CN102154487A - Reagent for detecting francisella tularensis and complex probe and fluorescent quantitative polymerase chain reaction (PCR) method for detecting francisella tularensis - Google Patents
Reagent for detecting francisella tularensis and complex probe and fluorescent quantitative polymerase chain reaction (PCR) method for detecting francisella tularensis Download PDFInfo
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Abstract
The invention provides a reagent for detecting francisella tularensis. The reagent comprises an upstream primer, a downstream primer, a fluorescent probe and a quenching probe, wherein the gene sequence FTF1 of the upstream primer is 5'-aagcaagtgttgactacagat-3', the gene sequence FTR1 of the downstream primer is 5'-caccaaagaaccatgttaaac-3', the gene sequence FPFT1 of the fluorescent probe is 5'-FAM-aatcatgttagtacccgctctgcca-p-3', and the gene sequence QPFT1 of the quenching probe is 5'-agcgggtactaacatgat-Dabcyl-3'. The invention also provides a complex probe and fluorescent quantitative PCR method for detecting the francisella tularensis by using the reagent. Compared with the traditional francisella tularensis detecting technology, the invention has the advantages of simple operation, high efficiency, quickness and high specificity.
Description
Technical field
The present invention relates to detect reagent of Francisella tularensis and the detection method of Francisella tularensis, relate in particular to combined probe Francisella tularensis sign gene and increase, and then utilize the method for combined probe fluorescent PCR technology for detection Francisella tularensis.
Background technology
Francisella tularensis (Francisella tularensis) is a kind of gram-negative coccobacillus, can cause the tularaemia (Tularemia) of infecting both domestic animals and human, be one of communicable pathogenic bacterium of tool, found this bacterium of zoogenetic infection more than 100 kinds at occurring in nature.Because of its route of transmission various, easily diffusion, strong toxicity and listed in category-A bio-terrorism preparation by US Centers for Disease Control and Prevention.The tularaemia lethality rate is very high, and the A type soil that virulence is the strongest draws hot subspecies under the situation that lacks effectively treatment, and infecting 10 bacteriums just can be fatal.Therefore detecting soil in time, accurately draws bacterium in time to treat for the tularaemia patient and prevents that diffusion from having great importance.The Francisella tularensis poor growth, can be by aerosol transmission, microbial culture diagnosis difficulty is bigger, usually adopt immunology or molecular biology method, as little aggegation experiment, enzyme linked immunological absorption, quick detection test paper bar, biosensor, PCR, nucleic acid hybridization detection, mass spectroscopy, gene chip etc.But up to the present also do not have a kind of sophisticated soil that is used for to draw the bacterium detection method, its major cause is that soil draws bacteria pathogenic strong, and is difficult for separation and Culture.
The discovery of conventional P CR technology and nucleic acid hybridization technique has promoted the development of Bacteria Detection technology greatly, but exist easy crossed contamination, can't quantitative analysis etc. defective.The real-time fluorescence quantitative PCR technology has solved the deficiency of conventional P CR technology, has become the one preferred technique of microorganism fast quantification detections such as bacterium.Quantitative PCR technique can be realized amplification and the synchronous detection of DNA in same pipe, and need not to carry out behind PCR gel electrophoresis analysis, has efficient, quick, special advantage, is highly suitable for the detection of single pathogenic agent.Taqman probe, molecular beacon, hybridization probe usefulness such as (Lightcycler) are widely used in the fields such as infectious disease pathogens monitoring, food safety, health quarantine.Develop examination technology, method and the reagent of the pathogenic micro-organism of quick, responsive, special, system, development is advanced, the pathogenic micro-organism of system separates and detection method, can be effectively breaking out and popularly carry out early warning, confirm fast transmissible disease, the port of control New Development transmissible disease is imported into, and can help to improve to public health emergency, breaks out the quick emergency reaction and the processing power of epidemic situation and unknown cause disease.
Summary of the invention
First purpose of the present invention is to provide a kind of reagent that detects Francisella tularensis at current conventional Francisella tularensis detection method imperfection also, can be used for detecting rapidly and accurately Francisella tularensis.For this reason, the present invention by the following technical solutions: it comprises following upstream primer, downstream primer, fluorescent probe and cancellation probe;
The gene order FTF1 of upstream primer is: 5 '-aagcaagtgttgactacagat-3 ', its gene order is shown in sequence table SEQ: NO 1;
The gene order FTR1 of downstream primer is: 5 '-caccaaagaaccatgttaaac-3 ', its gene order is shown in sequence table SEQ: NO 2;
The gene order FPFT1 of fluorescent probe is: 5 '-FAM-aatcatgttagtacccgctctgcca-P-3 ', its gene order is as sequence table SEQ: shown in the NO3;
The gene order QPFT1 of cancellation probe is: 5 '-agcgggtactaacatgat-Dabcyl-3 ', its gene order is shown in sequence table SEQ NO:4.
Another object of the present invention provides the method for a kind of combined probe fluorescence quantitative PCR detection Francisella tularensis, can detect Francisella tularensis rapidly and accurately.For this reason, the present invention is by the following technical solutions:
It adopts direct water-boiling method to extract the Francisella tularensis gene DNA: get the bacteria samples of 50 μ l, place boiling water bath to boil 10 minutes, centrifugal 2 minutes of 10000 * g gets 2 μ l supernatants as the amplification pcr template;
The PCR reaction system of described method is the Tris-HCl of 25 μ l:10mmol/L, and pH is 8.0; The KCl of 50mmol/L; Volume percent 3% methane amide; 6mmol/L MgCL
2The dATP of 100 μ mol/L, dCTP, dGTP, dTTP; 0.5 the above-mentioned upstream primer of μ mol/L, the above-mentioned downstream primer of 0.5 μ mol/L, 1.5U Taq enzyme, the above-mentioned fluorescent probe of 100nmol/L, the above-mentioned cancellation probe of 200nmol/L; The PCR response procedures is: the annealing temperature of pre-95 ℃ of 3min of sex change, 95 ℃ of 5s, amplification is 54 ℃ of 30s totally 40 circulations;
Described method also comprises following detection step: reaction system and the response procedures of amplification template by above-mentioned quantitative fluorescent PCR increased on the enforcement quantitative real time PCR Instrument, and carry out the amplification of negative control and positive control simultaneously, wherein negative control adopts non-Francisella tularensis, and positive control adopts the Francisella tularensis type strain; After reaction finishes,, just obtain the target fragment copy concentrations in the sample DNA with sample loops threshold value C (t) and the contrast of quantitative fluorescent PCR typical curve; Concentration according to Francisella tularensis in the target fragment copy concentrations judgement sample.
The present invention has advantage easy and simple to handle, efficient, quick, special with respect to traditional Francisella tularensis detection technique.Choosing the FopA gene is goal gene, coding Francisella tularensis outer membrane protein, this gene is conservative at Mark Lewis-Francis Pseudomonas camber, can be used as the diagnostic nucleic acid sign of Francisella tularensis, carry out the BLAST retrieval with whole genbank database, find that it does not have homology to the nucleotide sequences special and other species of the FopA gene order on the Francisella tularensis, has guaranteed the specificity of detection method.This method has shortened the detection time of Francisella tularensis greatly, and the detection by quantitative of sample is only needed can finish in about 2 hours, and is significant to the early diagnosis that soil draws.
Description of drawings
Fig. 1 a is the real-time amplification curve diagram of the same template of detection of combination 1 among the embodiment 1, and wherein, 1 is the real-time amplification curve of CMCC52502, and 2 is the real-time amplification curve of CMCC52503, and 3 is the real-time amplification curve of CMCC52504.
Fig. 1 b is the real-time amplification curve diagram of the same template of detection of combination 2 among the embodiment 1, and wherein, 1 is the real-time amplification curve of CMCC52502, and 2 is the real-time amplification curve of CMCC52503, and 3 is the real-time amplification curve of CMCC52504.
The sensitivity analysis of the plasmid reference material figure that increases in real time among Fig. 2 embodiment 2, wherein, 1 is 1 * 10
6The real-time amplification curve of individual copy/μ l, 2 is 1 * 10
5The real-time amplification curve of individual copy/μ l, 3 is 1 * 10
4The real-time amplification curve of individual copy/μ l, 4 is 1 * 10
3The real-time amplification curve of individual copy/μ l, 5 is 1 * 10
2The real-time amplification curve of individual copy/μ l, 6 is 1 * 10
1The real-time amplification curve of individual copy/μ l, 7 is 1 * 10
0The real-time amplification curve of individual copy/μ l.
Fig. 3 is the quantitative fluorescent PCR typical curve that the embodiment of the invention 2 is drawn.
Embodiment
The preparation of embodiment 1 primer and probe
1 target gene is selected
The outer membrane protein of FopA genes encoding Francisella tularensis, still there is anti-fopA antibody in the people in convalescent phase serum, and this gene is conservative at Mark Lewis-Francis Pseudomonas camber, can be used as the diagnostic nucleic acid sign of Francisella tularensis.Obtain this gene order according to the genbank database retrieval.
2PCR primer design and screening
Follow the combined probe principle of design,, designed two groups of primers and probe according to the FopA gene order.
Fluorescent probe 5 ' end mark fluorescent molecule FAM is as reporter group, and 3 ' end mark phosphoric acid is to block its extension.3 of cancellation probe ' end connects a quenching group Dabcyl.
In order from two groups of combined probes and combination of primers, to filter out a high combination of amplification efficiency, the PCR in real time analysis is carried out in these combinations, adopt Francisella tularensis CMCC52502, CMCC52503, CMCC52504 is for detecting sample, studies their increase efficient of same sample and determines best of breed.
The result is shown in table 1 and Fig. 1 a, Fig. 1 b, when adopting combination 1 to detect, CT value during its test sample is less, and fluorescence response intensity level Δ RFU is higher, illustrates that this combination amplification efficiency is higher, and it is low to make up 2 fluorescence response intensity level Δ RFU, illustrate that this combination amplification efficiency is also lower, therefore, in all experiments described later, all adopt combination 1.
Table 1 primer and probe combinations are to the influence of amplification efficiency
The sequence that makes up 1 primer and probe is as follows:
The gene order FTF1 of upstream primer is: 5 '-aagcaagtgttgactacagat-3 ';
The gene order FTR1 of downstream primer is: 5 '-caccaaagaaccatgttaaac-3 ';
The gene order FPFT1 of fluorescent probe is: 5 '-FAM-aatcatgttagtacccgctctgcca-P-3 ';
The gene order QPFT1 of cancellation probe is: 5 '-agcgggtactaacatgat-Dabcyl-3 '.
3. the preparation of detection probes and PCR primer
The chemosynthesis, mark and the purifying that are used to detect the oligonucleotide (comprising fluorescent probe, cancellation probe and PCR primer) of Francisella tularensis use Expedite 8909 dna synthesizers of American AB I company, the fluorescent probe fluorescein FAM mark of U.S. transgenomic company, the cancellation probe Dabcyl cancellation reagent mark of U.S. transgenomic company.
Detect actual application ability in order to investigate the present invention, the special blood simulated samples that has been equipped with the Francisella tularensis pollution is provided with pure bacterium simultaneously as positive control, and non-Francisella tularensis pathogenic bacterium are as negative control.
1. specimen preparation
(1). the preparation of contaminated blood and processing: the definite value Francisella tularensis is used the anticoagulation serial dilution, and making concentration is 1 * 10
6CFU/ml-1 * 10
1The contaminated blood sample of CFU/ml.Get each 1ml of blood that contains the different concns bacterium, centrifugal 10 minutes of 12000rpm, supernatant discarded adds 1ml erythrocyte cracked liquid (50mmol/LTrisHCL, 25mmol/L KCl, 5mmol/LMgCl
2, pH7.5, TKM liquid), thermal agitation mixing 2min, 12000rpm abandoned supernatant in centrifugal 10 minutes, added TKM liquid 1ml again, mixing, 12000rpm abandoned in centrifugal 10 minutes
(2). positive and negative contrast: in order to investigate the specificity of detection method, analyzed positive specificity reference material of 4 strain Francisella tularensis and the negative specificity reference material of 20 strains, seen Table 2 and table 3 with present method.Set up positive quality control product and negative quality control product during detection.
Table 2 positive strain relevant information
The negative specific strains relevant information of table 3
* ATCC: refer to American Type Culture Collection (ATCC), CMCC: refer to Chinese medicine DSMZ
2. the reaction system of quantitative fluorescent PCR and response procedures
The fluorescent PCR reaction system is 25 μ l, and composition is as follows after optimizing: the Tris-HCl of 10mmol/L, pH are 8.0; The KCl of 50mmol/L; 3% methane amide; 6mmol/L MgCl
2Concentration; The dATP of 100 μ mol/L, dCTP, dGTP, dTTP; 0.5 the above-mentioned upstream primer of μ mol/L, the above-mentioned downstream primer of 0.5 μ mol/L, 1.5U Taq enzyme, the above-mentioned fluorescent probe of 100nmol/L, the above-mentioned cancellation probe of 200nmol/L.The operating parameter of PCR instrument is: pre-95 ℃ of 3min of sex change.The annealing temperature of 95 ℃ of 5s, amplification is 54 ℃ of 30s, 40 circulations.
3. typical curve preparation
3.1 the preparation of plasmid DNA reference material: the mode by vitro recombination is inserted into the PMD18-T carrier with amplified fragments, and transformed into escherichia coli, extracting positive colony checks order, the clone that order-checking is correct, increase bacterium in 37 ℃ of shaking culture of airbath shaking table, culture extracts plasmid with the Promega plasmid extraction kit, and plasmid concentration is measured with ultraviolet spectrophotometer in sampling dilution back, and (concentration (copy/μ l)=OD by formula
260* extension rate * 50 * avogadros constant/double-chain length bp * 660) calculates the initial copy number of DNA.Be diluted to 1 * 10 with TE then
1, 1 * 10
2, 1 * 10
3, 1 * 10
4, 1 * 10
5, 1 * 10
6Six concentration of copy/μ l are classified, and 50 μ l/ manage packing ,-70 ℃ of preservations.
3.2 the preparation of plasmid DNA reference material typical curve: the PMD18-T plasmid that the clone is had target gene fragment is as the standard substance dna profiling, the sensitivity of plasmid standard and linear analysis: the plasmid reference material (1 * 10 of getting different DNA carrying capacity
0, 1 * 10
1, 1 * 10
2, 1 * 10
3, 1 * 10
4, 1 * 10
5, 1 * 10
6) each 2 μ l, carry out fluorescent PCR and detect, from Fig. 2, Fig. 3, table 4 as seen, present method can detect the plasmid DNA molecule of 10 copies.
Table 4 detects the sensitivity analysis of plasmid DNA
As Fig. 2, shown in Fig. 3, table 4, above-mentioned data are carried out regression analysis, obtain the PCR regression equation and be: Y=-2.8552X+40.378, relation conefficient is 0.9864.Experimental result shows, 1 * 10
1-1 * 10
6Between the copy scope, its corresponding CT value of sample copy number has good dependency, and therefore, present method can be to 1 * 10
1-1 * 10
6Template in the copy scope is carried out quantitatively.
4. the repeatability analysis of Jian Ceing
Adopt direct water-boiling method extracting solution to extract DNA strong positive quality control product, critical positive quality control product and negative quality control product, respectively get 2 μ l then and carry out the real-time fluorescence PCR detection, carrying out real-time fluorescence PCR detects, replication is 3 times between batch, replication is five times in batch, calculate batch interior and differences between batches, thereby the precision of detection method is examined, experimental result sees Table 5.
Table 5 detects the repeatability analysis of plasmid reference material
To the statistical results show of The above results, the precision of present method is fine, positive quality control product and negative quality control product different time measure three times and five repeated experiments of same time as a result the CV value all less than 5%.
5. sample detection
The blood simulated samples and the positive and negative check sample that pollute with above-mentioned Francisella tularensis extract DNA as template, increase on quantitative real time PCR Instrument by the reaction system and the response procedures of above-mentioned quantitative fluorescent PCR.Reaction system and amplification condition are consistent when doing typical curve.The results are shown in Table 6 in simulate blood, can detect 1 * 10
3The bacterium of CFU/ml, visible 4 strain Francisella tularensis results are positive for table 7, and all non-Francisella tularensis results are negative, and the result shows that the specificity of this method is 100%, has good detection specificity.
The detected result of table 6 analog sample
*ND：Not?Detectected
Table 7 specific detection result
*ND：Not?Detectected
＜110〉Zhejiang international travel health care center
＜120〉detect the reagent of Francisella tularensis and combined probe fluorescence quantitative PCR detection Francisella tularensis
Method
<130>
<160> 4
<170> PatentIn?version?3.5
<210> 1
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 1
aagcaagtgt?tgactacaga?t 21
<210> 2
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 2
caccaaagaa?ccatgttaaa?c 21
<210> 3
<211> 25
<212> DNA
＜213〉artificial sequence
<400> 3
aatcatgtta?gtacccgctc?tgcca 25
<210> 4
<211> 18
<212> DNA
＜213〉artificial sequence
<400> 4
agcgggtact?aacatgat 18
Claims (3)
1. detect the reagent of Francisella tularensis, it is characterized in that it comprises following upstream primer, downstream primer, fluorescent probe and cancellation probe;
The gene order FTF1 of upstream primer is: 5 '-aagcaagtgttgactacagat-3 ';
The gene order FTR1 of downstream primer is: 5 '-caccaaagaaccatgttaaac-3 ';
The gene order FPFT1 of fluorescent probe is: 5 '-FAM-aatcatgttagtacccgctctgcca-P-3 ';
The gene order QPFT1 of cancellation probe is: 5 '-agcgggtactaacatgat-Dabcyl-3 '.
2. the method for a combined probe fluorescence quantitative PCR detection Francisella tularensis, it is characterized in that: it adopts direct water-boiling method to extract the Francisella tularensis genomic dna: get 50 μ l bacteria samples, place boiling water bath to boil 10 minutes, centrifugal 2 minutes of 10000 * g gets 2 μ l supernatants as amplification template;
The PCR reaction system of described method is the Tris-HCl of 25 μ l:10mmol/L, and pH is 8.0; The KCl of 50mmol/L; Volume percent 3% methane amide; 6mmol/L MgCL2; The dATP of 100 μ mol/L, dCTP, dGTP, dTTP; 0.5 the described upstream primer of the claim 1 of μ mol/L, the described downstream primer of the claim 1 of 0.5 μ mol/L, 1.5U Taq enzyme, the described fluorescent probe of the claim 1 of 100nmol/L, the described cancellation probe of the claim 1 of 200nmol/L; The PCR response procedures is: the annealing temperature of pre-95 ℃ of 3min of sex change, 95 ℃ of 5s, amplification is 54 ℃ of 30s totally 40 circulations;
Described method also comprises following detection step: reaction system and the response procedures of amplification template by above-mentioned quantitative fluorescent PCR increased on the enforcement quantitative real time PCR Instrument, and carry out the amplification of negative control and positive control simultaneously, wherein negative control adopts non-Francisella tularensis, and positive control adopts the Francisella tularensis type strain; After reaction finishes,, just obtain the target fragment copy concentrations in the sample DNA with sample loops threshold value C (t) and the contrast of quantitative fluorescent PCR typical curve; Concentration according to Francisella tularensis in the target fragment copy concentrations judgement sample.
3. the method for a kind of fluorescence quantitative PCR detection Francisella tularensis as claimed in claim 2 is characterized in that: the quantitative fluorescent PCR typical curve adopts following steps to make:
The plasmid that the clone is had target gene fragment is as standard substance DNA cloning template, gets each 2 μ l of plasmid standard of different DNA carrying capacity, increases on the quantitative real time PCR Instrument implementing by the reaction system of above-mentioned quantitative fluorescent PCR and response procedures; Reaction finishes the cycle threshold C (t) of back according to each concentration that obtains, and adopts computer automatic drafting quantitative fluorescent PCR typical curve.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103468829A (en) * | 2013-09-25 | 2013-12-25 | 北京普利耐特生物科技有限公司 | Hepatitis B virus nucleic acid detection kit |
| CN110578011A (en) * | 2019-10-10 | 2019-12-17 | 中国检验检疫科学研究院 | Kit for rapidly detecting eupolyphaga |
| CN111394490A (en) * | 2020-05-15 | 2020-07-10 | 中国人民解放军军事科学院军事医学研究院 | CRISPR-Cas12a detection primer group for eupolyphaga and application thereof |
| CN113862384A (en) * | 2021-08-20 | 2021-12-31 | 江汉大学 | MNP (protein marker) marker locus of Francisella tularensis, primer composition, kit and application |
| CN117487941A (en) * | 2023-11-30 | 2024-02-02 | 广州维伯鑫生物科技有限公司 | Nucleic acid compositions, kits and methods for detecting Francisella tularensis |
-
2011
- 2011-02-28 CN CN2011100480062A patent/CN102154487A/en active Pending
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| 朱晓宇等: "《土拉菌的实验室检测及分析技术研究进展》", 《中国人兽共患病学报》 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103468829A (en) * | 2013-09-25 | 2013-12-25 | 北京普利耐特生物科技有限公司 | Hepatitis B virus nucleic acid detection kit |
| CN103468829B (en) * | 2013-09-25 | 2016-05-25 | 北京普利耐特生物科技有限公司 | A kind of hepatitis B virus nucleic acid kit that detects |
| CN110578011A (en) * | 2019-10-10 | 2019-12-17 | 中国检验检疫科学研究院 | Kit for rapidly detecting eupolyphaga |
| CN111394490A (en) * | 2020-05-15 | 2020-07-10 | 中国人民解放军军事科学院军事医学研究院 | CRISPR-Cas12a detection primer group for eupolyphaga and application thereof |
| CN113862384A (en) * | 2021-08-20 | 2021-12-31 | 江汉大学 | MNP (protein marker) marker locus of Francisella tularensis, primer composition, kit and application |
| CN113862384B (en) * | 2021-08-20 | 2023-12-22 | 江汉大学 | MNP (MNP) marking site of Francisella tularensis, primer composition, kit and application |
| CN117487941A (en) * | 2023-11-30 | 2024-02-02 | 广州维伯鑫生物科技有限公司 | Nucleic acid compositions, kits and methods for detecting Francisella tularensis |
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Application publication date: 20110817 |