CN102002529A - Fluorescent detection kit and detection method for Klebsiellapneumoniae carbapenamase - Google Patents

Abstract

Translated fromChinese

本发明提供了一种碳青霉烯酶肺炎克雷伯氏菌的荧光检测试剂盒及检测方法。所述试剂盒中特异性引物和荧光探针序列如下：上游引物：5′-ACT GGG CGC GCACCTA-3′，下游引物：5′-TCG CTGTGC TTG TCATCC TT-3′，荧光探针：5′-FAM-CCG TCT ACACCCGGG CGC C-TAMRA-3′，其中FAM为荧光报告基团，TAMRA为荧光淬灭基团。本发明的有益效果主要体现在：使用本发明检测试剂盒，碳青霉烯酶肺炎克雷伯氏菌的检测敏感性高，特异性好，降低了常规PCR扩增的假阳性率；可实现对碳青霉烯酶肺炎克雷伯氏菌的快速、准确、特异检测。The invention provides a carbapenemase Klebsiella pneumoniae fluorescence detection kit and detection method. The specific primers and fluorescent probe sequences in the kit are as follows: upstream primer: 5'-ACT GGG CGC GCACCTA-3', downstream primer: 5'-TCG CTGTGC TTG TCATCC TT-3', fluorescent probe: 5' -FAM-CCG TCT ACACCCGGG CGC C-TAMRA-3', wherein FAM is a fluorescent reporter group, and TAMRA is a fluorescent quencher group. The beneficial effects of the present invention are mainly reflected in: using the detection kit of the present invention, the detection sensitivity of carbapenemase Klebsiella pneumoniae is high, the specificity is good, and the false positive rate of conventional PCR amplification is reduced; Rapid, accurate and specific detection of carbapenemase Klebsiella pneumoniae.

Description

The fluorescence detection reagent kit and the detection method of carbapenem enzyme Klebsiella pneumonia

(1) technical field

The present invention relates to a kind of fluorescence detection reagent kit and detection method of carbapenem enzyme Klebsiella pneumonia.

(2) background technology

Antibiotic-resistant bacteria KPC is carbapenem enzyme Klebsiella pneumonia (Klebsiellapneumoniae carbapenamase) just, in the drug-fast Klebsiella Pneumoniae of a strain imipenum, be found, the 2f group that belongs to the Bush classification, the category-A of Ambler classification can the hydrolysis carbapenems, microbiotic such as penicillins, cephalosporins and aztreonam.At present existing 5 KPC hypotypes are discovered that both at home and abroad carbapenem enzyme Klebsiella pneumonia belongs to 2 types.The carbapenems medicine is generally used for tackling the infectation of bacteria that other microbiotic are unable to cope with, has anti-microbial activity very widely, because of resisting the hydrolysis of most of lactamases, so be usually used in producing super wide spectrum-lactamase (ESBL) and/or AmpC-lactamase (AmpC) bacterial strain that derepresses causes the treatment of severe infections.But the appearance of carbapenem resistance enterobacteria has brought very big difficulty to clinical treatment.After 2000, the KPC enzyme family is found in the New England and the area, Atlanta of the U.S. successively, mainly in klebsiella spp, also is found in other bacterial strains.Because enterobacteria is important clinically hospital infection bacterium, its resistance to the carbapenems antibacterials has brought very big difficulty for clinical anti-infective therapy.The KPC bacterium has been regarded as breaking through the organism of the last line of defense carbapenem antibiotic of clinical treatment, and carbapenem antibiotic does not all play effect to it.

It is reported, expression in Brazilian hygiene department on October 19th, 2010, by October 15, this KPC superbacteria caused 15 people's death in Brasilia, capital, other has 135 people infected, morbidity quantity since several in the past weeks number constantly increase sharply.U.S. sanitary person says that the almost all uncontrollable a kind of superbacteria of existing microbiotic has appeared at the U.S. 35 state hospitals, and wherein infecting at most appears in New York and New Jersey.This superbacteria with new gene also spreads at world's elsewhere.These bacteriums that United States Hospital is found infect the urgent patient especially easily, and 4 die rate up to 30% to 60%.The U.S. says the disease prevention and control center, though the KPC bacterium is the most common in New York and New Jersey, but its trace is all found in U.S. state over half.The KPC bacterium that U.S. various places hospital finds before 10 years has only 1% pair of carbapenem antibiotic to have resistance, and ratio surpasses 8% now.China KPC-2 enzyme gene is mainly seen in China East China, and other area rarely has report.Because the KPC bacterium produces greatly influence to clinical antibiotic use, therefore be necessary to set up a kind of carbapenem enzyme Klebsiella pneumonia (KPC) diagnostic method fast and effectively.

(3) summary of the invention

The present invention seeks to according to carbapenem enzyme Klebsiella pneumonia gene design primer and TaqMan probe, a kind of fluorescence detection reagent kit and detection method thereof that detects carbapenem enzyme Klebsiella pneumonia is provided, can realizes carbapenem enzyme Klebsiella pneumonia is carried out quick, accurate, special detection.

The technical solution used in the present invention is:

A kind of fluorescence detection reagent kit of carbapenem enzyme Klebsiella pneumonia, described test kit mainly comprises Auele Specific Primer and fluorescent probe, PCR damping fluid, deoxidation nucleoside triphosphate mixture and archaeal dna polymerase, and described Auele Specific Primer and fluorescent probe sequence are as follows:

Upstream primer: 5 '-ACTGGGCGCGCACCTA-3 '

Downstream primer: 5 '-TCGCTGTGCTTGTCATCCTT-3 '

Fluorescent probe: 5 '-FAM-CCGTCTACACCCGGGCGCC-TAMRA-3 '

Wherein FAM is the fluorescence report group, and TAMRA is the fluorescent quenching group.

Key of the present invention is the design of Auele Specific Primer and fluorescent probe sequence, other compositions in the test kit, can select by this area routine, this test kit can be used as the qualitative detection of carbapenem enzyme Klebsiella pneumonia, also can add standard substance and carry out detection by quantitative.

Preferably, also can comprise carbapenem enzyme Klebsiella pneumonia gene standard substance in the described test kit, described standard substance sequence is as follows: ggcacggcaa atgactatgc cgtcgtctgg cccactgggcgcgcacctat tgtgttggcc gtctacaccc gggcgcctaa caaggatgac aagcacagcgaggccgtcat cgccgctgcg gctagactcg cgctcgaggg.Carry out fluorescent PCR with the dna solution of the gradient concentration of standard substance and detect, can draw according to the relation of the logarithmic value of copy concentrations and standard substance Ct value and obtain typical curve, record the Ct value of sample DNA after, the reference standard curve can obtain the copy concentrations of sample DNA.

The invention still further relates to a kind of PCR detection method of carbapenem enzyme Klebsiella pneumonia, described method comprises:

(1) extracts sample to be tested DNA;

(2) get specificity amplification primer and specific probe, PCR damping fluid, deoxidation nucleoside triphosphate mixture and archaeal dna polymerase, add the negative control product respectively or testing sample DNA is made into the PCR reaction system, carry out pcr amplification reaction under equal conditions, reaction tubes places the quantitative fluorescence PCR instrument to carry out fluoroscopic examination;

Described Auele Specific Primer and fluorescent probe sequence are as follows:

Upstream primer: 5 '-ACTGGGCGCGCACCTA-3 '

Downstream primer: 5 '-TCGCTGTGCTTGTCATCCTT-3 '

Fluorescent probe: 5 '-FAM-CCGTCTACACCCGGGCGCC-TAMRA-3 '

Wherein FAM is the fluorescence report group, and TAMRA is the fluorescent quenching group;

Described negative control product can be selected by this area ordinary method, select non-target bacteria usually, as Vibrio parahemolyticus, shigella, salmonella, non-resistance Klebsiella pneumonia etc.

(3) select fluoroscopic examination model F AM fluorescence, baseline adjustment is got 3～15 round-robin fluorescent signals, with the vertex setting threshold line of threshold line just above normal negative control; If testing sample fluorescence growth curve surpasses threshold line, and is good logarithmic growth, then be judged as the positive.

For reaching the requirement of detection by quantitative, described method can be simultaneously with the standard substance dna solution of gradient concentration with sample DNA the same terms under carry out pcr amplification reaction, be that X-coordinate, standard substance Ct value are ordinate zou drawing standard curve with the logarithmic value of standard substance dna solution copy concentrations; The Ct value that records of DNA per sample, the reference standard curve obtains the copy concentrations of sample DNA; Described standard substance dna sequence dna is as follows: ggcacggcaa atgactatgc cgtcgtctgg cccactgggcgcgcacctat tgtgttggcc gtctacaccc gggcgcctaa caaggatgac aagcacagcgaggccgtcat cgccgctgcg gctagactcg cgctcgaggg.

Described PCR reaction conditions can be by with reference to the conventional fluorescence quantifying PCR method in this area, and preferred, described pcr amplification reaction condition is as follows: 95 ℃ ofsex change 2 minutes, 95℃ 15 seconds, 60 ℃ were carried out 40 cyclic amplifications in 30 seconds.

The PCR reaction solution is usually prepared (final concentration) by following composition: PCR buffer final concentration is 1 *, the primer of 0.3～0.5 μ M, the fluorescent probe of 0.1～0.3 μ M, the archaeal dna polymerase of 1～2.5U, the dNTPs of 0.2～0.4mM, usually get the template of 2 μ L (about 50ng/ μ l), the reaction cumulative volume is generally 20～50 μ L.PCR buffer final concentration is 1 *, be meant that each component final concentration is identical with 1 * PCR buffer in the damping fluid, adopt commercial 2 * PCR buffer usually, volumetric usage is 1/2 of a PCR reaction solution system cumulative volume.1 * PCR buffer is composed as follows: KCl 50mM, Tris-HCl 10mM, TritonX-1000.1%, MgCl₂1.5mM.

The present invention has set up the method for utilizing the TaqMan fluorescent quantitative PCR technique to detect carbapenem enzyme Klebsiella pneumonia, and clinical sample after testing, shows that this method is practical.Because present method has adopted the pcr amplification technology, the susceptibility that makes carbapenem enzyme Klebsiella pneumonia detect improves greatly, and because the application of fluorescent probe, make its specificity also improve greatly, reduce the false positive rate of conventional pcr amplification, made us can in few sample, obtain enough monitoring informations.

The present invention has adopted advanced at present specificity fluorescent probe hybridization quantitative PCR detection technique, reduces false positive as far as possible and disturb under the prerequisite that keeps hypersensitivity.Before the real-time fluorescence quantitative PCR detection technique occurs, no matter people are direct PCR or competitive PCR quantitatively to pcr template, basically all to pass through PCR product electrophoresis, again with electrophoresis result machine picture processing as calculated, determine what of final PCR product amount according to the brightness of electrophoretic band, or the PCR product of the tape label mode with ELISA detected, infer the amount of starting template more thus, but these methods in fact all belong to the sxemiquantitative level, because even the PCR condition has been optimized to optimum extent, the unstable of the operation of electrophoresis and subsequent step still can be brought influence to interpretation of result, thereby influences quantitatively this result.Appearance along with the real-time fluorescence quantitative PCR technology, people can accomplish the accurate quantification to pcr template veritably, this high sensitivity that has quantitatively not only kept conventional PCR, and because specificity fluorescent probe hybridization The Application of Technology makes the specificity of detected gene improve greatly.

Beneficial effect of the present invention is mainly reflected in: the detection sensitivity height of carbapenem enzyme Klebsiella pneumonia, and specificity is good, has reduced the false positive rate of conventional pcr amplification; Can realize quick, accurate, the special detection of carbapenem enzyme Klebsiella pneumonia.

(4) description of drawings

Fig. 1 detects for the real-time fluorescence quantitative PCR standard substance: carbapenem enzyme Klebsiella pneumonia real-time fluorescence quantitative PCR standard substance detected result is respectively 10 from left to right⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10¹Copies/ μ L standard substance.

Fig. 2 is the real-time fluorescence quantitative PCR typical curve.Typical curve is Y=-3.494 * lgX+34.916; Y: corresponding CT value; X: the copies of carbapenem enzyme Klebsiella pneumonia gene.

Fig. 3 is 5 routine clinical samples fluorescence quantitative PCR detection results of different concns, and the CT value is respectively 24.32,25.09,28.15,28.91 and 32.97, and copy number is respectively 6.58 * 10⁴Copies/ μ L, 3.09 * 10⁴Copies/ μ L, 7.41 * 10²Copies/ μ L, 2.25 * 10²Copies/ μ L and 0.003copies/ μ L.

(5) embodiment

The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

Embodiment 1:

1, material:

Bacterial genomes DNA extraction reagent is available from the precious biotechnology in Dalian company limited; Restriction enzyme is available from U.S. LTI/Gibco company, pGEM-T-Easy cloning system, Taq archaeal dna polymerase be available from U.S. Promega company, sequencing reagent, 377 type sequenators, Bio-Rad icycler PCR instrument, Rotor Gene Q 5-plex type quantitative PCR instrument (Qiagen company).

2, primer and probe design and synthetic:

With carbapenem enzyme Klebsiella pneumonia gene order (number of registration is AY034847) is template, uses Primer Express^TM(V2.0, American AB I company) software analysis TaqMan primer and probe site therefrom selected best of breed.

Standard substance PCR sequence:

Upstream primer: 5 '-GGCACGGCAAATGACTATGC-3 ',

Downstream primer: 5 '-CCCTCGAGCGCGAGTCTAGC-3 ',

Detect and be with the PCR sequence:

Upstream primer: 5 '-ACTGGGCGCGCACCTA-3 '

Downstream primer: 5 '-TCGCTGTGCTTGTCATCCTT-3 '

Fluorescent probe: 5 '-FAM-CCGTCTACACCCGGGCGCC-TAMRA-3 '

Wherein FAM is the fluorescence report group, and TAMRA is the fluorescent quenching group.

Synthetic by the farsighted bio tech ltd of Shanghai brightness.

3, examination criteria product preparation:

Adopt the position of ABI 394 oligonucleotide synthesizers, the oligonucleotide fragment of the synthetic 140bp of full gene according to standard substance PCR sequence in the carbapenem enzyme Klebsiella pneumonia gene order.Increase at the enterprising performing PCR of Bio-Rad icycler PCR instrument with standard substance upstream and downstream primer:

The PCR reaction solution is composed as follows:

2×PCR?buffer 10.0μL

Standard substance upstream primer (10 μ M) 1 μ L

Standard substance downstream primer (10 μ M) 1 μ L

Archaeal dna polymerase (5U/ μ L) 0.2 μ L

DNTPs (each 250mM) 1.6 μ L

(dATP, dTTP, dCTP, dGTP amount of substance were than 1: 1: 1: 1)

Template DNA (50ng/ μ L) 1 μ L

Water complements to 20 μ L.

The PCR condition is: 94 ℃ of sex change in 5 minutes, 94℃ 20 seconds, 55℃ 20 seconds, 72 ℃ were carried out 35 cyclic amplifications in 20 seconds, at last in 72 ℃ extend 5 minutes rearmounted 4 ℃.

The PCR product promptly inserts the pGEM-T-Easy cloning vector with cloning system after electrophoresis detection, and with positive colony through sequence verification.Reclaim the 140bp fragment, be standard substance, measure concentration and be converted into (copy number/volume).

4, result:

Through order-checking, above-mentioned standard product conform to expection fully, and the standard substance fragment sequence of recovery is as follows: ggcacggcaa atgactatgc cgtcgtctgg cccactgggc gcgcacctat tgtgttggccgtctacaccc gggcgcctaa caaggatgac aagcacagcg aggccgtcat cgccgctgcggctagactcg cgctcgaggg (SEQ ID No.4).

Embodiment 2: fluorescence quantitative PCR method detects clinical sample

1, sample detects:

5 routine manual simulation's samples adopt extracting genome DNA reagent to extract genomic dna, get 1.0 μ L respectively and do template, use downstream primer in the enterprising performing PCR amplification of Rotor Gene Q 5-plex type quantitative PCR instrument (Qiagen company) with detecting.

The PCR reaction solution is composed as follows:

2×PCR?buffer 10.0μL

Detect with upstream primer (10 μ M) 1 μ L

Detect with downstream primer (10 μ M) 1 μ L

Detect with fluorescent probe (10 μ M) 0.5 μ L

Archaeal dna polymerase (5U/ μ L) 0.2 μ L

DNTPs (each 250mM) 1.60 μ L

(dATP, dTTP, dCTP, dGTP amount of substance were than 1: 1: 1: 1)

Template DNA (50ng/ μ L) 1 μ L

Water complements to 20 μ L.

The PCR reaction conditions is: 95 ℃ ofpre-sex change 2 minutes, 95 ℃ 15 seconds, 60 ℃ were carried out 40 cyclic amplifications in 30 seconds.

Under the same terms, carry out the PCR detection with negative contrast of non-resistance Klebsiella pneumonia (ATCC 700603), with the vertex setting threshold line of threshold line just above normal negative control; If testing sample fluorescence growth curve surpasses threshold line, and is good logarithmic growth, then be judged as the positive, otherwise be judged as feminine gender.

Detect with the different concns standard substance under the same conditions simultaneously, and the drawing standard curve.

The measurement result of sample to be tested is handled the quantity that calculates the carbapenem enzyme Klebsiella pneumonia gene in the detection sample according to typical curve through instrument.

Carrying out PCR according to the method described above with Vibrio parahemolyticus (ATCC 17802), shigella (ATCC 12022), salmonella (ATCC 50035), streptococcus aureus (ATCC 25933), non-resistance Klebsiella pneumonia (ATCC 13883) and campylobacter jejuni (ATCC 33560) bacterial strain detects, detected result all is negative, and illustrates that the inventive method specificity is good.

2, sample detection result

The standard substance detected result is referring to Fig. 1, and typical curve is referring to Fig. 2.

5 routine clinical samples detect the carbapenem enzyme Klebsiella pneumonia positive, detected result is referring to Fig. 3: 5 routine positive clinical samples fluorescence quantitative PCR detection results, the CT value is respectively 24.32,25.09,28.15,28.91 and 32.97, and copy number is respectively 6.58 * 10⁴Copies/ μ L, 3.09 * 10⁴Copies/ μ L, 7.41 * 10²Copies/ μ L, 2.25 * 10²Copies/ μ L and 0.003copies/ μ L.

The present invention is described in conjunction with most preferred embodiment, yet after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (5)

Translated fromChinese

1.一种碳青霉烯酶肺炎克雷伯氏菌的荧光检测试剂盒，所述试剂盒主要包括特异性引物和荧光探针、PCR缓冲液、脱氧三磷酸核苷混合物和DNA聚合酶，其特征在于所述特异性引物和荧光探针序列如下：1. a fluorescent detection kit of carbapenemase Klebsiella pneumoniae, said kit mainly includes specific primers and fluorescent probes, PCR buffer, deoxynucleoside triphosphate mixture and DNA polymerase, It is characterized in that the sequences of the specific primers and fluorescent probes are as follows:上游引物：5′-ACTGGGCGCGCACCTA-3′Upstream primer: 5′-ACTGGGCGCGCACCTA-3′下游引物：5′-TCGCTGTGCTTGTCATCCTT-3′Downstream primer: 5′-TCGCTGTGCTTGTCATCCTT-3′荧光探针：5′-FAM-CCGTCTACACCCGGGCGCC-TAMRA-3′Fluorescent probe: 5′-FAM-CCGTCTACACCCGGGCGCC-TAMRA-3′其中FAM为荧光报告基团，TAMRA为荧光淬灭基团。Among them, FAM is a fluorescent reporter group, and TAMRA is a fluorescent quencher group.2.如权利要求1所述的试剂盒，其特征在于所述试剂盒中还包括碳青霉烯酶肺炎克雷伯氏菌基因标准品，所述标准品序列如下：ggcacggcaaatgactatgc cgtcgtctgg cccactgggc gcgcacctat tgtgttggcc gtctacacccgggcgcctaa caaggatgac aagcacagcg aggccgtcat cgccgctgcg gctagactcgcgctcgaggg。2. kit as claimed in claim 1, it is characterized in that also comprising carbapenemase Klebsiella pneumoniae gene standard item in said kit, described standard item sequence is as follows: ggcacggcaaatgactatgc cgtcgtctgg cccactgggc gcgcacctat tgtgttggcc gtctacacccgggcgcctaa caaggatgac aagcacagcg aggccgtcat cgccgctgcg gctagactcgcgctcgaggg.3.一种碳青霉烯酶肺炎克雷伯氏菌的PCR检测方法，所述方法包括：3. a PCR detection method of carbapenemase Klebsiella pneumoniae, said method comprising:(1)提取待测样本DNA；(1) Extract the DNA of the sample to be tested;(2)取特异性扩增引物及特异性探针、PCR缓冲液、脱氧三磷酸核苷混合物和DNA聚合酶，分别加入阴性对照品或待测样品DNA配成PCR反应体系，于同等条件下进行PCR扩增反应，反应管置于定量荧光PCR仪进行荧光检测；(2) Take specific amplification primers and specific probes, PCR buffer, deoxynucleoside triphosphate mixture and DNA polymerase, respectively add negative control substance or sample DNA to be tested to form a PCR reaction system, under the same conditions Carry out PCR amplification reaction, and the reaction tube is placed in a quantitative fluorescent PCR instrument for fluorescence detection;所述特异性引物和荧光探针序列如下：The specific primers and fluorescent probe sequences are as follows:上游引物：5′-ACTGGGCGCGCACCTA-3′Upstream primer: 5′-ACTGGGCGCGCACCTA-3′下游引物：5′-TCGCTGTGCTTGTCATCCTT-3′Downstream primer: 5′-TCGCTGTGCTTGTCATCCTT-3′荧光探针：5′-FAM-CCGTCTACACCCGGGCGCC-TAMRA-3′Fluorescent probe: 5′-FAM-CCGTCTACACCCGGGCGCC-TAMRA-3′其中FAM为荧光报告基团，TAMRA为荧光淬灭基团；Wherein FAM is a fluorescent reporter group, and TAMRA is a fluorescent quencher group;(3)选择荧光检测模式FAM荧光，基线调整取3～15个循环的荧光信号，以阈值线刚好超过正常阴性对照的最高点设定阈值线；若待测样品荧光增长曲线超过阈值线，并呈良好的对数增长，则判断为阳性。(3) Select fluorescence detection mode FAM fluorescence, baseline adjustment takes 3-15 cycles of fluorescence signals, set the threshold line with the threshold line just exceeding the highest point of the normal negative control; if the fluorescence growth curve of the sample to be tested exceeds the threshold line, and A good logarithmic growth is judged as positive.4.如权利要求3所述的方法，其特征在于所述方法同时以梯度浓度的标准品DNA溶液在与样品DNA相同条件下进行PCR扩增反应，以标准品DNA溶液拷贝浓度的对数值为横坐标、标准品Ct值为纵坐标绘制标准曲线；根据样品DNA测得的Ct值，对照标准曲线，获得样品DNA的拷贝浓度；所述标准品DNA序列如下：ggcacggcaa atgactatgccgtcgtctgg cccactgggc gcgcacctat tgtgttggcc gtctacaccc gggcgcctaacaaggatgac aagcacagcg aggccgtcat cgccgctgcg gctagactcg cgctcgaggg。4. method as claimed in claim 3, it is characterized in that described method carries out PCR amplification reaction with the standard sample DNA solution of gradient concentration simultaneously with sample DNA identical condition, with the logarithmic value of standard sample DNA solution copy concentration as The abscissa and the Ct value of the standard product are used to draw a standard curve on the ordinate; the Ct value measured according to the sample DNA is compared with the standard curve to obtain the copy concentration of the sample DNA; the DNA sequence of the standard product is as follows: aggccgtcat cgccgctgcg gctagactcg cgctcgaggg.5.如权利要求3或4所述的方法，其特征在于所述PCR扩增反应条件如下：95℃变性2分钟，95℃15秒、60℃30秒进行40个循环扩增。5. The method according to claim 3 or 4, characterized in that the PCR amplification reaction conditions are as follows: denaturation at 95°C for 2 minutes, 40 cycles of amplification at 95°C for 15 seconds and 60°C for 30 seconds.

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