological contamination, in access siemens test kit, 24-72 hour is cultivated, instrument result of determination at Siemens System.That is, before entering Siemens System and determining which kind of bacterial classification, first carried out dull and stereotyped primary dcreening operation, people eliminates the flat board that dull and stereotyped upper colony growth is less or it's too late containing mixed bacterium, or reviewer thinks the flat board contaminating miscellaneous bacteria.

Detect the 382 routine clinical midstream urine samples from infection from hospital, wherein every detected result display of 234 routine samples is negative findings, and 61 routine sample E. coli detections are positive.There is the liquid-phase chip of 14 routine samples to be defined as the intestinal bacteria positive in addition, but adopt the Siemens System detected result of gold standard to be shown as feminine gender.This is because hospital has carried out blood agar primary dcreening operation when carrying out Siemens System inspection, artificial judgement is also abandoned containing the sample (namely cannot detect mixed bacterium sample) of more than 2 and 2 bacterial classifications and the less sample (namely detection sensitivity is limited) (special sample 2) of colony number, or only selective advantage pattern detection (namely detection sensitivity is limited) (special sample 1), by contrast, adopt the liquid-phase chip system of the inventive method without primary dcreening operation and detect sensitive, liquid-phase chip can show low bacteria concentration or mixed bacterium positive findings.Further, the detection time of the inventive method only needs 2-3 hour, and without the need to primary dcreening operation sample, has efficient, easy advantage.

Conclusion

Sequential analysis due to 16S rRNA gene fundamentally have impact on the understanding of people to evolutionary relationship between bacterium, has all carried out 16S rRNA sequencing in the taxonomic identification of many microorganisms.But, not yet adopt 16S rDNA, pcr amplification in this area and differentiate the kind of microorganism in unknown sample by instrument fast.

Adopt pcr amplification that 16S rRNA fragment amplification is become 16S rDNA in this experiment.Then, by the 16S rDNA sequence of the multiple bacterium of comparison, find the high degree of specificity region of each bacterium, find at the two ends in high degree of specificity region the high conservative region of these bacterium design universal primer and make it be with biotin labeling.

This experiment is about the multipair primer of sequences Design of about 1500bp to 16S rDNA, Fig. 2-7 shows at about 900 ~ 1200bp place (the 975-1108bp place of such as Serratia marcesens 16S rDNA) of 16SrDNA design primer, can increase about 130-140bp fragment, high efficiency amplification can be obtained, and effective fragment of total length 16S rDNA universal primer 16S-FOR (SEQ ID NO:8) and 16S-REV (the SEQ ID NO:9) object bacteria that can not simultaneously increase and mixed bacterium sample, the requirement simultaneously detecting multiple bacterium can not be met.This illustrates, selects arbitrarily the so-called conserved sequence design primer in 16S rDNA, usually can not obtain target DNA product under the situation of mixed bacterium multiplex PCR.

Contriver finds through repeatedly testing and attempting: for concrete 8 kinds of clinical common pathogenic bacteria: intestinal bacteria (E.coli), Klebsiella Pneumoniae (K.peneumoniae, i.e. KPN), enterococcus faecalis (E.faecalis, i.e. EF), enterobacter cloacae (Enterobacter cloacae, i.e. ECL), Pseudomonas aeruginosa (P.Aeruginosa, i.e. PAE), proteus vulgaris (P.vulgaris, i.e. PV), Serratia marcescans (Serratia marcescens, i.e. SM), streptococcus aureus (Staphyloccocus aureus, i.e. SA), design upstream and downstream universal primer respectively, can be increased the significant DNA of each bacterium simultaneously.The design of this experiment primer solves the cross reaction that pathogenic bacterium in the past adopt the amplification of multiplex PCR to cause, and design of primers is simple, and cost reduces.

The design of capture probe is mainly for the high degree of specificity region of each bacterium in gained sequence after universal primer PCR amplification.(as amination) is modified so that the coupling of this probe and microballoon to 5 ' end of probe, (as color coding) microballoon of each probe conjugate one coding, microballoon signal after instrument identification PCR primer and probe microballoon are combined, thus realize the object that single reaction system detects multiple bacterial strain simultaneously.

Reaction system detection of the present invention is sensitive, high specificity, detection speed are fast, just testing process can be completed at 15min, liquid-phase chip technology changes the situation that general-purpose chip in the past needs to be fixed on chip by probe, chip cost is high, and only need an aseptic reaction system (as 96 porocyte culture plates), just once can complete multiple (as 96) sample tests, cost is low, efficiency is high, is the prefered method that batch samples detects.

Liquid-phase chip technology can with advantages such as its high-throughput, detection sensitivity are high, reproducible in the near future, and detects the advantages such as wide dynamic range and be widely used and each research field.

In addition, although the universal primer sequence in embodiment and capture probe are for specific clinical common pathogenic bacteria design and preparation, those of ordinary skill in the art design according to the present invention can determine variable region sequences used in the present invention in other pathogenic bacterium, and design and prepare corresponding universal primer sequence and capture probe, to realize the detection to other pathogenic bacterium.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (10)

1. obtain a method for the significant DNA sequence dna of pathogenic bacterium in sample, described pathogenic bacterium be selected from lower group of bacterium one or more: intestinal bacteria (E.coli), Klebsiella Pneumoniae (K.peneumoniae), enterococcus faecalis (E.faecalis), enterobacter cloacae (Enterobacter cloacae), Pseudomonas aeruginosa (P.Aeruginosa), proteus vulgaris (P.vulgaris), Serratia marcescans (Serratia marcescens), streptococcus aureus (Staphyloccocus aureus); Described method comprises:

I () obtains for the general upstream primer of sequence A CGCGAAGAA CCTTACC shown in SEQ ID NO:10 and the general downstream primer for sequence TGTTGGGTTAAGTCCCGCAACGA shown in sequence TGTTGGGTTAAGTCCCGCAACGAG shown in SEQ ID NO:11 or SEQ ID NO:12;

(ii) described general upstream primer and general downstream primer is adopted to carry out pcr amplification to described sample, thus obtain the significant DNA of described pathogenic bacterium, wherein, described significant DNA sequence dna is arranged in the 16S rDNA of described pathogenic bacterium, and comprises one or more pathogenic bacterium described specific sequence exclusive separately.

2. the method for claim 1, is characterized in that, described general upstream primer is 15 ~ 17 on all four sequences of continuous nucleotide sequence with sequence shown in SEQ IDNO:10; Described general downstream primer is the sequence with 15 ~ 23 of SEQ ID NO:11 or 12 continuous nucleotide sequence complete complementaries.

3. the method for claim 1, is characterized in that, described general upstream primer is the sequence of 15 ~ 17 continuous nucleotide sequence complete complementaries with sequence shown in SEQ IDNO:10; Described general downstream primer is and 15 ~ 23 of SEQ ID NO:11 or 12 on all four sequences of continuous nucleotide sequence.

4. detect a method for pathogenic bacterium in sample, described pathogenic bacterium be selected from lower group of bacterium one or more: intestinal bacteria (E.coli), Klebsiella Pneumoniae (K.peneumoniae), enterococcus faecalis (E.faecalis), enterobacter cloacae (Enterobacter cloacae), Pseudomonas aeruginosa (P.Aeruginosa), proteus vulgaris (P.vulgaris), Serratia marcescans (Serratia marcescens), streptococcus aureus (Staphyloccocus aureus); Described method comprises:

(A) method according to any one of claims 1 to 3 is adopted to obtain the significant DNA sequence dna of pathogenic bacterium described in sample;

(B) provide respectively for the corresponding specificity capture probe of the significant DNA sequence dna of described pathogenic bacterium;

(C) under hybridization conditions, the significant DNA sequence dna of described pathogenic bacterium is contacted with described specificity capture probe, and before described contact, when or afterwards with the mixture that significant DNA sequence dna and the described specificity capture probe of specificity capture probe described in different detectable label substance markers or described pathogenic bacterium are formed, obtain the hybrid product with different detectable;

(D) signal of the detectable be connected on described hybrid product is detected, to determine existence or the quantity of various pathogenic bacterium to be measured in sample.

5. method as claimed in claim 4, is characterized in that, described method is used for clinical sample detection, food test, food processing process detects or makeup detect, and is preferred for clinical sample and detects or food quarantine.

6. method as claimed in claim 4, it is characterized in that, described specificity capture probe is selected from:

A) for the specificity capture probe of the significant DNA sequence dna of intestinal bacteria shown in SEQ ID NO:21;

B) for the specificity capture probe of the significant DNA sequence dna of Klebsiella Pneumoniae shown in SEQ ID NO:22;

C) for the specificity capture probe of the significant DNA sequence dna of enterococcus faecalis shown in SEQ ID NO:23;

D) for the specificity capture probe of the significant DNA sequence dna of enterobacter cloacae shown in SEQ ID NO:24;

E) for the specificity capture probe of the significant DNA sequence dna of Pseudomonas aeruginosa shown in SEQ ID NO:25;

F) for the specificity capture probe of the significant DNA sequence dna of proteus vulgaris shown in SEQ ID NO:26;

G) for the specificity capture probe of the significant DNA sequence dna of Serratia marcescans shown in SEQ ID NO:27; With

H) for the specificity capture probe of the significant DNA sequence dna of streptococcus aureus shown in SEQ ID NO:28.

7. method as claimed in claim 4, it is characterized in that, the described detectable in step (C) is coding microball, and described coding microball adopts the marker coding that can produce different signal.

8. method as claimed in claim 4, is characterized in that, the described detection in step (D) adopts one or more systems or the instrument that are selected from lower group: liquid-phase chip detection system, flow cytometer or solid phase chip.

9. one kind for detect pathogenic bacterium and/or treatment pathogenic infection test kit, described pathogenic bacterium are be selected from one in lower group of bacterium or many two kinds: intestinal bacteria (E.coli), Klebsiella Pneumoniae (K.peneumoniae), enterococcus faecalis (E.faecalis), enterobacter cloacae (Enterobacter cloacae), Pseudomonas aeruginosa (P.Aeruginosa), proteus vulgaris (P.vulgaris), Serratia marcescans (Serratia marcescens), streptococcus aureus (Staphyloccocus aureus), described test kit comprises:

(i') for the general upstream primer of sequence shown in SEQ ID NO:10 and the general downstream primer for sequence shown in SEQ ID NO:11 or SEQ ID NO:12, described general upstream primer and general downstream primer is wherein adopted to carry out pcr amplification to described sample, the significant DNA of described pathogenic bacterium can be obtained, wherein, described significant DNA sequence dna is arranged in the 16S rDNA of described pathogenic bacterium, and comprises one or more pathogenic bacterium described specific sequence exclusive separately;

(ii') for the connector of the corresponding specificity capture probe of the significant DNA sequence dna of described pathogenic bacterium, various detectable or each species specificity capture probe and detectable;

(iii') one or more container and/or packing material;

(iv') optional, pcr amplification reagent;

(v') optional, for connecting the reagent of described specificity capture probe and detectable;

(vi') optional, the reagent of detection signal and/or instrument;

(vii') optional, working instructions;

(viii') optional, pathogenic bacterium therapeutical agent.

10. following substances detects the purposes in the test kit of pathogenic bacterium and/or treatment pathogenic infection in preparation:

I () is for the general upstream primer of sequence shown in SEQ ID NO:10 and the general downstream primer for sequence shown in sequence shown in SEQ ID NO:11 or SEQ ID NO:12, described general upstream primer and general downstream primer is wherein adopted to carry out pcr amplification to described sample, the significant DNA of described pathogenic bacterium can be obtained, wherein, described significant DNA sequence dna is arranged in the 16S rDNA of described pathogenic bacterium, and comprises one or more pathogenic bacterium described specific sequence exclusive separately;

(ii) for the connector of the corresponding specificity capture probe of the significant DNA sequence dna of described pathogenic bacterium, various detectable or each species specificity capture probe and detectable;

(iii) one or more container and/or packing material;

(iv) optional, pcr amplification reagent;

V () is optional, for connecting the reagent of described specificity capture probe and detectable;

(vi) optional, the reagent of detection signal and/or instrument;

(vii) optional, working instructions;

(viii) optional, pathogenic bacterium therapeutical agent.