CN102925574A - Primers special for detecting coxiella burnetii - Google Patents
Primers special for detecting coxiella burnetii Download PDFInfo
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- CN102925574A CN102925574A CN2012104430813A CN201210443081A CN102925574A CN 102925574 A CN102925574 A CN 102925574A CN 2012104430813 A CN2012104430813 A CN 2012104430813A CN 201210443081 A CN201210443081 A CN 201210443081A CN 102925574 A CN102925574 A CN 102925574A
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- isothermal amplification
- coxiella burnetii
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Abstract
The invention discloses primers special for detecting coxiella burnetii and provides a loop mediated isothermal amplification primer group for detecting the coxiella burnetii. The loop mediated isothermal amplification primer group comprises a primer 1, a primer 2, a primer 3 and a primer 4. Nucleotide sequences of the primer 1, the primer 2, the primer 3 and the primer 4 are respectively a sequence 1, a sequence 2, a sequence 3 and a sequence 4 in a sequence table in sequence. The primers special for detecting the coxiella burnetii have the advantages of being capable of performing amplification reaction only at constant temperature, free of special devices, high in specificity, rapid, efficient, capable of finishing the amplification reaction within 60 minutes, high in sensitivity, convenient and simple to identify and particularly suitable for rapid detection of clinical specimens.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of primer special that detects Coxiella burnetii.
Background technology
Coxiella burnetii is the rickettsia sample pathogenic agent that is separated to from Australian patient first nineteen thirty-five, belongs to obligate born of the same parents endoparasitism, Gram-negative acidophilic bacteria.The Coxiella burnetii associated diseases is called Q heat (Q fever).Q heat is divided into acute and chronic two kinds of Clinical types.Acute Q heat shows as acute influenza-like symptom, and take headache, heating, pneumonia as principal character, and the hot main manifestations of chronic Q is long-term fever, often with endocarditis, hepatitis, osteomyelitis etc.The infective dose of Coxiella burnetii is extremely low, and single pathogenic agent can be caused a disease, so this pathogenic agent is used as biological weapon, also may be used as the bio-terrorism agent by terroristic organization by some country.The nearly all country in Q pyreticosis example each continent of extend over the entire globe that has reported at present becomes one of the widest Amphixenosis of current distribution.Traditional regular-PCR and real time fluorescence quantifying PCR method etc. often need to take a long time or need specific instrument, are difficult to satisfy the needs of epidemic outbreaks quick diagnosis.
Ring mediated isothermal amplification method (loop-mediated isothermal amplification, LAMP) has under isothermal condition can be efficient, fast, the characteristics of high special, high-sensitive amplified target sequence.
Summary of the invention
An object of the present invention is to provide a kind of loop-mediated isothermal amplification (LAMP) primer group that detects Coxiella burnetii.
The loop-mediated isothermal amplification (LAMP) primer group of detection Coxiella burnetii provided by the invention is comprised of primer 1, primer 2, primer 3 and primer 4;
The nucleotide sequence of described primer 1, described primer 2, described primer 3 and described primer 4 is followed successively by respectively sequence 1, sequence 2, sequence 3 and the sequence 4 in the sequence table.
In the above-mentioned primer sets, the mol ratio of primer 1, primer 2, primer 3 and primer 4 is 1:1:8:8.
Another object of the present invention provides a kind of ring mediated isothermal amplification reagent that detects Coxiella burnetii.
The ring mediated isothermal amplification reagent of detection Coxiella burnetii provided by the invention comprises above-mentioned primer sets and ring mediated isothermal amplification damping fluid.
Above-mentioned ring mediated isothermal amplification reagent is comprised of above-mentioned primer sets, ring mediated isothermal amplification damping fluid and water;
Described primer 1 and the described primer 2 final concentration in described ring mediated isothermal amplification reagent is 5pmol/L,
Described primer 3 and described primer 4 final concentration in described ring mediated isothermal amplification reagent is 40pmol/L.
The 3rd purpose of the present invention provides a kind of loop-mediated isothermal amplification kit that detects Coxiella burnetii.
Test kit provided by the invention comprises above-mentioned primer sets or above-mentioned ring mediated isothermal amplification reagent.
Above-mentioned primer sets, above-mentioned ring mediated isothermal amplification reagent, the application of mentioned reagent box in preparation detection and/or auxiliary detection Coxiella burnetii product also are the scope of protection of the invention.
In the above-mentioned application, described detection is loop-mediated isothermal amplification, and the condition of described loop-mediated isothermal amplification is: 60-65 ℃ of reaction 30-90min is specially 63 ℃ of reaction 90min; 80 ℃ of 5min termination reactions again.
Above-mentioned detection and/or auxiliary detection Coxiella burnetii are for carrying out loop-mediated isothermal amplification reaction, detection ring mediated isothermal amplification reaction product with above-mentioned ring mediated isothermal amplification reagent to sample to be tested (Coxiella burnetii);
Above-mentioned detection ring mediated isothermal amplification reaction product can be judged by the following method:
1) analyze judgement by the turbidity of reaction solution: when nucleic acid synthesized in a large number, dNTP can separate out a large amount of pyrophosphate ions, the Mg in pyrophosphate ion and the LAMP system
2+(or Mn
2+) combination, produce magnesium pyrophosphate or manganese pyrophosphate precipitation, thereby the turbidity of the liquid that induces reaction changes; Based on this principle, can change according to the turbidity of reaction solution having judged whether that nucleic acid is synthetic in a large number, thereby whether judge templet is target sequence; If muddy, then positive, otherwise negative;
2) adopt real-time turbidimeter and the real-time detection reaction liquid of supporting program software thereof, if observe typical amplification curve on the turbidimeter in real time, then show sample to be tested from Coxiella burnetii, positive, on the contrary negative.
Of the present invention experimental results show that, the present invention has found 4 primer specials, adopt the ring mediated isothermal amplification method to carry out specific detection and quantitative analysis to Coxiella burnetii, primer of the present invention and method have following advantage: (1) only need in steady temperature with regard to the energy amplified reaction, not need specific installation; (2) specificity is high; (3) rapidly and efficiently, amplified reaction can be finished in 60 minutes; (4) highly sensitive; (5) evaluation is convenient and simple.Primer of the present invention is applicable to the rapid detection of clinical samples.
Description of drawings
Fig. 1 is real-time turbidimeter amplification and the amplification curve that the sensitivity of embodiment 2 detects.
Fig. 2 is the real-time turbidimeter amplification of the specific detection of embodiment 2.
Fig. 3 is real-time turbidimeter amplification (A) and the amplification curve (B) that the repeatability of embodiment 2 detects.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, such as the molecular cloning operational manual, or the condition of advising according to manufacturer.
Used inorganic chemical reagent and organic reagent all meet the requirement of molecular biology experiment.Primer is synthetic by Shanghai biotechnology company, the LAMP test kit: DNA Amplification Kit, Japanese Rong Yan company, CodeNo:LMP204.LAMP luciferase assay reagent: Fluorescent Detection Reagent, Japanese Rong Yan company, CodeNo:LMP221.LAMP reacts special-purpose centrifuge tube: Reaction Tube, Japanese Rong Yan company, CodeNo:LMP905.Japan's DNA extraction test kit: DNeasy Blood﹠Tissue Kit, QIAgen company product, Cat No.69506.The real-time turbidimeter of LA-320C, Japanese Rong Yan company product.
The part material is as follows among the following embodiment, the following identification number of the digitized representation of bracket:
Coxiella burnetii new bridge strain (1), rickettsia rickettsii (2), Rickettsia prowazeki (2), Rickettsia mooseri (2), Heilungkiang rickettsia (2), yersinia pestis (2), streptococcus aureus (3); The public can obtain from Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL.
E. coli bl21 (DE3) is (available from Novagen company, article No.: 70235-3).
1. detect the real-time fluorescence quantitative PCR of Coxiella burnetii, Chinese Amphixenosis's magazine, the 21st volume, the 8th phase, 652-655,2005.
2. real-time fluorescence quantitative PCR detects the rickettsial method of Spotted Fever and sets up PLA's medical journal, the 33rd volume, o. 11th, 1297-1299,2008.
3. the prokaryotic expression of vibrio cholerae outer membrane protein W and antigenicity analysis, military medicine, the 35th volume, the 10th phase, 746-748,2011.
Design and the preparation of embodiment 1, detection Coxiella burnetii primer special
Specific primer sequence according to the design of Coxiella burnetii new bridge strain whole genome sequence is as follows:
F3(sequence 1): 5 '-TTCAATAAGCGTTTTATCGCT-3 ';
B3(sequence 2): 5 '-ACAACCATTTAAACCGACAAG-3 ';
FIP(sequence 3): 5 '-AAGGCTTCCATCAGTGTGAATTATTTGAAGTTATCGAAAGATTGCGT-3 ';
BIP(sequence 4): 5 '-CGAATGGTGTCGCGTGTTTCCCCCTTTATCTCATTTCCGG-3 ';
The above-mentioned required primer of synthetic is used for the detection of following embodiment 2.
The application of embodiment 2, detection Coxiella burnetii primer special
One, the preparation of Coxiella burnetii sample
The chick embryo yolk sac film that the strain of Coxiella burnetii new bridge is infected adds meat soup and grinds, the yolk cyst membrane lapping liquid of preparation Coxiella burnetii infected chicken embryo.Adopt the urografic acid methylglucamine salt density gradient centrifugation to carry out purifying, the Coxiella burnetii behind the purifying is adopted DNeasy Blood﹠amp; Tissue Kit extracts tissue DNA, and concrete extracting method is seen the specification sheets of this test kit, adopts at last Nanodrop1000 to measure the concentration (ng/ μ L) of the thallus DNA that extracts.Coxiella burnetii new bridge pnca gene group size is 2Mbp, concentration (ng/ μ L) and genomic size (2Mbp) according to thallus DNA, calculate the mole number of the Coxiella burnetii new bridge strain thalline of purifying, calculate at last concentration (the individual genome/mL) of purified Coxiella burnetii new bridge strain.Use pure water that the thallus DNA that extracts is carried out gradient dilution, (concentration is respectively 1 * 10 to obtain the thallus DNA diluent of 7 kinds of different concns
6Individual genome/μ L, 1 * 10
5Individual genome/μ L, 1 * 10
4Individual genome/μ L, 1 * 10
3Individual genome/μ L, 1 * 10
2Individual genome/μ L, 1 * 10
1Individual genome/μ L, 1 * 10
0Individual genome/μ L).With 7 kinds of mycelium dilution liquid and the PBS(negative control that obtains) as 8 samples.
Sample 1: concentration is 1 * 10
6The Coxiella burnetii new bridge strain diluent of individual genome/μ L.
Sample 2: concentration is 1 * 10
5The Coxiella burnetii new bridge strain diluent of individual genome/μ L.
Sample 3: concentration is 1 * 10
4The Coxiella burnetii new bridge strain diluent of individual genome/μ L.
Sample 4: concentration is 1 * 10
3The Coxiella burnetii new bridge strain diluent of individual genome/μ L.
Sample 5: concentration is 1 * 10
2The Coxiella burnetii new bridge strain diluent of individual genome/μ L.
Sample 6: concentration is 1 * 10
1The Coxiella burnetii new bridge strain diluent of individual genome/μ L.
Sample 7: concentration is 1 * 10
0The Coxiella burnetii new bridge strain diluent of individual genome/μ L.
Sample 8:PBS damping fluid.
Two, primer special detects Coxiella burnetii (adopting the LAMP test kit)
1, sensitivity detects
Detect above-mentioned one DNA to be measured that obtains with the primer special among the embodiment 1, institute responds and all carries out in special-purpose centrifuge tube; DNA to be measured is sample 1-sample 8.
LAMP reaction system (25 μ l): the final concentration of F3 and B3 is 5pmol/L, the final concentration of BIP and FIP is 40pmol/L, 2 * reaction mixture (RM, Japan Rong Yan company, CodeNo:LMP204) 12.5 μ l, Bst archaeal dna polymerase 1 μ l, DNA1 μ l to be measured, the deionized water constant volume of nuclease free and DNAzyme; Above concentration is the final concentration in the system.
The LAMP reaction system is 63 ℃ of reactions 90min, then 80 ℃ of 5min termination reactions.
The turbidimeter amplification curve is seen Fig. 1 in real time, and wherein A is amplification, and B is corresponding amplification curve, can find out, sample 1 is positive to sample 5, and sample 6 is negative to sample 8.
The sensitivity of this presentation of results primer of the present invention and method is 100 genome/reaction systems.
The outer above-mentioned LAMP reaction system of above-mentioned 4 primers or removing template can be used as the component in the test kit that detects Coxiella burnetii.
2, specific detection
Detect the specificity that 7 kinds of non-Coxiella burnetii genomic dnas are used for estimating this primer special with the primer special among the embodiment 1,7 kinds of non-Coxiella burnetiis are as follows respectively: rickettsia rickettsii, Rickettsia prowazeki, Rickettsia mooseri, Heilungkiang rickettsia, yersinia pestis, gold-coloured staphylococci, e. coli bl21 (DE3).The DNA concentration of sample to be tested is 10
4Individual genome/μ L.
Detection method and system such as above-mentioned 1.
The result as shown in Figure 2, wherein Coxiella burnetii new bridge strain (Fig. 2-1), rickettsia rickettsii (Fig. 2-2), Rickettsia prowazeki (Fig. 2-3), Rickettsia mooseri (Fig. 2-4), Heilungkiang rickettsia (Fig. 2-5), yersinia pestis (Fig. 2-6), streptococcus aureus (Fig. 2-7), e. coli bl21 (DE3) (Fig. 2-8); Can find out that the LAMP amplification of Coxiella burnetii new bridge pnca gene group DNA is positive on scheming, the LAMP amplification of other genomic dnas and negative control is all negative.
This presentation of results primer of the present invention and method have good specificity.
3, repeatability detects
Detecting sample 2(concentration with 4 primer special duplicate detection among the embodiment 1 is 1 * 10
5The Coxiella burnetii new bridge strain diluent of individual genome/μ L).Multiplicity is 8 times.
Detection method and system such as above-mentioned 1 the results are shown in Figure 3,1-8 and are respectively 8 times of repetition, and A is amplification, and B is corresponding amplification curve; Can find out that the result of 8 detections is all positive on scheming, amplification curve does not have obvious difference, illustrates that this primer special has higher repeatability.
Claims (7)
1. detect the loop-mediated isothermal amplification (LAMP) primer group of Coxiella burnetii, formed by primer 1, primer 2, primer 3 and primer 4;
The nucleotide sequence of described primer 1, described primer 2, described primer 3 and described primer 4 is followed successively by respectively sequence 1, sequence 2, sequence 3 and the sequence 4 in the sequence table.
2. primer sets according to claim 1, it is characterized in that: the mol ratio of described primer 1, described primer 2, described primer 3 and described primer 4 is 1:1:8:8.
3. detect the ring mediated isothermal amplification reagent of Coxiella burnetii, comprise claim 1 or 2 described primer sets and ring mediated isothermal amplification damping fluid.
4. ring mediated isothermal amplification reagent according to claim 3, it is characterized in that: described ring mediated isothermal amplification reagent is comprised of claim 1 or 2 described primer sets, ring mediated isothermal amplification damping fluid and water;
Described primer 1 and the described primer 2 final concentration in described ring mediated isothermal amplification reagent is 5pmol/L,
Described primer 3 and described primer 4 final concentration in described ring mediated isothermal amplification reagent is 40pmol/L.
5. detect the loop-mediated isothermal amplification kit of Coxiella burnetii, comprise claim 1 or 2 described primer sets or claim 3 or 4 described ring mediated isothermal amplification reagent.
6. claim 1 or 2 described primer sets, claim 3 or 4 described ring mediated isothermal amplification reagent, the application of the described test kit of claim 5 in preparation detection and/or auxiliary detection Coxiella burnetii product.
7. application according to claim 6 is characterized in that: described detection is loop-mediated isothermal amplification, and the condition of described loop-mediated isothermal amplification is: 60-65 ℃ of reaction 30-90min, again 80 ℃ of 5min termination reactions.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110468222A (en) * | 2019-09-16 | 2019-11-19 | 中国人民解放军军事科学院军事医学研究院 | The special primer for checking of one group of China's Rana amurensis |
| CN112280877A (en) * | 2020-11-09 | 2021-01-29 | 中国人民解放军军事科学院军事医学研究院 | CRISPR-Cas13a system for detecting coxiella burnetii nucleic acid |
-
2012
- 2012-11-08 CN CN2012104430813A patent/CN102925574A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| REKHA SESHADRI等: "Complete genome sequence of the Q-fever pathogen Coxiella burnetii", 《PNAS》 * |
| 何玲等: "环介导等温扩增技术在动物病原检测中的应用", 《广东畜牧兽医科技》 * |
| 徐丽丽等: "Q热LAMP检测方法的建立及应用", 《全国动物生理生化第十二次学术交流会论文摘要汇编》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110468222A (en) * | 2019-09-16 | 2019-11-19 | 中国人民解放军军事科学院军事医学研究院 | The special primer for checking of one group of China's Rana amurensis |
| CN110468222B (en) * | 2019-09-16 | 2022-08-30 | 中国人民解放军军事科学院军事医学研究院 | Special detection primers for rickettsiae in China Heilongjiang |
| CN112280877A (en) * | 2020-11-09 | 2021-01-29 | 中国人民解放军军事科学院军事医学研究院 | CRISPR-Cas13a system for detecting coxiella burnetii nucleic acid |
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Application publication date: 20130213 |