CN102312010A - Rapid detection primers, kit and detection method for C.parapsilosis with loop-mediated isothermal amplification - Google Patents
Rapid detection primers, kit and detection method for C.parapsilosis with loop-mediated isothermal amplification Download PDFInfo
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- CN102312010A CN102312010A CN201110303598A CN201110303598A CN102312010A CN 102312010 A CN102312010 A CN 102312010A CN 201110303598 A CN201110303598 A CN 201110303598A CN 201110303598 A CN201110303598 A CN 201110303598A CN 102312010 A CN102312010 A CN 102312010A
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- isothermal amplification
- mediated isothermal
- ring mediated
- candida parapsilosis
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Abstract
The invention provides rapid detection primers, a kit and a detection method for C.parapsilosis with loop-mediated isothermal amplification (LAMP). The LAMP rapid detection primers for C.parapsilosis comprise a pair of inner primers and a pair of outer primers, wherein, the sequences of the inner primers are shown as the SEQ ID No.1 and SEQ ID NO.2, the sequences of the outer primers are shown as the SEQ ID No.3 and SEQ ID NO.4. By utilizing the invention to detecting C.parapsilosis, the advantages of high sensitivity, strong singularity, rapid detection and simple operation are achieved.
Description
Technical field
The invention belongs to the detection technique scope of biological fungi, be specifically related to rapid detection primer and the test kit and the detection method of Candida parapsilosis ring mediated isothermal amplification.
Background technology
Ring mediated isothermal amplification (loop-mediated isothermal amplification; Be called for short LAMP) be to 6 specificity sites on the target sequence; Design 2 pairs of special primers; And through having the active archaeal dna polymerase of strand displacement, special under constant temperature, efficiently, the new technology of DNA amplification apace.Amplified production is the loop-stem structure and the mixtures that encircle the dna fragmentation of Cauliflower spline structures that a series of reverse multiple target sequences constitute more.Existing prior art is applied to cytomegalovirus with LAMP, mycobacterium, salmonella, Paracoccidioides brasiliensis, the detection of African trypanosome etc.The key of its specificity and high efficiency is the selection and the primer design of target gene.
In recent years, along with the intracorporeal indwelling conduit increases, the spectrum microbiotic, immunosuppressor, glucocorticosteroid, being widely used of antineoplastic chemotherapy medicine reaches extensively carrying out of organ transplantation, and the ratio of fungi bloodstream infection increases thereupon, and candidemia is occupied an leading position.Publication number is the one Chinese patent application (application number: 200810157907.3) disclose loop-mediated isothermal amplification fast detection method of producing candida albicans fungi of CN101381775.And the LAMP detection method of Candida parapsilosis does not still have report.
Summary of the invention
The rapid detection primer and test kit and the detection method that the purpose of this invention is to provide the Candida parapsilosis ring mediated isothermal amplification.
The rapid detection primer of Candida parapsilosis ring mediated isothermal amplification provided by the invention; Comprise inner primers and a pair of outer primer; Wherein, With shown in the SEQ ID NO 2, the sequence of described a pair of outer primer is shown in SEQ ID NO 3 and SEQ ID NO 4 like SEQ ID NO 1 for the sequence of described inner primers.
The quick detection kit of Candida parapsilosis ring mediated isothermal amplification provided by the invention contains the rapid detection primer and the loop-mediated isothermal amplification reaction reagent of described Candida parapsilosis ring mediated isothermal amplification.
The method for quick of Candida parapsilosis ring mediated isothermal amplification provided by the invention; It is characterized in that; Detected sample is extracted DNA through cultivating the back; Get dna profiling, utilize the rapid detection primer of the described Candida parapsilosis ring mediated isothermal amplification of claim 1 to carry out ring mediated isothermal amplification, carry out interpretation then.
The mol ratio of mix primer SEQ ID NO 1～4 each sequence is 8: 8: 1 in the reaction system of ring mediated isothermal amplification: 1.
The reaction system of ring mediated isothermal amplification is formed as follows: the Tris-HCl of 20mM pH8.8,10mM KCL, 10mM (NH
4)
2SO
4, 2mM MgSO
4, 0.1%Triton X-100,3.6 μ l mix primer, 8U bstDNA polysaccharase, 1.5 μ l dna profilings, 1.2mM dNTP, the 0.8M trimethyl-glycine adds H
2O to 20 μ l
Can carry out interpretation through turbidity, the color of SYBR green I fluorescent reaction or the bar belt shape of agarose gel electrophoresis.
Utilize the present invention that Candida parapsilosis is detected to have the susceptibility height, high specificity, detect advantage fast.
Embodiment
Key of the present invention is target gene selection and design of primers.Contriver of the present invention selects one section special sequence in the Candida parapsilosis conserved sequence as target gene, and it is peculiar in GenBank, to determine that it is Candida parapsilosis institute after the inquiry comparison.To this sequence, adopt primerexplorer V3 software, carry out design of primers.Software provides 1000 groups of candidate's primers.The following 2 pairs of primers of final acquisition, primer sequence is:
SEQ?ID?NO?1：
5’-GCCTACCAAAGCAAAGTTTTCAAAA-GAATGAAAAGTGCTTAACTGC-3’；
SEQ?ID?NO?2：
5’-GGCCTGCCAGAGATTAAACTC-CCGTTGTTGAAAGTTTTGAC-3’；
SEQ?ID?NO?3：
5’-TAGGTGAACCTGCGGAAG-3’；
SEQ?ID?NO?4：
5’-GATGCGAGAACCAAGAG-3’。
Experiment confirm uses the specificity and the susceptibility of this group primer detection Candida parapsilosis all better.
Further specify through the method for quick of embodiment below Candida parapsilosis ring mediated isothermal amplification provided by the invention.
1. experiment material
(1) selection of experimental strain
Experimental strain has the reference culture of known Candida parapsilosis and the candidiasis of identifying through routine.And the method that adopts this experiment to set up detects the unknown candidiasis in the clinical Blood culture bottle.
(2) reagent buying
The bstDNA polysaccharase is from New England Biolabs, and trimethyl-glycine is from Sigma company, and lyticase is from Solarbio, and the synthetic of primer accomplished by giving birth to worker's biotechnology ltd.
2.DNA extract
1) simplified method: for pure bacterium (reference culture of known Candida parapsilosis and the candidiasis of identifying through routine), the bacterium to be checked of fresh culture is processed bacteria suspension, 100 ℃ were boiled 10 minutes, extracted supernatant, and-20 ℃ of preservations are continued to employ.Test kit method: adopt the MyLab pastoris genomic dna to extract test kit (Beijing Mei Laibo medical science and technology ltd), according to the process specifications extraction DNA of producer.
2) bacterium to be checked in the Blood culture bottle then adopts alkali wash to extract.Concrete steps: at first in 1ml alkaline wash (0.5M NaOHand 0.05M Sodium Citrate), add 0.5ml hemoculture liquid, fully behind the mixing in 13000 revolutions per seconds centrifugal 5 minutes, abandon supernatant, get deposition; Adding 0.5M Tris-HCl (pH8.0,0.5ml), 13000 revolutions per seconds are centrifugal 5 minutes; Abandon supernatant again, get deposition, add Tris-EDTA (0.1ml; 10mM Tris-HCl pH8.0 contains 1mM EDTA) and lyticase (20 μ l 0.5mg/ml) deposited 1 hour in 30 ℃, again in 100 ℃ of heating 1 hour; Freezing dissolving twice, at last in 13000 revolutions per seconds centrifugal 15 minutes, get supernatant-20 a ℃ preservation and continue to employ.
3.LAMP step
(1) primer preparation: all primers are configured to 10 μ M use liquid, again with SEQ ID NO 1～4 in 8: 8: 1: 1 ratio is formulated as mix primer.
(2) reaction system: reaction system amounts to 20 μ l.Comprise: the Tris-HCl of 20mM pH8.8,10mM KCL, 10mM (NH
4)
2SO
4, 2mM MgSO
4, 0.1%TritonX-100,3.6 μ l mix primer, 8U bstDNA polysaccharase, 1.5 μ l dna profilings, 1.2mM dNTP, the 0.8M trimethyl-glycine adds H
2O to 20 μ l.
(3) LAMP reaction: the reaction tubes that will include 20 μ l reaction systems is in 64 ℃ of thermostat module heating 60 minutes, and then be heated to 80 ℃ 10 minutes, with the deactivation of bstDNA polysaccharase.
(4) interpretation as a result: owing to produce a large amount of by products one white magnesium pyrophosphate deposition in the LAMP reaction, get final product result of determination through visual inspection or turbidometer, limpid person is negative in the pipe of reaction back, and muddiness or adularescent deposition person are positive in the pipe; Also can finish the back in reaction and in reaction tubes, add fluorescent substance SYBR green I, fluorescent substance is reacting positive by the orange green that becomes; Reaction product in agarose gel electrophoresis, is run out of stepped band person and is reacting positive, and no band person is negative.
This method has the following advantages
(1) susceptibility is high: extract cerevisiae dna according to the test kit method, make it in the end reaction system, have the DNA amount that is equivalent to 101 fungal cells and can obtain positive findings.
(2) rapid detection:, only need about 2 hours to interpretation as a result from pure bacterium colony DNA extraction.
(3) high specificity: this experiment primer only carries out specific amplification to Candida parapsilosis, detects Candida albicans, Oidium tropicale, Candida glabrata through parallel test; Candida krusei, Cryptococcus neoformans, koumiss yeast; Streptococcus aureus, intestinal bacteria are negative findings.
(4) required instrument is simple: key instrument equipment only needs a thermostatically heating module or a constant water bath box to get final product.
Need to prove,, guarantee working specification, prevent to pollute, to avoid generation false positive as a result because susceptibility is high.
Effect of the present invention has also obtained the checking of a large amount of clinical cases, is illustrated below.
Example 1, the male sex, 45 years old; Clinical diagnosis is obstructive jaundice, carcinoma of gallbladder postoperative, and its hemoculture sample changes kind of Sha Baoruo culture medium culturing after 2 days after the hemoculture appearance report positive; Get pure bacterium colony and be formulated as (1.8-2.2) Maxwell unit bacteria suspension in 0.45%NaCl liquid (PH5.5-7.2); Select Vitek II YST identification card for use, press relating operation rules operations Vitek II instrument after, after 24 hours, be diagnosed as Candida parapsilosis and infect.We are then positive in the report of hemoculture appearance, after smear is shown as fungi infestation, directly from Blood culture bottle, extract DNA; With Candida parapsilosis Auele Specific Primer provided by the invention (sequence is respectively shown in SEQ ID NO 1～4), adopt above-mentioned LAMP method to detect its DNA, after reaction finishes; In reaction tube, add SYBR green I; Result's colour developing is confirmed as Candida parapsilosis in view of the above and is infected for green, from extracting DNA to LAMP reaction and 4～5 hours interpretation times spent.
Example 2, the male sex, 84 years old; Clinical diagnosis is a cerebral infarction, and its hemoculture sample is after the hemoculture appearance report positive, through the Sha Baoruo culture medium culturing after 3 days; Getting single bacterium colony commentaries on classics plants in CHROMagar candidiasis color developing culture medium and corn-tween 80 substratum; Color developing culture medium is shown as blueness as a result, and corn-tween shows pseudohypha, is diagnosed as Oidium tropicale.With example 1, we are positive in the report of hemoculture appearance, after smear is shown as fungi infestation, directly in Blood culture bottle, extract DNA, detect with the LAMP method, and reaction result is negative, can get rid of Candida parapsilosis and infect.
Claims (6)
1. the rapid detection primer of Candida parapsilosis ring mediated isothermal amplification; Comprise inner primers and a pair of outer primer; It is characterized in that; With shown in the SEQ ID NO 2, the sequence of described a pair of outer primer is shown in SEQ ID NO 3 and SEQ ID NO 4 like SEQ ID NO 1 for the sequence of described inner primers.
2. the quick detection kit of Candida parapsilosis ring mediated isothermal amplification is characterized in that, this test kit contains the rapid detection primer and the loop-mediated isothermal amplification reaction reagent of the described Candida parapsilosis ring mediated isothermal amplification of claim 1.
3. the method for quick of Candida parapsilosis ring mediated isothermal amplification; It is characterized in that; Detected sample is extracted DNA through cultivating the back; Get dna profiling, utilize the rapid detection primer of the described Candida parapsilosis ring mediated isothermal amplification of claim 1 to carry out ring mediated isothermal amplification, carry out interpretation then.
4. the method for quick of Candida parapsilosis ring mediated isothermal amplification according to claim 3; It is characterized in that SEQ ID NO 1～4 each sequence that the mix primer in the reaction system of ring mediated isothermal amplification is 10 μ M by concentration separately was in 8: 8: 1: 1 ratio is formulated.
5. the method for quick of Candida parapsilosis ring mediated isothermal amplification according to claim 3 is characterized in that, in the reaction system of the ring mediated isothermal amplification of 20 μ l, contains following composition: the Tris-HCl of 20mM pH8.8,10mM KCL, 10mM (NH
4)
2SO
4, 2mM MgSO
4, 0.1%Triton X-100,3.6 μ l mix primer, 8U bstDNA polysaccharase, 1.5 μ lDNA templates, 1.2mM dNTP, the 0.8M trimethyl-glycine adds H
2O to 20 μ l.
6. the method for quick of Candida parapsilosis ring mediated isothermal amplification according to claim 3 is characterized in that, carries out interpretation through turbidity, the color of SYBR green I fluorescent reaction or the bar belt shape of agarose gel electrophoresis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110303598 CN102312010B (en) | 2011-10-09 | 2011-10-09 | Rapid detection primers, kit and detection method for C.parapsilosis with loop-mediated isothermal amplification |
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|---|---|---|---|
| CN 201110303598 CN102312010B (en) | 2011-10-09 | 2011-10-09 | Rapid detection primers, kit and detection method for C.parapsilosis with loop-mediated isothermal amplification |
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| CN102312010A true CN102312010A (en) | 2012-01-11 |
| CN102312010B CN102312010B (en) | 2013-02-20 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838824A (en) * | 2016-06-08 | 2016-08-10 | 广州医科大学附属第三医院 | Kit, primer and method for detecting candida tropicalis |
| CN105950740A (en) * | 2016-05-30 | 2016-09-21 | 中国人民解放军第二军医大学 | LAMP (loop-mediated isothermal amplification) kit for early diagnosis of candida krusei and special primer |
| CN106521010A (en) * | 2016-12-28 | 2017-03-22 | 上海速创诊断产品有限公司 | LAMP primer composition used for detecting candida parapsilosis and LAMP detection kit and application method thereof |
Citations (3)
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|---|---|---|---|---|
| WO2008135931A2 (en) * | 2007-05-04 | 2008-11-13 | STAB VIDA, Investigação e Serviços em Ciências Biológicas, Lda | Kit and method for the detection and identification of clinically relevant yeasts, using an isothermal dna amplification followed by the hybridisation to species- specific oligonucleotide probes, and respective applications |
| CN101381775A (en) * | 2008-10-15 | 2009-03-11 | 山东出入境检验检疫局检验检疫技术中心 | Loop-mediated isothermal amplification fast detection method of producing candida albicans fungi |
| CN102031289A (en) * | 2010-08-18 | 2011-04-27 | 李国辉 | DNA (deoxyribose nucleic acid) probe and gene chip for detecting candida parapsilosis and applications thereof |
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2011
- 2011-10-09 CN CN 201110303598 patent/CN102312010B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008135931A2 (en) * | 2007-05-04 | 2008-11-13 | STAB VIDA, Investigação e Serviços em Ciências Biológicas, Lda | Kit and method for the detection and identification of clinically relevant yeasts, using an isothermal dna amplification followed by the hybridisation to species- specific oligonucleotide probes, and respective applications |
| CN101381775A (en) * | 2008-10-15 | 2009-03-11 | 山东出入境检验检疫局检验检疫技术中心 | Loop-mediated isothermal amplification fast detection method of producing candida albicans fungi |
| CN102031289A (en) * | 2010-08-18 | 2011-04-27 | 李国辉 | DNA (deoxyribose nucleic acid) probe and gene chip for detecting candida parapsilosis and applications thereof |
Non-Patent Citations (1)
| Title |
|---|
| 牟兆钦: "复合PCR检测医学常见常见真菌的研究", 《中国人兽共患病杂志》, vol. 18, no. 2, 31 December 2002 (2002-12-31), pages 92 - 95 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105950740A (en) * | 2016-05-30 | 2016-09-21 | 中国人民解放军第二军医大学 | LAMP (loop-mediated isothermal amplification) kit for early diagnosis of candida krusei and special primer |
| CN105950740B (en) * | 2016-05-30 | 2019-12-10 | 中国人民解放军第二军医大学 | LAMP kit for early diagnosis of candida krusei and special primer thereof |
| CN105838824A (en) * | 2016-06-08 | 2016-08-10 | 广州医科大学附属第三医院 | Kit, primer and method for detecting candida tropicalis |
| CN106521010A (en) * | 2016-12-28 | 2017-03-22 | 上海速创诊断产品有限公司 | LAMP primer composition used for detecting candida parapsilosis and LAMP detection kit and application method thereof |
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| CN102312010B (en) | 2013-02-20 |
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