CN102181565B - Fluorescence PCR (Polymerase Chain Reaction) rapid detection primer and kit for super bacteria - Google Patents
Fluorescence PCR (Polymerase Chain Reaction) rapid detection primer and kit for super bacteria Download PDFInfo
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- CN102181565B CN102181565B CN 201110122489 CN201110122489A CN102181565B CN 102181565 B CN102181565 B CN 102181565B CN 201110122489 CN201110122489 CN 201110122489 CN 201110122489 A CN201110122489 A CN 201110122489A CN 102181565 B CN102181565 B CN 102181565B
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Abstract
The invention relates to a fluorescence PCR (Polymerase Chain Reaction) rapid detection primer and kit for super bacteria. The primer comprises a forward primer with a sequence shown as SEQ ID NO: 1 and a reverse primer with a sequence shown as SEQ ID NO: 2. According to sequences of NDM1 (New Delhi Metallobetalactamase) genes of different super bacteria, a specific primer is obtained through molecular biological analysis; and the conserved gene sequence is shared by different bacteria with super drug resistance, so that reliability of the NDM1 genes from different sources is detected at the species level. Target genes can be directly amplified from clinical samples such as bacteria-containing body fluid, foods, body fluid culture and the like of a patient without being influenced by culture conditions and physiological conditions of the bacteria, so that the method is more accurate than the conventional identification method. In addition, fluorescence labeling is adopted for the primer and the kit; and compared with the normal PCR technology, the rapid detection primer and the kit have the advantages that: a gel electrophoresis method does not require to be used for observation; and closed detection of an integrated detection flow is realized.
Description
Technical field
The invention belongs to the disease detection technical field, be specifically related to a kind of superbacteria fluorescent PCR rapid detection primer and comprise the test kit of this primer.
Background technology
There is a large amount of natural drug resistance genes in occurring in nature, and these genes were in the metabolic regulation network complicated in the original host originally, is playing the part of the effect of signal conduction, gene regulating.But when they shifted in the new host of entering through gene level after, because these drug resistant genes are difficult to incorporate the metabolism network in the new host, variation had taken place in its function, shows as antimicrobial resistance.And human microbiotic abuse, environmental pollution as screening pressure, are selected and are evolved that these are integrated with the drug resistant gene germ, make the latter finally become superbacteria.
And the NDM1 gene is the crucial Disease-causing gene of superbacteria, and under the effect of this gene, superbacteria has possessed superpower drug-resistant performance.The NDM1 gene is found after deliberation and can in different types of pathogenic thalline, be shifted through the form of plasmid vector, along with the numerous realization multiplication of the expansion of thalline quantity.2010; The severe infection disease that caused by superpower drug-resistant bacteria has appearred in a plurality of in the world countries; This kind bacterium can be resisted at present broad sense, the most powerful antibiotic treatment, and patient with severe symptoms's treatment is had no way of doing it, and has formed the serious situation that no medicine can be cured.Show through research, in multiple superbacteria body, found a kind of unknown general character gene of mystery, and facilitate multiple pathogenic bacteria just superbacteria to possess powerful chemical sproof factor be exactly this NDM1 gene that was named as afterwards.Even more serious is to judge that tentatively the NDM1 gene is present on the plasmid of bacterium, can freely propagate in mikrobe.Research finds that also this NDM1 is present in India and the Pakistani case widely.Carry the intestinal bacteria and the Klebsiella Pneumoniae of this drug resistant gene, present most microbiotic are comprised that lactams, quinolones, aminoglycoside antibiotics all have resistance." Horizontal Gene Transfer " can take place by plasmid in NDM1 between mikrobe, possibly cause the whole world of this germ to spread.Present case confirms that also NDM1 resistant organism case presents the situation that spreads to Europe from South Asia.
And in conventional detection, generally just judge through drug sensitive experiment whether mikrobe has resistance to a certain microbiotic, and this method is time-consuming, and complex operation can not satisfy the needs of disease control; Simultaneously requirement can be fast again, sensitive detects whether there is superbacteria in food, the water source for the high hazardness of superbacteria, prevents the harm of superbacteria to human body or cultivated animals.
Summary of the invention
The fluorescent PCR rapid detection primer and the test kit that the purpose of this invention is to provide superbacteria, promptly a kind of can whether the existence through detection NDM-gene judged the fluorescent primer that whether has superbacteria in the test sample.
Fluorescent primer of the present invention comprises:
1) sequence is that upstream primer and the sequence of SEQ ID NO:1 are the downstream primer of SEQ ID NO:2;
2) to 1) in primer to through conversion, insert, disappearance or 5 ' add the formed primer of base is right, and primer to the product of amplification under rigorous condition with 1) the amplified production of primer hybridize;
3) 1) or 2) design obtains on the amplified fragments of the primer of describing primer is right.
The fluorescent PCR quick detection kit of superbacteria of the present invention comprises following component:
1) the PCR system premixture of 2 times of concentration;
2) upstream primer and downstream primer, concentration are respectively 10 μ mol/L;
3) optical dye of 20 times of concentration;
4) DNA sample/reporter plasmid, 10 μ g/ μ L;
5) distilled water.
Comprise in above-mentioned DNA sample/reporter plasmid that sequence is the fragment of SEQ ID NO:3.
The present invention is according to the sequence of the NDM1 gene of different superbacterias; Obtained Auele Specific Primer through molecular biological analysis; This conservative gene sequence is common for having super chemical sproof different strain, to guarantee to detect from the level of planting the safety of the NDM1 gene of different sources.Directly amplified target gene from clinical samples such as patient's mycetome liquid, food and body fluid culture does not receive the influence of culture condition and bacterium physiological status, and more traditional authentication method is more accurate.In addition, primer of the present invention adopts fluorescent mark, and the regular-PCR compared with techniques needn't be observed with gel electrophoresis method, has realized that the incorporate sealing of testing process detects.
Embodiment
Fluorescent primer of the present invention is according to the sequence of the NDM1 gene of different superbacterias, has obtained Auele Specific Primer through molecular biological analysis, and this primer comprises:
1) sequence is that upstream primer and the sequence of SEQ ID NO:1 are the downstream primer of SEQ ID NO:2;
2) through conversion, insert, disappearance or 5 ' add the formed primer of base is right, and primer to the product of amplification under rigorous condition with 1) the amplified production of primer hybridize;
3) 1) or the amplified fragments of 2 primers of describing on the primer that obtains of design.
For 2) in the primer described, be included in 5 of primer ' added the different primer of the restriction endonuclease identification formed length of fragment, and the base replacement, insert, the formed variation primer of deletion.These are that the upstream primer of SEQ ID NO:1 and the primer that downstream primer derived that sequence is SEQ ID NO:2 also can be used for detecting superbacteria by sequence, promptly 2) the nucleotide fragments that primer amplified of deriving described in can be under rigorous condition and 1) the amplified production of primer hybridize; Described rigorous condition is with reference to the DIG efficient DNA mark of Roche company and the hybridization conditions in the detection Starter Kit1 handbook.
Primer of the present invention is used for the fluoroscopic examination superbacteria, and each component composition is following in the PCR reaction system:
The pcr amplification program:
(1) 94 ℃ of preparatory sex change 3min;
(2) 94 ℃ of sex change 30s; 55 ℃ of annealing, 30s, 72 ℃ are extended 30s, circulate 40 times.
(3) add at last do solubility curve (95 ℃ of 15sec, 60 ℃ of 15sec, 95 ℃, 15sec)
Primer of the present invention is used to detect superbacteria, and testing process is following:
1, the designs specificity Oligonucleolide primers is used for the chimeric fluorescent PCR detection of optical dye;
Structure can use the reporter plasmid of upstream primer and downstream primer amplification, as the positive control plasmid, prevents false negative result.
The used reporter plasmid of the present invention can be selected Klebsiella pneumonia ADB65 (Klebsiella pneumoniae strain ADB65) NDM1 (GeneBank:HQ259057.1) gene fragment of synthetic for use; Its sequence is SEQ ID NO:3, this amplified fragments is inserted the PMC-T carrier obtain the applied reporter plasmid of the present invention.
2, with fluorescent primer of the present invention as primer, be template with the tested bacteria genomic dna, carry out the specific amplification of goal gene;
Use the fluorescent PCR appearance to carry out the real-time fluorescence luminous intensity measurement in the amplification procedure; And finish the back in the PCR reaction and measure the segmental melting temperature (Tm) of institute's synthetic DNA; Data transmission is crossed fluorescent PCR appearance software kit to the computer expert to be analyzed; Can observe the NDM1 gene is carried out the fluorescence that specific amplification produces, and obtain the melting curve (main peak 90.5 ± 0.5) that the specific fragment that produces holds that increases, then prove to have the NDM1 gene in the testing sample NDM1; As only observe fluorescent signal, and melting curve does not meet the distinctive melting curve of NDM1 gene, and can observe the melting curve of positive control reporter plasmid fluorescent signal and generation thereof, then prove not have the NDM1 gene in the testing sample; As other results occur and then show check failure this time, be not sure of whether there is the NDM1 gene, need check again.
Embodiment 1: effect detection of the present invention
Auele Specific Primer of the present invention detects superbacteria through fluorescent PCR report fluorescent signal; Use the fluorescent PCR appearance to carry out the real-time fluorescence luminous intensity measurement in the amplification procedure; And finish the back in the PCR reaction and measure the segmental melting temperature (Tm) of institute's synthetic DNA; Data transmission is crossed fluorescent PCR appearance software kit to the computer expert to be analyzed; Can observe the NDM1 gene is carried out the fluorescence that specific amplification produces, and obtain the melting curve (main peak 90.5 ± 0.5) that the specific fragment that produces holds that increases NDM1.The result shows that primer of the present invention and test kit can detect the superbacteria that carries the NDM1 gene accurately, can not produce false negative.
Embodiment 2: negative control
With primer of the present invention and test kit; By detection step of the present invention 11 kinds of bacterial classifications that do not carry the NM1 gene such as intestinal bacteria, vibrio alginolyticus, streptococcus aureus, Salmonellas, Salmonella choleraesuls and Pseudomonas aeruginosa are carried out fluoroscopic examination; Use the fluorescent PCR appearance to carry out the real-time fluorescence luminous intensity measurement in the amplification procedure; And finish the back in the PCR reaction and measure the segmental melting temperature (Tm) of institute's synthetic DNA; Data transmission is crossed fluorescent PCR appearance software kit to the computer expert to be analyzed; Do not detect the melting curve that conforms to the distinctive melting curve of NDM1 gene (main peak 90.5 ± 0.5), and can observe the melting curve of positive control reporter plasmid fluorescent signal and generation thereof.Thereby proof false positive results can not occur when the bacterium of not carrying the NDM1 gene is detected.
Claims (3)
1. the fluorescent PCR rapid detection primer of a superbacteria is characterized in that, it is the downstream primer of SEQ ID NO:2 that described primer includes upstream primer and the sequence that sequence is SEQ ID NO:1.
2. the fluorescent PCR quick detection kit of superbacteria comprises following component:
1) the PCR system premixture of 2 times of concentration;
2) upstream primer and downstream primer, concentration are respectively 10 μ mol/L;
3) optical dye of 20 times of concentration;
4) DNA sample/reporter plasmid, 10 μ g/ μ L;
5) distilled water;
Wherein upstream primer and downstream primer are the primer described in the claim 1.
3. test kit as claimed in claim 2 is characterized in that comprising in above-mentioned DNA sample/reporter plasmid that sequence is the fragment of SEQ ID NO:3.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201110122489 CN102181565B (en) | 2011-05-12 | 2011-05-12 | Fluorescence PCR (Polymerase Chain Reaction) rapid detection primer and kit for super bacteria |
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| CN 201110122489 CN102181565B (en) | 2011-05-12 | 2011-05-12 | Fluorescence PCR (Polymerase Chain Reaction) rapid detection primer and kit for super bacteria |
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| CN102181565A CN102181565A (en) | 2011-09-14 |
| CN102181565B true CN102181565B (en) | 2012-12-19 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101948920B (en) * | 2010-09-10 | 2013-05-08 | 杨瑞馥 | Positive control template for PCR detection on drug resistance gene, preparation method and kit thereof |
| CN102002528B (en) * | 2010-11-22 | 2012-09-26 | 浙江省疾病预防控制中心 | Fluorescence detection kit and detection method of antibiotic resistance NDM-1 (New Delhi Metallo-beta-lactamase 1) gene |
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