CN100427610C - Authenticating gram positive bacteria species and method for testing drug resistant gene and dedicating kit - Google Patents
Authenticating gram positive bacteria species and method for testing drug resistant gene and dedicating kit Download PDFInfo
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- CN100427610C CN100427610C CNB2005100644349A CN200510064434A CN100427610C CN 100427610 C CN100427610 C CN 100427610C CN B2005100644349 A CNB2005100644349 A CN B2005100644349A CN 200510064434 A CN200510064434 A CN 200510064434A CN 100427610 C CN100427610 C CN 100427610C
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Abstract
The present invention discloses a method for gram-positive bacterium species identification and drug resistance gene detection and a special kit thereof. The kit comprises a primer pair, a gram-positive bacterium species detection specific gene probe and a drug resistance gene probe, wherein the primer pair is used for amplifying drug resistance genes. In the method, the kit is used for the species identification and the drug resistance gene detection. The method of the present invention has the steps that 1, primers arranged in the kit are used for the PCR amplification on the drug resistance genes and the species specific genes of the strains to be detected; 2, amplified products are hybridized with the gram-positive bacterium species detection specific gene probe and the drug resistance gene probe for determining the bacterium species of the strains to the detected and the drug resistance genes in the strains to be detected. The kit of the present invention has the characteristics of high integration degree, high sensitivity, wide application range, high specificity, stable detected results and high reliability.
Description
Technical field
The present invention relates to the gram positive bacterium kind identifies and drug resistant gene detection method and dedicated kit thereof.
Background technology
One. the significance that Bacteria Identification and resistance detect
Bacterium is the pathogenic agent that causes most of infectious diseases, causes that the pathogenic bacterium that the clinical bacteria sexuality is dyed comprise aerophil and anerobe, is divided into gram-positive microorganism and negative bacterium two big classes, and every class is divided into coccus and bacillus again.Aerobic gram-positive cocci and gram negative bacillus are clinical modal two class pathogenic bacterium.Gram-positive microorganism is isostructural different owing to cell walls with Gram-negative bacteria, thereby causes medicine that also there are significant difference in its mechanism of action and resistance mechanism thereof, and the clinical application to this two bacterioid has very big difference clinically.
Human infection's bacterial spectrum is not unalterable.As time goes on, the bacterium that causes infection is also taking place to change.Eighties of last century, preponderate with suis the thirties.Along with the use of 40-50 age penicillin and sulfonamide, streptococcus aureus has replaced the The main pathogenic fungi of suis as hospital infection.After this use the cynnematin and the aminoglycoside medicaments of narrow spectrum, and replaced streptococcus aureus by gram negative bacillus the seventies.The wide spectrum cephalosporin antibacterial application of phase at the beginning of the eighties latter stage seventies, the use of various conduits and the application of immunosuppressor, although gram negative bacillus still in the highest flight, there is other bacterium to become the important pathogen of infection gradually again, as coagulase negative staphylococcus and faecalis etc., and various resistant organism incidence is more and more higher.Up to the present, the infection rate of gram positive bacterium has the trend that increases year by year, can account for the 30-40% of whole infectation of bacteria, and wherein, coagulase negative staphylococcus, streptococcus aureus and faecalis can account for more than 95% of gram positive bacterium.
Infectation of bacteria can occur in almost any position of human body, and disease can be primary infection, also can be secondary infection, as the septicemia after the serious burn, also has a kind of infection that obtains in hospital, is called hospital infection or ward infection.Usually name relevant infection according to the happening part of infectious diseases clinically, but the infection at same position can be caused by different pathogenic agent, its symptom can be different with the reaction to medicine, for example antibacterials are only effective to infectation of bacteria, and antibacterials of the same race have different killing actions to different types of bacterium.In addition, pathogenic micro-organism can be the germ that human body is had virulence, and can be have (or low) toxic conditioned pathogen yet.Therefore etiological examination, i.e. Bacteria Identification is the treatment infectation of bacteria, rationally uses the basis of antibacterials.
The treatment of infectious diseases mainly relies on antibacterials.But along with being extensive use of of antibacterials, pathogenic bacteria constantly increases the resistance of common antibacterials, and the treatment of drug-fast bacteria infection has become a global difficult problem.
Microbiotic is most widely used antibacterials in the hospital clinical, is controlling, is preventing and treat in the various infectious diseases to play a significant role.Yet since nineteen thirty-nine, microbiotic came out, over particularly past 10 years, the bacterial resistance rate increases rapidly, and was especially serious during nosocomial infection.Effectively treatment means is fewer and feweri, and M ﹠ M increases.
According to the preliminary statistics, microbiotic has reached kind more than 200 at present, but how long their " life-span " does not all continue.Almost each microbiotic can both and destroy by many kind opposings in the bacterium.The faecalis of for example anti-beta-lactam and aminoglycoside has produced the resistance to vancomycin again.In a single day this bacterial strain is infected by the high risk population, just may can select for use by antibiotic-free.Bacterial strain in the streptococcus aureus more than 90% has produced β-Nei Xiananmei, adds a part of bacterial strain and carries effable mecA gene, and these bacterial strains are all no longer responsive to all β-Nei Xiananleikangshengsus, but also expand many other class microbiotic to.Therefore but vancomycin just becomes unique medicine for treatment.Under the situation of a large amount of use vancomycins, the bacterial strain of vancomycin resistance might appear.In September, 2002, the drug resistance of vancomycin streptococcus aureus of an example by the vanA gene mediated reported by the U.S., caused global relevant scholar and doctor's [the Tenover F that shows great attention to, Weigel L, Appelbaum P, et al.Vancomycin-resistant Staphylococcus aureus isolate from apatient in Pennsylvania.ANTIMICRO.AGENTS CHEMOTH.2004,48:275-280].
The continuous appearance of various resistant organisms can cause serious consequence, for example, causes that operative treatment failure, complication increase, infection and recurrence, hospital stays prolong, the usage quantity increase of expensive microbiotic and other medicine etc.Persister also spreads in the world along with the high speed development of international trade and tourism.Understand appearance reason, the mechanism of drug tolerant bacteria, grasping and using correct detection method is in time to find Resistant strain, effectively prevent and control the key of drug tolerant bacteria diffusion.Grasp bacterial resistance mechanism timely and accurately, significant to the development of instructing clinical rational drug use and new antibacterials.
Directly bacterial detection is to different antibiotic susceptibility (being drug-resistant phenotype), be that the direct and the most the most frequently used method of Drug Resistance Detection is carried out in present laboratory, but recall rate is subjected to influence of various factors such as the pH of measuring method, incubation temperature, time, substratum and concentration bigger, therefore, must learn its measuring method and carry out strictness control, otherwise be difficult to obtain correct result.
Drug-resistant phenotype detection method commonly used has: (1) disk diffusion method, judge sensitivity, intermediary or resistance to measure inhibition zone diameter, this method is subjected to the influence of many factors, as inoculation bacterium amount, preincubate time, antibiotic content and diffusive force, plate thickness etc.(2) dilution method can be measured certain medicine to detecting the minimal inhibitory concentration (MIC) of bacterium.Its shortcoming is consuming time, effort.(3) E test is to be released by Sweden AB Biodisk company in 1988, and this method can directly be measured the MIC of medicine to bacterium to be measured in conjunction with dilution method and diffusion ratio juris and characteristics.Its shortcoming is to cost an arm and a leg, and the result is subjected to multiple factor affecting as disk diffusion method.(4) instrumentation and automatic assay are as Vitek system, MicroScan system etc.By detecting turbidity or the fluorescence intensity of fluorescent indicator or the hydrolysis reaction sentence read result of fluorogenic substrate, the automatization assessing instrument also has certain limitation.At first the selection of medicine lacks handiness, secondly is resistance, especially the quick medicine-sensitive plate that is difficult to detect some special bacterium, and its reliability is not good.But in 3-4 hour, be difficult to reach detection level as some bacterium and chemical sproof bacterium of delay expression that produce inducible enzyme.Low anti-VanB type also can cause omission because of incubation time is short in faecalis.In addition, it is separating process that above susceptibility detection method and bacterium kind are identified, therefore obtains the long time of bacteriology report needs that portion contains susceptibility detection and bacterium kind.And susceptibility detects and to identify with kind and to combine, and will make the result more accurate, thereby provides more more Useful Informations for clinical treatment.
Except that the method for above-mentioned detection drug-resistant phenotype, drug sensitive test also can detect its drug resistant gene type by means of molecular biology method.Detect the mecA gene as mould being have " gold standard " that drug-fast staphylococcus aureus MRSA (Methicilin-Resistant Staphylococcus aureus) detects as polymerase chain reaction (PCR), real time PCR detects MRSA etc.Compare with conventional phenotype susceptibility detection method, the advantage of molecular biology method is that quick and precisely when MIC was in drug-fast threshold value, molecular biology method can be treatment strong foundation is provided.But conventional molecular Biological Detection index is single, thereby practicality is relatively poor.
Two. the main resistance problem of current gram-positive microorganism
In recent years, aerobic gram positive bacteria infection proportion in bacterial infection cause of disease bacterium is and increases trend, mainly contains penicillin resistant streptococcus pneumoniae, methicillin-resistant staphylococcus, vancomycin-resistant enterococcus and the gram-positive cocci of anti-macrolide antibiotics the etc.
1. methicillin-resistant staphylococcus (MRSA)
From 1961 since Britain finds the first MRSA [Stewart, G.T., and R.J.Holt.1963.Evolution of natural resistance to the newer penicillin.Br.Med.J.1:308-311], its infection rate constantly rises.In China, the report MRSA of the hospital infection rate that has is up to [the challenge Chinese journal of medical examination that the check of Zhou Huiping clinical bacteriology faces, 1999,22:12-14.] more than 70%.Thought in the past that coagulase negative staphylococcus (CNS) was the non-pathogenic bacteria of commensalism in human skin and mucous membrane.In recent years, being extensive use of of medical embedded device such as ductus venosus particularly, the hospital infection pathogenic bacteria that this bacterium is become have important clinical significance.Account for the 9%[Kloos of nosocomial infection, W.E., and T.L.Bannerman.1994.Update on clinical significance ofcoagulase-negative Staphylococci.Clin.Microbiol.Rev.7:117-140.].MRSA and methicillin resistant coagulase negative staphylococcus (MRCNS) increase just with surprising rapidity, the infection that the ever-increasing staphylococcus of the resistance of β-Nei Xiananleikangshengsu is caused has become serious day by day clinical problem [Couto I, Melo-Cristino J, Fernandes ML, et al.Unusually large number ofmethicillin-resistant Staphylococcus aureus clones in a Portuguese hospital.J Clin Microbiol, 1995,33:2032-2035.].Staphylococcus mainly contains 2 aspects to the antibiotic resistance of β-amides:
A. produce β-Nei Xiananmei.Staphylococcus mainly is to produce penicillinase by plasmid-mediated blaZ gene to the resistance of penicillin G, thereby the outer penicillin of hydrolysis born of the same parents reduces the drug level in the born of the same parents and produces resistance.Because penicillin can induce bacterium to produce a large amount of enzymes, so even use heavy dose of penicillin can not treat infection due to such bacterium producing multi enzyme preparation.
B. produce and the extremely low penicillin-binding protein PBP2a of β-Nei Xiananleikangshengsu avidity.This is the main resistance mechanism of MRSA, by the mecA gene mediated on the karyomit(e), and a kind of PBP2a that reduces with β-Nei Xiananleikangshengsu avidity of this genes encoding, thus produce resistance.(penicillin binding proteins PBPs) plays katalysis to penicillin-binding protein in bacteria cell wall is synthetic.β-Nei Xiananleikangshengsu combines with enzyme site on the bacteria cell wall PBPs, thereby the blocking-up bacteria cell wall is synthetic, suppresses the growth of bacterium.Responsive streptococcus aureus has 4 kinds of PBPs, i.e. PBP1,2,3 and 4.β-Nei Xiananleikangshengsu can combine simultaneously with PBP1,2,3 and bring into play maximum antibacterial effect, so PBP1,2,3 is referred to as necessary PBPs.Remove above-mentioned 4 kinds of PBPs, MRSA can produce a kind of extra PBP, be called PBP2a (PBP2 '), in the therapeutic dose scope of most of β-Nei Xiananleikangshengsus, must combine and inactivation with medicine by PBPs, keep active but PBP2a does not combine with medicine, continue to exercise its function, thereby show resistance.MecA is positioned on the chromosomal DNA, can insert in other karyomit(e)s or the plasmid under the transposon effect, resistance is propagated, and can be obtained new resistance by gene recombination.
2. penicillin-fast streptococcus pneumoniae (PRSP)
From 1967 at Australian reported first penicillin resistant streptococcus pneumoniae (penicillin-resistantpneumococcus, PRP) [Hamsman D, Bullen MM.A resistant pneumococcus.Lancet.1967,2:264-265.] since, various countries have all found penicillin-fast S. pneumoniae strains, resistant rate has nothing in common with each other, from 1.3% to 57.8%, and higher [the Sessegolo J F of children's resistant rate, Levin ASS, Levy CE, et al.Distribution of serotypes and antimicrobial resistance of Streptococcuspneumoniae strains isolated in Brazil from 1988 to 1992.J Clin Microbiol, 1994,32:906-911.].Streptococcus pneumoniae is the main pathogenic bacterium of children's community infection, also be especially the first pathogenic bacterium [the Musher DM.Infections caused by Streptococcus pneumoniae:Clinical spectrum of the elderly community infection that is grown up, pathogenesis, immunity and treatment.Clin Infect Dis, 1992,14:801-809; Marrie TJ.Community-acquired pneumonia.Clin Infect Dis, 1994,18:501-515.].The appearance of PRP brings severe challenge to treatment.Recently data shows, PRP and multidrug resistant strain continue to increase [Baquero F.Pneumococcal resistance to β-lactam antibiotics:A globalgeographic overview.Microbial Drug Resistance in the whole world, 1995,2:115].
Streptococcus pneumoniae mainly is that particularly as the PBP2 of penicillin target spot, these modified proteins reduce the affinity of penicillin, thereby penicillin is produced resistance owing to there is the modified forms of PBPs to the resistance of penicillin.Streptococcus pneumoniae produces 6 PBPs, and its encoding gene is high conservative in sensitive strain, but the encoding gene of PBP1a, the PBP2b of low affinity and PBP2x (pbp-1a, pbp-2b, pbp-2x) in some zone have the height variability.These region of variability come from other relevant kinds, especially transform from Streptococcus viridans to obtain.These genes can make PBPs change, thereby reduce with the avidity of penicillin.Find simultaneously, generally crossing drug resistant also can occur to (except the vancomycins) such as cynnematin, tsiklomitsin, erythromycin, paraxin, Streptomycin sulphates, thereby become multidrug resistant streptococcus pneumoniae the drug-fast bacterial strain of penicillin height.
3. the faecalis of high aminoglycoside-resistant and vancomycin resistance
Faecalis is the normal microflora in human skin, the upper respiratory tract, digestive tube and the urogenital tract, when causing flora imbalance for a certain reason, the infection at above-mentioned position be can cause, even urinary tract infections, suppurative abdominal infection, septicemia and endocarditis caused as primary lesion.Faecalis is one of The main pathogenic fungi of present nosocomial infection, the person's that often causes the immunologic hypofunction infection, and sum frequency takes place and accounts for second (12%) in it.The report of China bacterial drug resistance monitoring center, in whole nation bacterial drug resistance monitoring net (participating in unit is 57 tame Grade III Class A hospitals) the main clinical isolates in 2002, enterococcus faecalis accounts for the 8th, faecium account for the 11st [horse is more. Li Jingyun. Zhang Xinmei. tension force. Hu Changqin. the clinical common bacteria resistance monitoring of the few great .2002 of gold. Chinese laboratory medicine magazine .2004.27 (1): 38-45].Faecalis is multi-drug resistant bacteria and can obtains and transmit resistance, is one of treatment difficult problem of current bacterial infection.The clinical drug combination of often taking is treated.Clinical isolating faecalis antimicrobial resistance is more and more serious, and especially anti-high-level aminoglycoside antibiotics and vancomycin-resistant enterococcus increase gradually.Faecalis since the high anti-gentamicin of the reported first seventies in 20th century, faecalis constantly increases the high-level resistance of aminoglycoside medicaments such as gentamicin, and the resistant rate of faecium is apparently higher than enterococcus faecalis [Wang Qingtao, Xu Yingchun, Wang Hui, Deng. on-site investigation of faecalis resistance and anti-infective medication are inquired into. Chinese journal of medical examination, 1999,22:154-156.].Its resistance mechanism is as follows:
A. aminoglycoside modifying enzyme
At present, the aminoglycoside modifying enzyme of finding in faecalis relevant with the anti-gentamicin of height has 3 kinds: bifunctional enzyme 6 '-O-aminoglycoside Transacetylase-2 "-N-aminoglycoside phosphotransferase (AAC (6 ')-APH (2 ")) and two kinds of phosphotransferase APH (2 ")-Id, APH (2 ")-Ib.Wherein bifunctional enzyme AAC (6 ')-APH (2 ") is the most important aminoglycoside modifying enzyme of finding so far, faecalis to the high resistance of gentamicin nearly all by this enzyme mediation.This enzyme is by aac (6 ')-Ie-aph (2 ")-Ia coding; mainly be present in faecalis; in the gram-positive microorganisms such as streptococcus aureus; be positioned at (similar) Tn4001 or (similar) Tn5281[Simjee S on plasmid and the karyomit(e); Manzoor SE; Fraise AP; et al.Nature of transposon-mediated high-level gentamicin resistance inEnterococcus faecalis isolated in the United Kingdom.J Antimicrob Chemother; 2000; 45:565-575.] etc. in the structure, can quicken its diffusion between bacterium by the transfer of plasmid or transposon.Adenylyl transferase AAD (6 ') is another kind of important aminoglycoside modifying enzyme, and by the aadE genes encoding, mediation is to the resistance of Streptomycin sulphate.
B. the resistance of vancomycin property of medicine
Found the first strain vancomycin-resistant enterococcus (vancomycin-resistantenterococcus in the London from 1988, VRE) since, the enterococcal quantity of glycopeptide resistance is on the increase, even the serious phenomenon that its resistance shifts to other bacteriums occurred.The molecular biology research of VRE vancomycin resistance mechanism shows, VRE is bunch to be caused by the drug resistant gene that is positioned on karyomit(e) or the plasmid to the resistance majority of vancomycin.Difference according to the resistance level of vancomycin and teicoplanin and drug resistant gene bunch can be divided into vanA with glycopeptide resistance faecalis, vanB, vanC, vanD, 6 kinds of genotype such as vanE and vanG.VRE is not only to drug resistance of vancomycin, but also β-Nei Xiananleikangshengsu, aminoglycoside antibiotics are had natural resistance.Glycopeptide antibiotics such as vancomycin combines with peptidoglycan precursor small peptide C end D-Ala-D-Ala specificity on the bacteria cell wall, thereby suppresses transpeptidation reaction, makes the bacterium can not the synthetic cell wall and death.In above several VRE drug-resistant types, have only vanA and vanB type to have important clinical significance.VanA and vanB type VRE not only have the higher resistance of vancomycin property of medicine, and its resistance determining factor can shift, and are to cause to infect and the popular main pathogen.
4. the Macrolide resistance of gram-positive microorganism
Macrolide-lincomycin class-streptogramine B (Macrolide-Lincosamide-Streptogramin B, MLS
B) being widely used in the treatment of staphylococcal infections, its resistance is also in continuous increase.Be mainly ermA, the 23S rRNA methylase of ermB and ermC genes encoding (erythromycin resistance methylase), this enzyme change 23S rRNA go up Macrolide, lincomycin class and chain sun mycin B in conjunction with target spot, thereby cause resistance to the positive mycin B of Macrolide, lincomycin class and chain.The change of rrna target site shows as the crossing drug resistant (MLS to Macrolide, lincomycin class and chain sun mycin B
BDrug-resistant phenotype), it is the main resistance mechanism of Macrolide, wherein ermA and ermC mainly are present in the staphylococcus, reported that ermA mainly is present in the streptococcus aureus, and ermC mainly is present in [Jung-A.Lim among the CNS (coagulase negative staphylococcus), Ae-Ran Kwon, Sook-Kyung Kim, Yunsop Chong, Kungwon Lee and Eung-Chil Choi.Prevalence of resistance to macrolide, lincosamide and streptogramin antibiotics in Gram-positive cocci isolated in aKorean hospital.JAC.2004.49:489-495.].ErmB mainly is present in faecalis and the suis.
Drug-fast another mechanism of Macrolide is the mechanism of pumping, mainly by mefA/E (macrolide efflux) and msrA/B (macrolide and streptogramin resistance) gene mediated, these two pump genes are different, the pump of mef genes encoding only pumps Macrolide, and the pump of msr genes encoding all produces resistance to Macrolide and chain sun mycin.Macrolide resistance more common [Ross in coagulase negative staphylococcus by the msrA gene mediated, J.I., E.A.Eady, J.H.Cove, W.J.Cunliffe, S.Baumberg, and J.C.Wootton.1990.Inducible erythromycin resistance in staphylococci is encoded by a member of ATP-binding transport super-gene family.Mol.Microbiol.4:1207-1214.].The Macrolide resistance of mefA gene mediated is more common [Sutcliffe in suis then, J., Tait-Kamradt, A.and Wondrack, L. (1996) .Streptococcus pneumoniae and Streptococcus pyogenesresistant to macrolides but sensitive to clindamycin:a common resistance patternmediated by an efflux system.Antimicrobial Agents and Chemotherapy.40,1817-24.] [Characterization of a genetic element carrying the macrolide efflux gene mef (A) in Streptococcus pneumoniae Antimicrob.Agents Chemother.44 (9), 2585-2587 (2000)].
In sum, clinical medicine has proposed more urgent requirement to bacteriological analysis.From taking sample to often needing 3～4 days to clinical with clear and definite bacteriological analysis result (etiological diagnosis and antibacterials sensitization test), taking of having is longer at present, and this has brought a difficult problem just for clinical timely treatment.The clinical bacteriology check will be satisfied an urgent demand of clinical treatment, will take effective measures, shortens report time, increases reliability and strengthens the dependency of clinical treatment to bacteriologic test, to obtain optimum curative effect.
For clinical application, Bacteria Identification and corresponding Drug Resistance Detection are to complement each other, and be indispensable.Routine inspection means to bacterium comprise morphological examination clinically, separation and Culture evaluation and drug sensitive experiment etc.These method complex operations, segmentation carry out, very long and poor accuracy consuming time.Microorganism is identified automatically and the susceptibility system has begun to be applied to the Clinical microorganism check, this phenomenon is taken on a new look to some extent at large hospital, but it still has the intrinsic defective fully based on the phenotypic evaluation scheme of microbial culture.Be badly in need of a kind of quick, accurate, easy and can detect the detection method of the multinomial different indexs of multiple Different Kinds of Pathogens or same cause of disease simultaneously clinically.Undoubtedly, biochip technology [Fodor SP based on gene test, et al.1991.Science.251:767-773], fast with its analysis speed, can the parallel detection of many indexs, required reagent and sample size be few etc., and advantage has adapted to this needs, for addressing these problems the technological means that provides new.
More existing patents also utilize biochip technology to carry out bacteriological detection, but they have following limitation:
1. can only be used for the bacterium kind identify (as Li Junwen etc., a kind of oligonucleotide probe that is used for detecting simultaneously various bacteria, application number 01120290.4; Li Junwen etc., a kind of dna microarray that detects common pathogen in the water, application number 01120291.2; Ma Wei etc. are based on the biochip that the common clinical pathogenic bacteria of the single-minded sequence of 16S rDNA gene kind is diagnosed, application number 02136567.9) or bacterial resistance detection (Liu Yuan etc. adopt biochip technology to detect drug resistant gene, application number 01120441.9).
2. claimed range is extensive, and not at special population, practicality is relatively poor.
3. these patents do not provide a cover effective concrete detection scheme, so its feasibility is relatively poor.As (Liu Yuan etc. adopt biochip technology to detect drug resistant gene, application number 01120441.9).
Summary of the invention
The purpose of this invention is to provide a kind of gram positive bacterium kind identifies and the drug resistant gene detection kit.
Gram positive bacterium kind provided by the present invention is identified and the drug resistant gene detection kit, comprise that the primer of the kind specific gene of the gram positive bacterium bacterial strain to be measured that increases and drug resistant gene is right and detect described Gram-positive bacteria strain kind specific gene probe and resistance gene probe.
Described gram positive bacterium is meant the bacterial strain that is accredited as Gram-positive through gramstaining.Described drug resistant gene is meant the drug resistant gene of gram-positive microorganism.Described primer is meant and is used to carry out PCR, the nucleotide sequence of particularly multiple asymmetric PCR amplification.Described probe is meant the nucleotide sequence of hybridizing with above-mentioned PCR product.
Described gram positive bacterium bacterial strain to be measured comprises staphylococcus, faecalis and suis.
Described kind specific gene can be 16S rRNA gene or msrC.
MsrC is the kind specific gene of faecium.
Described drug resistant gene comprises blaZ (Staphylococcus), mecA (Staphylococcus), aac (6 ')-Ie-aph (2 ")-Ia (Staphylococcus and enterococcus spp); pbp-1a; pbp-2b; pbp-2x (streptococcus pneumoniae); aadE (enterococcus spp); vanA (enterococcus spp), vanB (enterococcus spp), ermA (Staphylococcus), ermB (streptococcus and enterococcus spp), ermC (Staphylococcus), mefA (streptococcus) and msrA genes such as (Staphylococcus).
The gene specific sequence length of the forward primer of described primer centering and reverse primer is 15-50 Nucleotide, is preferably 15-35 Nucleotide.
Described test kit also comprises a universal primer, described universal primer size is a 15-35 Nucleotide, its sequence is from the dna sequence dna of the species far away with the bacterium sibship (as Arabidopis thaliana etc.), and confirms as and all unmatched irrelevant sequence of all DNA of bacteria sequences through sequence alignment.But the amplification efficiency of each target gene of balance, and the generation of promotion strand PCR product.
5 ' end of the forward primer of the kind specific gene of described amplification gram positive bacterium bacterial strain to be measured and the primer centering of drug resistant gene or reverse primer connects the above universal primer sequence.
For increasing the amount of marked product, 5 ' end of described universal primer is by fluorescent mark; 5 ' the end that is connected the forward primer of the above universal primer sequence or reverse primer is by fluorescent mark.
Described universal primer has the nucleotide sequence of sequence 2 in the sequence table.
The nucleotide sequence that the forward primer of the 16S rRNA gene of described amplification gram positive bacterium bacterial strain to be measured has sequence 3 in the sequence table, the nucleotide sequence that the reverse primer of the 16S rRNA gene of described amplification gram positive bacterium bacterial strain to be measured has sequence 4 in the sequence table.
Described amplification Gram-positive bacteria strain kind specific gene primer is right to the primer that also can be amplification msrC.The nucleotide sequence that the forward primer of described amplification msrC has sequence 35 in the sequence table, reverse primer has the nucleotide sequence of sequence 36 in the sequence table.
The nucleotide sequence that the forward primer of described amplification blaZ has sequence 7 in the sequence table, reverse primer has the nucleotide sequence of sequence 8 in the sequence table; The nucleotide sequence that the forward primer of amplification mecA has sequence 9 in the sequence table, reverse primer has the nucleotide sequence of sequence 10 in the sequence table; Amplification aac (6 ')-Ie-aph (2 ")-forward primer of Ia has the nucleotide sequence of sequence 11 in the sequence table, and reverse primer has the nucleotide sequence of sequence 12 in the sequence table; The nucleotide sequence that the forward primer of amplification pbp-1a has sequence 13 in the sequence table, reverse primer has the nucleotide sequence of sequence 14 in the sequence table; The nucleotide sequence that the forward primer of amplification pbp-2b has sequence 15 in the sequence table, reverse primer has the nucleotide sequence of sequence 16 in the sequence table; The nucleotide sequence that the forward primer of amplification pbp-2x has sequence 17 in the sequence table, reverse primer has the nucleotide sequence of sequence 18 in the sequence table; The nucleotide sequence that the forward primer of amplification aadE has sequence 19 in the sequence table, reverse primer has the nucleotide sequence of sequence 20 in the sequence table; The nucleotide sequence that the forward primer of amplification vanA has sequence 21 in the sequence table, reverse primer has the nucleotide sequence of sequence 22 in the sequence table; The nucleotide sequence that the forward primer of amplification vanB has sequence 23 in the sequence table, reverse primer has the nucleotide sequence of sequence 24 in the sequence table; The nucleotide sequence that the forward primer of amplification ermA has sequence 25 in the sequence table, reverse primer has the nucleotide sequence of sequence 26 in the sequence table; The nucleotide sequence that the forward primer of amplification ermB has sequence 27 in the sequence table, reverse primer has the nucleotide sequence of sequence 28 in the sequence table; The nucleotide sequence that the forward primer of amplification ermC has sequence 29 in the sequence table, reverse primer has the nucleotide sequence of sequence 30 in the sequence table; The nucleotide sequence that the forward primer of amplification mefA has sequence 31 in the sequence table, reverse primer has the nucleotide sequence of sequence 32 in the sequence table; The nucleotide sequence that the forward primer of amplification msrA has sequence 33 in the sequence table, reverse primer has the nucleotide sequence of sequence 34 in the sequence table.
It is right that described test kit also comprises as the increase primer of 23S rRNA gene of gram positive bacterium bacterial strain to be measured of interior target.
The nucleotide sequence that the forward primer of the 23S rRNA gene of described amplification gram positive bacterium bacterial strain to be measured has sequence 5 in the sequence table, the nucleotide sequence that the reverse primer of the 23S rRNA gene of described amplification gram positive bacterium bacterial strain to be measured has sequence 6 in the sequence table.
The size of described probe is 15-50 Nucleotide.
5 ' end of described probe connects goes up 10-35 oligomerization dT, and preferred the connection gone up 12-25 oligomerization dT.
Described detection Gram-positive bacteria strain kind specific gene probe can be the probe that detects the special 16S rRNA gene probe of gram-positive microorganism kind or detect msrC.
The probe of described detection msrC is for detecting the kind specific gene probe of faecium.
The special 16S rRNA gene probe of described detection gram-positive microorganism kind comprises the detection Staphylococcus, streptococcus aureus, coagulase negative staphylococcus, enterococcus spp, enterococcus faecalis, faecium and Enterococcus durans and Hai Shi faecalis, Enterococcus gallinarum and enterococcus avium and E. casselflavus are conciliate sugared faecalis, streptococcus, streptococcus pneumoniae, the probe of the 16S rRNA gene of streptococcus pyogenes and streptococcus agalactiae.
The nucleotide sequence that the probe of the 16S rRNA gene of described detection Staphylococcus has sequence 37 in the sequence table; The nucleotide sequence that the probe of the 16S rRNA gene of described detection streptococcus aureus has sequence 38 in the sequence table; The nucleotide sequence that the probe of the 16S rRNA gene of described detection coagulase negative staphylococcus has sequence 39 in the sequence table; The nucleotide sequence that the probe of the 16S rRNA gene of described detection enterococcus spp has sequence 40 in the sequence table; The nucleotide sequence that the probe of the 16S rRNA gene of described detection enterococcus faecalis has sequence 41 in the sequence table; The nucleotide sequence that the probe of described detection faecium and Enterococcus durans and the enterococcal 16S rRNA of Hai Shi gene has sequence 42 in the sequence table; Described detection Enterococcus gallinarum and enterococcus avium and E. casselflavus are conciliate the nucleotide sequence that the probe of sugared enterococcal 16S rRNA gene has sequence 43 in the sequence table; The nucleotide sequence that the probe of the 16S rRNA gene that described detection of streptococcus belongs to has sequence 44 in the sequence table; The nucleotide sequence that the probe of the 16S rRNA gene of described detection streptococcus pneumoniae has sequence 45 in the sequence table; The nucleotide sequence that the probe of the 16S rRNA gene of described detection streptococcus pyogenes has sequence 46 in the sequence table; The nucleotide sequence that the probe of the 16S rRNA gene of described detection streptococcus agalactiae has sequence 47 in the sequence table.
The nucleotide sequence that the probe of described detection msrC has sequence 48 in the sequence table.
Described test kit also comprises the general probe of bacterial detection 16S rRNA gene, the general probe of the general probe of gram positive bacterium 16S rRNA gene and gram negative bacterium 16S rRNA gene.
The nucleotide sequence that the general probe of described bacterial detection 16S rRNA gene has sequence 63 in the sequence table, the general probe of described detection gram positive bacterium 16S rRNA gene has the nucleotide sequence of sequence 64 in the sequence table or sequence 1, the nucleotide sequence that the general probe of described detection gram negative bacterium 16S rRNA gene has sequence 65 in the sequence table.
Described detection resistance gene probe comprises detection blaZ, mecA, aac (6 ')-Ie-aph (2 ")-Ia, pbp-1a, pbp-2b, pbp-2x, aadE, vanA, vanB, ermA, ermB, ermC, the probe of mefA and msrA.
The nucleotide sequence that the probe of described detection blaZ has sequence 49 in the sequence table; The nucleotide sequence that the probe of described detection mecA has sequence 50 in the sequence table; Described detection aac (6 ')-Ie-aph (2 ")-probe of Ia has the nucleotide sequence of sequence 51 in the sequence table; The nucleotide sequence that the probe of described detection pbp-1a has sequence 52 in the sequence table; The nucleotide sequence that the probe of described detection pbp-2b has sequence 53 in the sequence table; The nucleotide sequence that the probe of described detection pbp-2x has sequence 54 in the sequence table; The nucleotide sequence that the probe of described detection aadE has sequence 55 in the sequence table; The nucleotide sequence that the probe of described detection vanA has sequence 56 in the sequence table; The nucleotide sequence that the probe of described detection vanB has sequence 57 in the sequence table; The nucleotide sequence that the probe of described detection ermA has sequence 58 in the sequence table; The nucleotide sequence that the probe of described detection ermB has sequence 59 in the sequence table; The nucleotide sequence that the probe of described detection ermC has sequence 60 in the sequence table; The nucleotide sequence that the probe of described detection mefA has sequence 61 in the sequence table; The nucleotide sequence that the probe of described detection msrA has sequence 62 in the sequence table.
Described test kit also comprises the general probe of detection as interior target bacterium 23S rRNA gene.
The nucleotide sequence that the general probe of described bacterial detection 23S rRNA gene has sequence 66 in the sequence table.
Described test kit also comprises surface chemistry Quality Control probe, hybridization Quality Control probe, Quality Control probe and hybridization blanks such as negative control probe.
The size of described Quality Control probe is 15-35 Nucleotide.
The nucleotide sequence that described surface chemistry Quality Control probe has sequence 67 in the sequence table, the nucleotide sequence that hybridization Quality Control probe has sequence 68 in the sequence table, negative control probe has the nucleotide sequence of sequence 69 in the sequence table.
Described hybridization blank is that to contain mass percentage concentration be 50% dimethyl sulfoxide (DMSO) (DMSO) aqueous solution.
Described probe and hybridization blank are fixed on the carrier.
The slide that described carrier is silicon chip, modify with various functional groups or with various functional group deutero-films.
Described carrier is preferably the slide that has aldehyde groups.
Described test kit also comprises the reaction solution that carries out PCR reaction and molecular hybridization.
Contain hybridization Quality Control reporter probe in the described molecular hybridization reaction solution.
The nucleotide sequence of described hybridization Quality Control reporter probe for hybridizing with the hybridization Quality Control probe that is fixed on the carrier, its 5 ' end is by fluorescent mark.
The nucleotide sequence that described hybridization Quality Control reporter probe has sequence 70 in the sequence table.
Second purpose of the present invention provides a kind of method that above-mentioned gram positive bacterium kind is identified and the drug resistant gene detection kit is carried out evaluation of gram positive bacterium kind and drug resistant gene detection of utilizing.
Gram positive bacterium kind provided by the present invention is identified and the drug resistant gene detection method, be may further comprise the steps:
1) utilizes primer PCR in the described test kit increase the kind specific gene and the drug resistant gene of gram positive bacterium bacterial strain to be measured;
2) detection gram-positive microorganism kind specific gene probe and the resistance gene probe in amplified production that step 1) is obtained and the described test kit hybridized, and determines the kind and the contained drug resistant gene thereof of gram positive bacterium bacterial strain to be measured.
Described PCR is multiple asymmetric PCR.
In two forward and reverse primers of the kind specific gene of described pcr amplification gram positive bacterium bacterial strain to be measured and the primer centering of drug resistant gene, the concentration of a primer of 5 ' the above universal primer sequence of end connection is 1-20 times of another primer.
The concentration of described universal primer be the kind specific gene of described pcr amplification gram positive bacterium bacterial strain to be measured and drug resistant gene primer centering two forward and reverse primer concentrations 1-50 doubly.
Described pcr amplification temperature cycle is divided into two stages: each temperature cycle of fs is made up of sex change, annealing and three steps of extension, comprises 10-30 temperature cycle; Each temperature cycle of subordinate phase is made up of sex change and two steps of extension, comprises 10-30 temperature cycle.
The elongating temperature of described subordinate phase is 60-75 ℃, is preferably 70 ℃.
Gramstaining is very easy (＜10min) routine clinical detection, after the present invention separates positive bacteria and negative bacterium fast by dyeing, detect with gene chip again, what the present invention is directed to is modal gram-positive microorganism and important drug resistant gene thereof, has tangible clinical specific aim and practical value.In addition, the present invention is incorporated into Bacteria Identification and Drug Resistance Detection together, can detect common gram positive bacterium kind and important drug resistant gene thereof simultaneously.This helps shortening detection time greatly and strengthens clinical practice, for the spread and epidemic of the rational use of drug of infectation of bacteria and personalized treatment, control Resistant strain etc., has crucial meaning.
Gram positive bacterium kind of the present invention identifies with drug resistant gene detection method and dedicated kit thereof to have integrated degree height, highly sensitive, applied range, and the specificity height, detected result is stable, high reliability features.
Description of drawings
Fig. 1 is the probe of embodiment 1 synoptic diagram of arranging;
Fig. 2 is the fluoroscopic examination result of embodiment 1 hybridization;
Fig. 3 is the probe of embodiment 2 synoptic diagram of arranging;
Fig. 4 is the fluoroscopic examination result of embodiment 2 hybridizations;
Fig. 5 is the probe of embodiment 3 synoptic diagram of arranging;
Fig. 6 is the fluoroscopic examination result of embodiment 3 hybridizations;
Embodiment
Method among the following embodiment is ordinary method if no special instructions.
In multiple asymmetric PCR amplification system of the present invention, archaeal dna polymerase, dNTP, Mg
2+Concentration is identical with conventional PCR with components such as reaction buffers, and can be optimized according to different reactions.Difference is the primer that uses: a gene-specific primer is identical with conventional PCR, and adds the preceding paragraph and the irrelevant oligonucleotide tail of target sequence to be amplified at 5 ' end of another gene specific primer.The different genes Auele Specific Primer can add the identical sequence tail.Also can add an extra universal primer in the reaction system, the general irrelevant sequence tail of its sequence and gene-specific primer is identical, and the concentration of universal primer is higher than gene-specific primer.The pcr amplification temperature cycle is divided into two stages (referring to table 6): the fs is with conventional PCR, comprise sex change, annealing and three steps of extension, annealing temperature can correspondingly be adjusted according to the Tm value of specific gene primer, and similarly, the extension time also can be according to the expanding fragment length adjustment.After 10-30 temperature cycle, begin subordinate phase immediately, 10-30 temperature cycle.The subordinate phase temperature cycle has only sex change and extends two steps, and the temperature of extension is 60-75 ℃.In the amplified reaction of a preceding 10-30 temperature cycle, because annealing temperature and gene-specific primer Tm value are suitable, two gene-specific primers can carry out the common PCR amplification.And, have only gene-specific primer (longer, the Tm value of its total length the is higher) extension of can annealing of tailing, thereby reach the purpose of preparation strand in 10-30 the temperature cycle in back.Universal primer can have following effect: in a preceding 10-30 temperature cycle, it can participate in second amplified reaction that circulation is later, the amplification efficiency of different targets during the balance multiplex amplification; In the whole process of amplification, because universal primer concentration is higher, can be with the gene-specific primer that adds universal sequence, increase the concentration difference with corresponding primer, thereby further help asymmetric amplification and strand generation.Can greatly improve the signal of hybridization.Wherein, gram positive bacterium kind of the present invention is identified primer such as the table 1 that comprises with the drug resistant gene detection kit.
Table 1 gram positive bacterium kind of the present invention is identified the primer that comprises with the drug resistant gene detection kit
Table 2 gram positive bacterium kind of the present invention is identified the specific probe that comprises with the drug resistant gene detection kit
*Y=T or C
Technology and condition that probe (table 2) and PCR product are hybridized are known to the skilled person in this area.For example hybridization conditions is as follows: the rigorous condition of moderate, and promptly at 50-60 ℃, 5 * SSC hybridization is after 1-2 hour, and with solution 2 * SSC, 0.1%SDS, pH8.0 washs, and uses the distilled water room temperature washing then 2 minutes.As highly rigorous hybridization conditions, also may use higher temperature to hybridize (for example at 65-70 ℃).
For highly selective, preferably use relatively low salt concn and/or higher temperature condition, for example to about 0.15mol/L, temperature is from about 50 ℃ to about 65 ℃ from about 0.02mol/L for salt concn.
In a preferred embodiment of the invention, what use in detection is reverse hybridized technology, be about to probe stationary on carrier, suitable carriers is silicon chip, the slide of modifying with various functional groups and with various functional groups (for example nitro) deutero-films (for example nylon membrane, nitrocellulose filter etc.) preferably, most preferably is the slide that has aldehyde groups.The process mark of sample will be derived from, after the sex change of preferably fluorescently-labeled amplification PCR product, hybridize with the probe that is fixed on the slide, in this process, select temperature, ionic strength, pH and other buffer conditions (to hybridize according to the melting temperature (Tm) of the length of nucleic acid probe, composition and expection heterozygote (being the PCR product of mark and combining of probe) referring to Wahl, G.M., etc., Proc.Natl.Acad.Sci.USA.76 (1979) 3683-3687).
In order to detect gram positive bacterial strain kind to be measured and contained drug resistant gene thereof, the hybridization signal of reply target nucleic acid and probe hybridization is for example analyzed, typically, hybridization signal is by fluorescent scanning instrument, for example GenePix4000B scanner (AxonInstruments, Inc., CA, USA) detect, and (for example, Genepix3.0) hybridization signal is analyzed by appropriate software.
Further set forth the present invention below in conjunction with embodiment.
1, detects target and primer, probe
The detection target of this test kit comprises: the streptococcus aureus of Staphylococcus, coagulase negative staphylococcus, the enterococcus faecalis of enterococcus spp, faecium, the streptococcus pneumoniae of streptococcus, and gene mecA, aac (6 ')-Ie-aph (2 ")-Ia, vanA, vanB, pbp-1a/ pbp-2b/ pbp-2x, ermA, ermB, ermC, mefA and msrA.
Select for use and detect corresponding primer of target and probe, as table 3, table 4 (details such as table 1, table 2).
Table 3 gram positive bacterium is identified and Drug Resistance Detection gene chip kit the primer (31) totally
Table 4 gram positive bacterium is identified and the used probe of Drug Resistance Detection gene chip kit (27) totally
| Identify probe | The probe numbering | The resistance probe | The probe numbering | The Quality Control probe | The probe numbering |
| Staphylococcus | PBB-0201053 | mecA | PBB-0204633 | Surface chemistry Quality Control (QC) | PBB-0201090 |
| Streptococcus aureus | PBB-0201002 | aac6 | PBB-0204643 | Hybridization Quality Control (EC) | PBB-0204652 |
| Coagulase negative staphylococcus (CNS) | PBB-0201115 | vanA | PBB-0204645 | 23S rRNA gene (interior mark IC) | PBB-0204655 |
| Enterococcus spp | PBB-0201094 | vanB | PBB-0204647 | Negative control (NC) | PBB-0204656 |
| Enterococcus faecalis | PBB-0201081 | pbp-1a | PBB-0204635 | Bacterium general (BU) | PBB-0201001 |
| Faecium (msrC gene) | PBB-0201113 | pbp-2b | PBB-0204637 | Gram positive bacterium (G+) | PBB-0201024 |
| Streptococcus | PBB-0201005 | pbp-2x | PBB-0204639 | Blank (BC) | 50％DMSO |
| Streptococcus pneumoniae | PBB-0201078 | ermA | PBB-0204661 | ||
| ermC | PBB-0204663 | ||||
| ermB | PBB-0204665 | ||||
| mefA | PBB-0204666 | ||||
| msrA | PBB-0204668 |
Primer and probe are synthetic by Shanghai Bo Ya biotech company.The TAMRA fluorescent mark of part primer 5 ' end and probe 5 ' end is amido modified also to be finished by Shanghai Bo Ya biotech company.
2, the preparation of gram positive bacterium evaluation and Drug Resistance Detection gene chip
1) has the matrix preparation of aldehyde groups
Glass matrix is soaked in the washing lotion ambient temperature overnight.With tap water flushing cleaning the acid solution on the glass matrix, distilled water flushing three times, rinsed with deionized water once, more once with deionized water rinsing.The centrifuge dripping glass matrix, 110 ℃ of dryings 15 minutes, the finish-drying glass matrix.Glass matrix is immersed in 95% ethanol of 1%APTES (isopropylamine base-triethoxyl silane), at room temperature used the shaking table jog 1 hour.With the glass matrix that 95% ethanol clean is crossed, wash once earlier, post rinse is once.Washed glass matrix is put into vacuum drying oven, be evacuated to maximum scale (0.08Mpa to-0.1Mpa), close breather valve, handled 20 minutes for 110 ℃.Then cold to the glass matrix of room temperature is soaked in 12.5% glutaraldehyde solution (400ml 12.5% glutaraldehyde solution: the glutaraldehyde of 100ml 50%, 300ml phosphate buffered saline buffer (1mol/L NaH
2PO
430ml, 2.628g NaCl), adjust pH to 7.0) and, jog is 4 hours under the room temperature.Glass matrix is taken out from glutaraldehyde solution, 3 * SSC rinsing once, deionized water rinsing twice, centrifuge dripping, drying at room temperature.
2) the preparation surface is fixed with the glass matrix (gene chip) of probe
Probe in the table 4 is dissolved among 50% the DMSO, final concentration is 10 μ mol/L.The point sample instrument of employing Cartesian (Cartesian Technologies, Inc., CA, USA) according to pattern (10 row * 12 row) point sample of Fig. 1, every kind of probe repeats 4 points of point sample at every turn.The glass matrix that point is good is placed at room temperature and is spent the night with dry glass matrix, under the room temperature glass matrix is soaked twice in 0.2% SDS then, and each 2 minutes, vibration.With glass matrix with deionized water rinsing twice, rinsed with deionized water once, centrifuge dripping.Glass matrix is transferred to NaBH
4Solution (1.0gNaBH
4Be dissolved among 300ml 1 * PBS, add the 100ml dehydrated alcohol again), room temperature shaking table jog 5 minutes.With glass matrix once, rinsed with deionized water twice, each 1 minute, centrifuge dripping with deionized water rinsing.
3, microbial culture and nucleic acid extraction
7 strain gram positive bacterium bacterial strains in the use table 5 are tested.Wherein external drug sensitive test result is the experimental result according to American National Instrument Committee for Clinical Laboratory Standards (NCCLS) standard method gained.
The Given information of table 5 bacterial strain to be detected
Annotate:
aMedicine: P--penicillin; The OX--Oxazacillin; The FOX--cefoxitin; The GM--gentamicin; The CM--clindamycin; ER--erythromycin; The VAN--vancomycin.
bStaphylococcus and faecalis data are antibacterial circle diameter (mm);
cThe suis data are MIC value (μ g).
In Bechtop, the bacterium streak inoculation is inverted flat board to separate single bacterium colony in MH (Mueller Hinton Agar Medium) culture medium flat plate in incubator, hatches 24h for 35 ℃.
Take by weighing 10mg G1145 type granulated glass sphere and 40mg G1152 type granulated glass sphere (Sigma) in an aseptic 1.5ml centrifuge tube, in pipe, add 25 μ l, 1 * TE.After sterilization on the flame and cooling, gripping toothpick or tip head pick a single bacterium colony from the MH flat board of culturing bacterium with tweezers, and repeated friction is stayed in the pipe bacterium as far as possible fully in the pipe granulated glass sphere at the end, build the centrifuge tube lid.Hand-held centrifuge tube, (TDX-1 type, Beijing is sensible) maximum dynamics sustained oscillation 5min on turbine mixer.Then with 95 ℃ of water-bath 5min of centrifuge tube, put 4 ℃ standby.
4, nucleic acid amplification
Multiple asymmetric PCR reaction system composed as follows: 1 * MasterMix (sky, Beijing is the epoch); The multiple asymmetric PCR universal primer PMB_0408047 of 1 μ mol/L; 16S rRNA gene specific primer PMB-0201042 (0.5 μ mol/L) and PMB-0201002 (0.2 μ mol/L); All the other 14 pairs of gene-specific primers in the table 3 (tailed primer 0.1 μ mol/L, not tailed primer 0.04 μ mol/L); Bacterium liquid or 3 μ l sterilized waters (as blank) after the vibration of 3 μ l granulated glass spherees.The cumulative volume of reaction is 25 μ l.
PCR carries out on PTC-200 (MJ Research Inc.) thermal cycler, adopts the multiple asymmetric PCR amplification thermal cycling program of table 6.
The multiple asymmetric PCR amplification of table 6 thermal cycling program
5, chip hybridization and signal detection
The preparation of hybridization reaction solution is as shown in table 7.
Table 7 hybridization reaction solution
At hybridizing box (HybriCassettes
TM, the CapitalBio Corporation) and the interior 200 μ l distilled water that add, to prevent the hybridization reaction solution evaporation, the surface is fixed with the glass matrix and the cover plate (SmartCover of reactant
TM, the CapitalBio Corporation) be positioned in the hybridizing box.Hybridization reaction solution be heated to 95 ℃ keep making the abundant sex change of PCR product in 5 minutes after, place mixture of ice and water to cool off immediately.Get 13 μ l hybridization reaction solutions by in the space between adding cover plate of the aperture on the cover plate and the glass matrix, build hybridizing box, hybridized 90 minutes for 54 ℃.Take out chip, immerse 2 * SSC, in the solution of 0.2%SDS, room temperature shaking table jog 5 minutes.Again with chip rinsed with deionized water twice, each 2 minutes, centrifuge dripping.
The chip fluorescent signal adopts brilliant
LuxScan
TM-10K/B laser confocal scanning instrument and software kit thereof (CapitalBio Corporation) detect, and testing conditions is: wavelength (wavelength), 555nm; PMT, 80%; Power (power), 80%.
6, interpretation of result
The chip hybridization result as shown in Figure 2.The gained result all is consistent with bacterial strain Given information in the table 5;
The 26001-chip detection is a streptococcus aureus, is consistent with Given information.Chip does not detect the target drug resistant gene, and that external drug sensitive test result also shows corresponding medicine is all responsive;
The TR558-chip detection is a streptococcus aureus, is consistent with Given information.Chip detection goes out mecA, aac (6 ')-Ie-aph (2 ")-Ia, ermA, four kinds of drug resistant genes of ermC; analyze this bacterial strain and should be all drug-fast MRSA bacterial strains of Macrolide such as lincomycin class, erythromycin such as aminoglycosides such as penicillin, Oxazacillin, cefoxitin, gentamicin, clindamycins, be consistent with external drug sensitive test result;
The J175-chip detection is a coagulase negative staphylococcus, is consistent with Given information.Chip detection goes out mecA, aac (6 ')-Ie-aph (2 ")-Ia, ermC, four kinds of drug resistant genes of msrA, analyzes to be similarly all drug-fast MRSCN bacterial strain of all above-mentioned 6 kinds of medicines, also is consistent with external drug sensitive test result;
The M9-chip detection is an enterococcus faecalis, is consistent with Given information.Chip detection goes out aac (6 ')-Ie-aph (2 ")-Ia gene, analyzes aminoglycoside height resistances such as this bacterial strain reply gentamicin, and is consistent with external drug sensitive test result.Chip also detects the ermB gene, but faecalis is mostly to the Macrolide natural drug resistance, and the treatment of infecting for faecalis is generally without Macrocyclolactone lactone kind medicine clinically, so the ermB gene detects index mainly as streptococcic one; And NCCLS is also based on this reason, and unmatchful faecalis detects the chemical sproof standard method of Macrolide, therefore faecalis do not carried out the external drug sensitive test of Macrolide;
The YY7b-chip detection is a faecium, is consistent with Given information.Chip detection goes out the vanA gene, illustrates that this bacterial strain should be rarer drug resistance of vancomycin faecalis (VRE), and external drug sensitive test result has also confirmed this point.In addition, chip does not detect aac (6 ')-Ie-aph (2 ")-Ia gene, analyzes aminoglycoside sensitivities such as this bacterial strain reply gentamicin, and is same consistent with external drug sensitive test result;
The 31111-chip detection is a streptococcus pneumoniae, is consistent with Given information.Chip detection goes out pbp-1a, pbp-2b, the isogenic probe positive of pbp-2x, illustrates that the hypervariable region of these genes does not morph, so to the penicillins sensitivity, and external drug sensitive test result also shows the penicillin sensitivity.In addition, chip does not detect ermB and mefA gene, analyzes this bacterial strain reply Macrolide sensitivity, and is consistent with external drug sensitive test result yet;
The PS53-chip detection is a streptococcus pneumoniae, is consistent with Given information.Chip detection pbp-1a, pbp-2b, the isogenic probe feminine gender of pbp-2x illustrate that variation has taken place in the hypervariable region of these genes, so this bacterial strain should be the drug-fast PRSP of penicillins, and external drug sensitive test result also shows the penicillin resistance.Chip also detects ermB and mefA gene, illustrates that this bacterial strain also tackles the Macrolide resistance, still is consistent with external drug sensitive test result.
More than analyze and to illustrate that the result of the result of present method gained and routine clinical method gained is identical.This illustrates that gram positive bacterium provided by the present invention identifies and the Drug Resistance Detection gene chip kit, the kind of common gram positive bacterium in the identification and detection target specifically, and detect drug resistant gene in its contained detection target specifically.
1, detects target and primer, probe
The detection target of this test kit comprises: the enterococcus faecalis of enterococcus spp, faecium, faecium and Enterococcus durans and Hai Shi faecalis, Enterococcus gallinarum and enterococcus avium and E. casselflavus are conciliate sugared faecalis, and drug resistant gene aac (6 ')-Ie-aph (2 ")-Ia, aadE, vanA and vanB.
Select for use and detect corresponding primer of target and probe, as table 8, table 9 (details such as table 1, table 2).
Table 8 faecalis is identified and Drug Resistance Detection gene chip kit the primer (15) totally
Table 9 faecalis is identified and the used probe of Drug Resistance Detection gene chip kit (27) totally
| Identify probe | The probe numbering | The resistance probe | The probe numbering | The Quality Control probe | The probe numbering |
| Staphylococcus | PBB-0201053 | aac6 | PBB-0204643 | Surface chemistry Quality Control (QC) | PBB-0201090 |
| Streptococcus | PBB-0201005 | aadE | PBB-0204641 | Hybridization Quality Control (EC) | PBB-0204652 |
| Enterococcus spp | PBB-0201094 | vanA | PBB-0204645 | 23S rRNA gene (interior mark IC) | PBB-0204655 |
| Enterococcus faecalis | PBB-0201081 | vanB | PBB-0204647 | Negative control (NC) | PBB-0204656 |
| Faecium (msrC gene) | PBB-0201113 | Bacterium general (BU) | PBB-0201001 | ||
| Dung intestines/durable/Hai Shi faecalis | PBB-0201104 | Gram positive bacterium (G+) | PBB-0201101 | ||
| The sugared faecalis of quail chicken/bird/lead and yellow-collation/separate | PBB-0201110 | Gram negative bacterium (G-) | PBB-0201023 | ||
| Blank (BC) | 50％DMSO |
Primer and probe are synthetic by Shanghai Bo Ya biotech company.The TAMRA fluorescent mark of part primer 5 ' end and probe 5 ' end is amido modified also to be finished by Shanghai Bo Ya biotech company.
2, the preparation of faecalis evaluation and Drug Resistance Detection gene chip
Method is substantially with embodiment 1.Pattern (10 row * 8 row) point sample (104 expression probe PBB_0201104 among Fig. 3,110 expression probe PBB_0201110) according to Fig. 3.
3, microbial culture and nucleic acid extraction
5 strain faecalis bacterial strains in the use table 10 are tested.Wherein external drug sensitive test result is the experimental result according to NCCLS standard method gained.
The Given information of table 10 bacterial strain to be detected
Microbial culture and method for extracting nucleic acid are with embodiment 1.
4, nucleic acid amplification
Multiple asymmetric PCR reaction system composed as follows: 1 * MasterMix (sky, Beijing is the epoch); The multiple asymmetric PCR universal primer PMB_0408047 of 1 μ mol/L; 16S rRNA gene-specific primer PMB-0201042 (0.25 μ mol/L) and PMB-0201002 (0.25 μ mol/L); All the other 6 pairs of gene-specific primers in the table 8 (tailed primer 0.05 μ mol/L, not tailed primer 0.05 μ mol/L); Bacterium liquid or 1 μ l sterilized water (as blank) after the vibration of 1 μ l granulated glass sphere.The cumulative volume of reaction is 25 μ l.
PCR carries out on PTC-200 (MJ Research Inc.) thermal cycler, adopts the multiple asymmetric PCR amplification thermal cycling program of table 6.
5, chip hybridization and signal detection
Method is with embodiment 1.
6, interpretation of result
The chip hybridization result as shown in Figure 4.The gained result all is consistent with bacterial strain Given information in the table 10.This illustrates that gram positive bacterium kind provided by the present invention identifies and the drug resistant gene detection kit, enterococcal kind in the identification and detection target specifically, and detect drug resistant gene in its contained detection target specifically.
1, detects target and primer, probe
The detection target of this test kit comprises: the streptococcus aureus of Staphylococcus, coagulase negative staphylococcus, and gene mecA, blaZ, aac (6 ')-Ie-aph (2 ")-Ia, ermA/ermC and msrA.
Select for use and detect corresponding primer of target and probe, as table 11, table 12 (details such as table 1, table 2).
Primer and probe are synthetic by Shanghai Bo Ya biotech company.The TAMRA fluorescent mark of part primer 5 ' end and probe 5 ' end is amido modified also to be finished by Shanghai Bo Ya biotech company.
2, the preparation of staphylococcus evaluation and Drug Resistance Detection gene chip
Method is substantially with embodiment 1.Pattern (10 row * 8 row) point sample according to Fig. 5.
Table 11 staphylococcus is identified and Drug Resistance Detection gene chip kit the primer (17) totally
Table 12 staphylococcus is identified and the used probe of Drug Resistance Detection gene chip kit (27) totally
| Identify probe | The probe numbering | The resistance probe | The probe numbering | The Quality Control probe | The probe numbering |
| Staphylococcus | PBB-0201053 | mecA | PBB-0204633 | Surface chemistry Quality Control (QC) | PBB-0201090 |
| Streptococcus aureus | PBB-0201002 | blaZ | PBB-0204631 | Hybridization Quality Control (EC) | PBB-0204652 |
| Coagulase negative staphylococcus (CNS) | PBB-0201115 | aac6 | PBB-0204643 | 23S rRNA gene (interior mark IC) | PBB-0204655 |
| Enterococcus spp | PBB-0201094 | ermA | PBB-0204661 | Negative control (NC) | PBB-0204656 |
| Streptococcus | PBB-0201005 | ermC | PBB-0204663 | Bacterium general (BU) | PBB-0201001 |
| msrA | PBB-0204668 | Gram positive bacterium (G+) | PBB-0201101 | ||
| Gram negative bacterium (G-) | PBB-0201023 | ||||
| Blank (BC) | 50％DMSO |
3, microbial culture and nucleic acid extraction
4 strain aureus strains in the use table 13 are tested.Wherein external drug sensitive test result is the experimental result according to NCCLS standard method gained.
The Given information of table 13 bacterial strain to be detected
Microbial culture and method for extracting nucleic acid are with embodiment 1.
4, nucleic acid amplification
Multiple asymmetric PCR reaction system composed as follows: 1 * MasterMix (sky, Beijing is the epoch); The multiple asymmetric PCR universal primer PMB_0408047 of 1 μ mol/L; 16S rRNA gene-specific primer PMB-0201042 (0.5 μ mol/L) and PMB-0201002 (0.25 μ mol/L); All the other 7 pairs of gene-specific primers in the table 11 (tailed primer 0.05 μ mol/L, not tailed primer 0.05 μ mol/L); Bacterium liquid or 1 μ l sterilized water (as blank) after the vibration of 1 μ l granulated glass sphere.The cumulative volume of reaction is 25 μ l.
PCR carries out on PTC-200 (MJ Research Inc.) thermal cycler, adopts the multiple asymmetric PCR amplification thermal cycling program of table 6.
5, chip hybridization and signal detection
Method is substantially with embodiment 1.
6, interpretation of result
The chip hybridization result as shown in Figure 6.The gained result all is consistent with bacterial strain Given information in the table 13.This illustrates that gram positive bacterium kind provided by the present invention identifies and the drug resistant gene detection kit, staphylococcic kind in the identification and detection target specifically, and detect drug resistant gene in its contained detection target specifically.
Sequence table
<160>70
<210>1
<211>35
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
tttttttttt ttaaggggca tgatgatttg acgtc 35
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
ggtttcggat gttacagcgt 20
<210>3
<211>40
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
ggtttcggat gttacagcgt agagtttgat cctggctcag 40
<210>4
<211>17
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
aaggaggtga tccagcc 17
<210>5
<211>40
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>5
ggtttcggat gttacagcgt aacggtccta aggtagcgaa 40
<210>6
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>6
ggctcctacc tatcctgtac a 21
<210>7
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>7
caacgtctaa aagaactagg aga 23
<210>8
<211>41
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>8
ggtttcggat gttacagcgt tagtcttttg gaacaccgtc t 41
<210>9
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>9
gatggctatc gtgtcacaat c 21
<210>10
<211>40
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>10
ggtttcggat gttacagcgt tgagttgaac ctggtgaagt 40
<210>11
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>11
agccttggga agatgaagtt 20
<210>12
<211>42
<212>DNA
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<220>
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ggtttcggat gttacagcgt gccacactat cataaccact ac 42
<210>13
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gtggttttca atttcctcgt c 21
<210>14
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ggtttcggat gttacagcgt ctaaacaagg tcggactcaa c 41
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tgctacatac tgagccaact 20
<210>16
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ggtttcggat gttacagcgt atatggtcca aacagcctta g 41
<210>17
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tcagtgctaa aacaggggaa 20
<210>18
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ggtttcggat gttacagcgt tcatcccaac gttacttgag t 41
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agccggagga tatggaatta tt 22
<210>20
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ggtttcggat gttacagcgt aaaagtttct cccacaaatc ctc 43
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accaaatcag gctgcagta 19
<210>22
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ggtttcggat gttacagcgt ctaatacgat caagcggtca at 42
<210>23
<211>19
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acgcttacct accctgtct 19
<210>24
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ggtttcggat gttacagcgt aagatcaaca cgagcaagc 39
<210>25
<211>21
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cctgtcggaa ttggttttta g 21
<210>26
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ggtttcggat gttacagcgt cggtaaaccc ctctgagaat a 41
<210>27
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taacgacgaa actggctaaa at 22
<210>28
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ggtttcggat gttacagcgt tggtatggcg ggtaagtttt 40
<210>29
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agtaatgcca atgagcgttt t 21
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ggtttcggat gttacagcgt ggtgtaattt cgtaactgcc a 41
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cagggcaagc agtatcatta a 21
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ggtttcggat gttacagcgt caaacggagt ataagagtgc t 41
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tacttgaagc tatttaccac ca 22
<210>34
<211>41
<212>DNA
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ggtttcggat gttacagcgt taatttcgtt ctttccccac c 41
<210>35
<211>20
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<223>
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gaacatatcc gcaaacaagg 20
<210>36
<211>44
<212>DNA
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<220>
<223>
<400>36
ggtttcggat gttacagcgt catctaacag caacacatta cttg 44
<210>37
<211>32
<212>DNA
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<220>
<223>
<400>37
tttttttttt tttcctccat atctctgcgc at 32
<210>38
<211>32
<212>DNA
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<220>
<223>
<400>38
tttttttttt ttagaagcaa gcttctcgtc cg 32
<210>39
<211>34
<212>DNA
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<220>
<223>
<400>39
tttttttttt ttggagcaag ctccttrtct gttc 34
<210>40
<211>30
<212>DNA
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<220>
<223>
<400>40
tttttttttt ttgtttccaa gtgttatccc 30
<210>41
<211>30
<212>DNA
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<220>
<223>
<400>41
tttttttttt ttccctctga tgggtaggtt 30
<210>42
<211>34
<212>DNA
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<220>
<223>
<400>42
tttttttttt ttcctttcaa atcaaaacca tgcg 34
<210>43
<211>33
<212>DNA
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<220>
<223>
<400>43
tttttttttt ttcggtgaaa gaaaaagcgt tcg 33
<210>44
<211>31
<212>DNA
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<220>
<223>
<400>44
tttttttttt ttgttagccg tccctttctg g 31
<210>45
<211>30
<212>DNA
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<220>
<223>
<400>45
tttttttttt ttgtgatgca agtgcacctt 30
<210>46
<211>38
<212>DNA
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<220>
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tttttttttt ttttactaac atgcgttagt ctctctta 38
<210>47
<211>39
<212>DNA
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<220>
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tttttttttt ttactaacat gtgttaatta ctcttatgc 39
<210>48
<211>38
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<220>
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tttttttttt ttggaatcct tctctctccg aaagtaag 38
<210>49
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<212>DNA
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<220>
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tttttttttt ttctgctttc ggtaagactt taaataaact t 41
<210>50
<211>36
<212>DNA
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<220>
<223>
<400>50
tttttttttt tttatccacc ctcaaacagg tgaatt 36
<210>51
<211>39
<212>DNA
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<220>
<223>
<400>51
tttttttttt ttattggagt aaaggaattg gtacaagat 39
<210>52
<211>40
<212>DNA
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<400>52
tttttttttt ttacttgctc catatttttt gtctgattcg 40
<210>53
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<212>DNA
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tttttttttt ttgcagtacc caagccatat tcgc 34
<210>54
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tttttttttt ttagtgatga tgttggctgc tgctatt 37
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<212>DNA
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tttttttttt ttgcaatgaa ttttggaatg taacacctt 39
<210>56
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tttttttttt ttgtctagcc cgtgtggata tgt 33
<210>57
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tttttttttt ttacggtatc ttccgcatcc atc 33
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tttttttttt ttatagtaaa cccaaagctc gttgc 35
<210>59
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tttttttttt ttcttggata ttcaccgaac actagg 36
<210>60
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tttttttttt ttttggaaat tatcgtgatc aacaagtt 38
<210>61
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<400>61
tttttttttt ttattggtgt gctagtggat cgtc 34
<210>62
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tttttttttt ttgcaaatgg catactatcg tcaact 36
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tttttttttt ttgctgcctc ccgtaggagt 30
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tttttttttt ttgggcatga tgatttgacg tc 32
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tttttttttt ttagggccat gatgacttga cg 32
<210>66
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<221>misc-feature
<222>(14)
＜223〉y=t or c
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tttttttttt ttayggggtc tttccgtcct gt 32
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tcacttgctt ccgttgagg 19
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tttttttttt ttcctcaacg gaagcaagtg at 32
<210>69
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tttttttttt ttgttgcttc tggaatgagt ttgct 35
<210>70
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atcacttgct tccgttgagg 20
Claims (30)
1, the gram positive bacterium kind is identified and the drug resistant gene detection kit, comprise that the primer of the kind specific gene 16S rRNA gene of the gram positive bacterium bacterial strain to be measured that increases or msrC and drug resistant gene blaZ, mecA, aac (6 ')-Ie-apb (2 ")-Ia, pbp-1a, pbp-2b, pbp-2x, aadE, vanA, vanB, ermA, ermB, ermC, mefA and msrA is right and detect the probe of above-mentioned Gram-positive bacteria strain kind specific gene and drug resistant gene;
The right base sequence of the primer of described amplification 16S rRNA gene is shown in SEQ ID NO:3 and SEQ ID NO:4; The right base sequence of the primer of described amplification msrC is shown in SEQ ID NO:35 and SEQ ID NO:36; The right base sequence of the primer of described amplification blaZ is shown in SEQ ID NO:7 and SEQ ID NO:8; The right base sequence of the primer of described amplification mecA is shown in SEQ ID NO:9 and SEQ ID NO:10; The right base sequence of the primer of described amplification aac (6 ')-Ie-aph (2 ")-Ia is shown in SEQ ID NO:11 and SEQ ID NO:12; The right base sequence of the primer of described amplification pbp-1a is shown in SEQ ID NO:13 and SEQ ID NO:14; The right base sequence of the primer of described amplification pbp-2b is shown in SEQ ID NO:15 and SEQ ID NO:16; The right base sequence of the primer of described amplification pbp-2x is shown in SEQ ID NO:17 and SEQ ID NO:18; The right base sequence of the primer of described amplification aadE is shown in SEQ ID NO:19 and SEQ ID NO:20; The right base sequence of the primer of described amplification vanA is shown in SEQ ID NO:21 and SEQ ID NO:22; The right base sequence of the primer of described amplification vanB is shown in SEQ ID NO:23 and SEQ ID NO:24; The right base sequence of the primer of described amplification ermA is shown in SEQ ID NO:25 and SEQ ID NO:26; The right base sequence of the primer of described amplification ermB is shown in SEQ ID NO:27 and SEQ ID NO:28; The right base sequence of the primer of described amplification ermC is shown in SEQ ID NO:29 and SEQ ID NO:30; The right base sequence of the primer of described amplification mefA is shown in SEQ ID NO:31 and SEQ ID NO:32; The right base sequence of the primer of described amplification msrA is shown in SEQ ID NO:33 and SEQ ID NO:34;
The probe of the 16S rRNA gene that described detection gram-positive microorganism kind is special is the probe of the detection Staphylococcus of base sequence shown in SEQ IDNO:37, the probe of the detection streptococcus aureus of base sequence shown in SEQ ID NO:38, the probe of the detection coagulase negative staphylococcus of base sequence shown in SEQ ID NO:39, the probe of the detection enterococcus spp of base sequence shown in SEQ ID NO:40, the probe of the detection enterococcus faecalis of base sequence shown in SEQ ID NO:41, the detection faecium of base sequence shown in SEQ ID NO:42, the enterococcal probe of Enterococcus durans and Hai Shi, the detection Enterococcus gallinarum of base sequence shown in SEQ ID NO:43, enterococcus avium, E. casselflavus is conciliate sugared enterococcal probe, the probe that the detection of streptococcus of base sequence shown in SEQID NO:44 belongs to, the probe of the detection streptococcus pneumoniae of base sequence shown in SEQ ID NO:45, the probe of the probe of the detection streptococcus pyogenes of base sequence shown in SEQ ID NO:46 and the detection streptococcus agalactiae of base sequence shown in SEQ ID NO:47;
The base sequence of the probe of described detection msrC is shown in SEQ ID NO:48;
The probe of described detection drug resistant gene is the probe of the detection blaZ of base sequence shown in SEQ ID NO:49, the probe of the detection mecA of base sequence shown in SEQ ID NO:50, the probe of detection aac (6 ')-Ie-aph of base sequence shown in SEQ ID NO:51 (2 ")-Ia; the probe of the detection pbp-1a of base sequence shown in SEQ ID NO:52; the probe of the detection pbp-2b of base sequence shown in SEQ ID NO:53; the probe of the detection pbp-2x of base sequence shown in SEQ ID NO:54; the probe of the detection aadE of base sequence shown in SEQ ID NO:55; the probe of the detection vanA of base sequence shown in SEQ ID NO:56, the probe of the detection vanB of base sequence shown in SEQ ID NO:57, the probe of the detection ermA of base sequence shown in SEQ ID NO:58, the probe of the detection ermB of base sequence shown in SEQ ID NO:59, the probe of the detection ermC of base sequence shown in SEQ ID NO:60, the probe of the probe of the detection mefA of base sequence shown in SEQ ID NO:61 and the detection msrA of base sequence shown in SEQ ID NO:62.
2, test kit according to claim 1 is characterized in that: described gram positive bacterium bacterial strain to be measured is staphylococcus, faecalis and suis.
3, test kit according to claim 1 and 2, it is characterized in that: described test kit also comprises a universal primer, described universal primer size is a 15-35 Nucleotide, its sequence from the dna sequence dna of bacterium sibship species far away, and confirm as and all unmatched irrelevant sequence of all DNA of bacteria sequences through sequence alignment.
4, test kit according to claim 3 is characterized in that: 5 ' end of the forward primer of the kind specific gene of described amplification gram positive bacterium bacterial strain to be measured and the primer centering of drug resistant gene or reverse primer connects the above universal primer sequence.
5, test kit according to claim 4 is characterized in that: 5 ' end of described universal primer is by fluorescent mark; 5 ' the end that is connected the forward primer of the above universal primer sequence or reverse primer is by fluorescent mark.
6, test kit according to claim 5 is characterized in that: the base sequence of described universal primer is shown in SEQ ID NO:2.
7, test kit according to claim 1 and 2 is characterized in that: described test kit comprises that also the primer of 23S rRNA gene of the gram positive bacterium bacterial strain to be measured that increases is right.
8, test kit according to claim 7, it is characterized in that: the base sequence of the forward primer of the 23S rRNA gene of described amplification gram positive bacterium bacterial strain to be measured is shown in SEQ ID NO:5, and the base sequence of the reverse primer of the 23S rRNA gene of described amplification gram positive bacterium bacterial strain to be measured is shown in SEQ ID NO:6.
9, test kit according to claim 1 and 2, it is characterized in that: described test kit also comprises the general probe of bacterial detection 16S rRNA gene, the general probe of the general probe of gram positive bacterium 16S rRNA gene and gram negative bacterium 16S rRNA gene.
10, test kit according to claim 9, it is characterized in that: the base sequence of the general probe of described bacterial detection 16S rRNA gene is shown in SEQ ID NO:63, the base sequence of the general probe of described detection gram positive bacterium 16S rRNA gene is shown in SEQ ID NO:64, and the base sequence of the general probe of described detection gram negative bacterium 16SrRNA gene is shown in SEQ ID NO:65.
11, test kit according to claim 1 and 2 is characterized in that: described test kit also comprises the general probe of bacterial detection 23S rRNA gene.
12, test kit according to claim 11 is characterized in that: the base sequence of the general probe of described bacterial detection 23S rRNA gene is shown in SEQ ID NO:66.
13, test kit according to claim 12 is characterized in that: described test kit also comprises Quality Control probe and hybridization blank; Described Quality Control probe comprises surface chemistry Quality Control probe, hybridization Quality Control probe and negative control probe.
14, test kit according to claim 13 is characterized in that: the size of described Quality Control probe is 15-35 Nucleotide.
15, test kit according to claim 14, it is characterized in that: the base sequence of described surface chemistry Quality Control probe is shown in SEQ ID NO:67, the base sequence of hybridization Quality Control probe is shown in SEQ ID NO:68, and the base sequence of negative control probe is shown in SEQ ID NO:69.
16, test kit according to claim 15 is characterized in that: described hybridization blank is that to contain mass percentage concentration be 50% dimethyl sulphoxide aqueous solution.
17, test kit according to claim 16 is characterized in that: described probe and hybridization blank are fixed on the carrier.
18, test kit according to claim 17 is characterized in that: the slide that described carrier is silicon chip, modify with various functional groups or with various functional group deutero-films.
19, test kit according to claim 18 is characterized in that: described carrier is the slide that has aldehyde groups.
20, test kit according to claim 1 and 2 is characterized in that: described test kit also comprises the reaction solution that carries out PCR reaction and molecular hybridization.
21, test kit according to claim 20 is characterized in that: contain hybridization Quality Control reporter probe in the described molecular hybridization reaction solution.
22, test kit according to claim 21 is characterized in that: the nucleotide sequence of described hybridization Quality Control reporter probe for hybridizing with the described hybridization Quality Control probe that is fixed on the carrier, its 5 ' end is by fluorescent mark.
23, test kit according to claim 22 is characterized in that: the base sequence of described hybridization Quality Control reporter probe is shown in SEQ ID NO:70.
24, utilize the method that the described gram positive bacterium kind of arbitrary claim is identified and the drug resistant gene detection kit is carried out evaluation of gram positive bacterium kind and drug resistant gene detection among the claim 1-23, may further comprise the steps:
1) utilizes primer PCR in the described test kit increase the kind specific gene and the drug resistant gene of gram positive bacterium bacterial strain to be measured;
2) detection gram-positive microorganism kind specific gene probe and the resistance gene probe in amplified production that step 1) is obtained and the described test kit hybridized, and determines the kind and the contained drug resistant gene thereof of gram positive bacterium bacterial strain to be measured.
25, method according to claim 24 is characterized in that: described PCR is multiple asymmetric PCR.
26, method according to claim 25, it is characterized in that: in two forward and reverse primers of the primer centering of described pcr amplification gram positive bacterium bacterial strain to be measured kind specific gene and drug resistant gene, the concentration of a primer of 5 ' the above universal primer sequence of end connection is 1-20 times of another primer.
27, method according to claim 26 is characterized in that: the concentration of described universal primer be described pcr amplification gram positive bacterium bacterial strain to be measured kind specific gene and drug resistant gene primer centering two forward and reverse primer concentrations 1-50 doubly.
28, method according to claim 27 is characterized in that: described pcr amplification temperature cycle is divided into two stages: each temperature cycle of fs is made up of sex change, annealing and three steps of extension, comprises 10-30 temperature cycle; Each temperature cycle of subordinate phase is made up of sex change and two steps of extension, comprises 10-30 temperature cycle.
29, method according to claim 28 is characterized in that: the elongating temperature of described subordinate phase is 60-75 ℃.
30, method according to claim 29 is characterized in that: the elongating temperature of described subordinate phase is 70 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100644349A CN100427610C (en) | 2005-04-15 | 2005-04-15 | Authenticating gram positive bacteria species and method for testing drug resistant gene and dedicating kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100644349A CN100427610C (en) | 2005-04-15 | 2005-04-15 | Authenticating gram positive bacteria species and method for testing drug resistant gene and dedicating kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1687459A CN1687459A (en) | 2005-10-26 |
| CN100427610C true CN100427610C (en) | 2008-10-22 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100644349A Expired - Fee Related CN100427610C (en) | 2005-04-15 | 2005-04-15 | Authenticating gram positive bacteria species and method for testing drug resistant gene and dedicating kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100427610C (en) |
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| CN110066882B (en) * | 2019-04-27 | 2020-08-07 | 浙江大学 | Specific primer and probe combination for detecting drug resistance genes of five glycopeptide drugs in enterococcus and application |
| CN112852938B (en) * | 2021-03-29 | 2022-06-21 | 中国农业大学 | Primer group for gram-positive bacterium drug-resistant gene high-throughput amplicon sequencing and application |
| CN114891908A (en) * | 2022-06-25 | 2022-08-12 | 上海交通大学医学院附属瑞金医院 | Characteristic gene combination, kit and sequencing method for predicting antibiotic drug sensitive phenotype of enterococcus faecium |
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| CN1369271A (en) * | 2002-02-05 | 2002-09-18 | 卿成让 | Bee larve extract and its preparing process |
| CN1396270A (en) * | 2001-07-16 | 2003-02-12 | 军事医学科学院卫生学环境医学研究所 | DNA microarray for detecting frequently countered pathogenic bacteria in water |
| CN1396269A (en) * | 2001-07-16 | 2003-02-12 | 军事医学科学院卫生学环境医学研究所 | Oligonucleotide probe for detecting different bacteria at same time |
| CN1397649A (en) * | 2002-08-19 | 2003-02-19 | 上海华冠生物芯片有限公司 | Biochip base on 16S rDNA gene for diagnosing frequently encountered pathogenic bacteria |
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| CN1396270A (en) * | 2001-07-16 | 2003-02-12 | 军事医学科学院卫生学环境医学研究所 | DNA microarray for detecting frequently countered pathogenic bacteria in water |
| CN1396269A (en) * | 2001-07-16 | 2003-02-12 | 军事医学科学院卫生学环境医学研究所 | Oligonucleotide probe for detecting different bacteria at same time |
| CN1369271A (en) * | 2002-02-05 | 2002-09-18 | 卿成让 | Bee larve extract and its preparing process |
| CN1397649A (en) * | 2002-08-19 | 2003-02-19 | 上海华冠生物芯片有限公司 | Biochip base on 16S rDNA gene for diagnosing frequently encountered pathogenic bacteria |
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