CN104988237A - Pneumonia mycoplasma nucleic acid rapid detection method - Google Patents
Pneumonia mycoplasma nucleic acid rapid detection method Download PDFInfo
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- CN104988237A CN104988237A CN201510436255.7A CN201510436255A CN104988237A CN 104988237 A CN104988237 A CN 104988237A CN 201510436255 A CN201510436255 A CN 201510436255A CN 104988237 A CN104988237 A CN 104988237A
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Abstract
The invention provides a pneumonia mycoplasma nucleic acid rapid detection method. According to the provided method, an isothermal amplification technology is utilized, six different areas in the conserved region of pneumonia mycoplasma genes are selected, four primers are designed, at the same time, under the assistance of DNA polymerase with a strand displacement function, the nucleic acid isothermal amplification can be achieved; the amplification product will give off a fluorescence signal after being combined with a fluorescence dye, the fluorescence signal can be read by a detection instrument in real time, the result can be determined according to the amplification curve, and thus the pneumonia mycoplasma detection on a sample can be realized. The provided kit programs the operation steps; the pneumonia mycoplasma detection on phlegm and swab sample is realized, moreover, the operation becomes more convenient and controllable, the detection is carried out in an enclosed tube, at the same time, a stabilizing liquid is added to prevent the formation of aerogel, the contamination problem is solved, and the result is more reliable. The provided kit has the advantages of rapidness, high sensitivity, simple operation, and low cost, and thus is very suitable for on-site detection and basic medical institutions.
Description
Technical field
The present invention relates to biology techniques field, be specifically related to a kind of mycoplasma pneumoniae nucleic acid method for quick.
Background technology
Mycoplasma pneumoniae (Mycoplasma pneumoniae, MP), the variforms such as diameter is 125-150 nm, similar with the size of myxovirus, containing DNA and RNA, lacks cell walls, spherical in shape, shaft-like, thread, gram's staining is negative.Mycoplasma pneumoniae (MP) be in infant, one of a kind of its healthy pathogenic agent of a kind of main harm caused due to respiratory tract infection that adolescence easily occurs.MP diagnostic method mainly comprises classical culture method, serology body detection method and nucleic acid detection method etc. on the market at present.Isolated culture is that diagnosis MP infects the most reliable method, but MP separation and Culture needs 3-4 week, and low, the consuming time length of sensitivity, technical requirements are high.Serologic detection (comprising cold agglutination test, enzyme linked immunosorbent assay (ELISA), Immunogold-labeling method and immunofluorescence technique etc.) could be applied after need waiting until body generation antibody has certain hysteresis quality.Current multiplex ELISA measures anti-MP antibody, and immunology detection technology is fast and convenient with low cost, but requires the monoclonal antibody of high quality high stable, otherwise accuracy is inadequate, only can as auxiliary detection means.Therefore can provide fast, Gao Min, the emphasis that special, easy, reliable, detection technique that is that be applicable to clinical application has become research both at home and abroad.Detection of nucleic acids common are nucleic acid hybridization assay and round pcr, the former degrees of sensitivity, specific degree are strong, but need to apply more equipment to assist, therefore be difficult to promote, the latter to detecting the sensitivity of MP DNA, specific degree is high, but because of its program relative complex, requires high to laboratory environment, the problems such as fluorescence PCR method apparatus expensive, also make this technology popularization make slow progress.Therefore, a kind of method of early diagnosis that is easy, quick, effective, that be suitable for basic unit's popularization is found extremely important.
Summary of the invention
For solving the problem, the invention provides a kind of mycoplasma pneumoniae nucleic acid method for quick, detection method step is as follows:
(1) sample process, estimate the amount of sputum sample, add the NaOH solution of 4% of 2-4 times of volumes, fully mix, room temperature leaves standstill liquefaction 10 min-20 min, makes it fully liquefy; Swab sample is directly stored in swab conserving liquid;
(2) sample washing, sputum (or the swab washing fluid) sample getting liquefaction is transferred in centrifuge tube, and centrifugal segregation supernatant collection precipitates, with the washing precipitation of sample washings, centrifugal collecting precipitation;
(3) nucleic acid extraction, by the resuspended above-mentioned precipitation of sample lysate, mixing, 100 DEG C of temperature bath cracking 5-15 min, be placed in immediately on ice, then centrifugal, supernatant liquor is sample form DNA;
(4) application of sample reaction, takes out detector tube, marks negative control pipe, detector tube and positive control pipe respectively, adds negative solution, step (3) supernatant liquor and positive solution successively respectively;
(5) upper machine runs, and puts into Fluorescent reader after being mixed by the experiment tube of step (4), sets 60 –, 65 DEG C of bar lower part insulation 30-60 min; Add fluorescence dye in advance in amplification system, can fluorescent signal be sent after gene-amplification product is combined with fluorescence dye, read fluorescent signal in real time by detecting instrument, according to amplification Reading determination result;
(6) result interpretation, if instrument amplification reading is in " S " type or imperfect " S " type, then result interpretation is positive; If instrument amplification reading is "-" type, then result interpretation is negative.
In above-mentioned steps, should according to positive control pipe instrument amplification reading in " S " type during result interpretation, negative control pipe instrument amplification reading is in "-" type, otherwise should judge that this experiment is invalid.
The usefulness of this invention is: test kit sequencing of the present invention operation steps, achieve the detection to mycoplasma pneumoniae in sputum and swab sample, make operating process easy and controlled, testing process adopts stopped pipe to detect simultaneously, and adding again stable liquid prevents and treats aerocolloidal formation, solve pollution problem, result is more reliable.Test kit of the present invention because of fast, Gao Min, simple to operate, low cost and other advantages is applicable to Test Field and basic medical unit is applied.
Embodiment
Below, in conjunction with embodiment, the present invention is described further:
The technical scheme of the inventive method is: the invention provides a kind of mycoplasma pneumoniae nucleic acid method for quick, detection method step is as follows:
(1) sample process, estimate the amount of sputum sample, add the NaOH solution of 4% of 2-4 times of volumes, fully mix, room temperature leaves standstill liquefaction 10 min-20 min, makes it fully liquefy; Swab sample is directly stored in swab conserving liquid;
(2) sample washing, sputum (or the swab washing fluid) sample getting liquefaction is transferred in centrifuge tube, and centrifugal segregation supernatant collection precipitates, with the washing precipitation of sample washings, centrifugal collecting precipitation;
(3) nucleic acid extraction, by the resuspended above-mentioned precipitation of sample lysate, mixing, 100 DEG C of temperature bath cracking 5-15 min, be placed in immediately on ice, then centrifugal, supernatant liquor is sample form DNA;
(4) application of sample reaction, takes out detector tube, marks negative control pipe, detector tube and positive control pipe respectively, adds negative solution, step (3) supernatant liquor and positive solution successively respectively;
(5) upper machine runs, and puts into Fluorescent reader after being mixed by the experiment tube of step (4), sets 60 –, 65 DEG C of bar lower part insulation 30-60 min; Add fluorescence dye in advance in amplification system, can fluorescent signal be sent after gene-amplification product is combined with fluorescence dye, read fluorescent signal in real time by detecting instrument, according to amplification Reading determination result;
(6) result interpretation, if instrument amplification reading is in " S " type or imperfect " S " type, then result interpretation is positive; If instrument amplification reading is "-" type, then result interpretation is negative.
In above-mentioned steps, should according to positive control pipe instrument amplification reading in " S " type during result interpretation, negative control pipe instrument amplification reading is "-" type, otherwise should judge that this experiment is invalid.
The constant-temperature amplification method that in the present invention, test kit uses is a kind of brand-new nucleic acid amplification method, can realize 10 in 15-60 minute
9-10
10amplification doubly, fluorescence dye is added in advance in amplification system, fluorescent signal can be sent after gene-amplification product is combined with fluorescence dye, fluorescent signal is read in real time by detecting instrument, according to amplification Reading determination result, thus the mensuration realized mycoplasma pneumoniae in sample, there is feature that is simple, quick, high specificity.This test kit utilizes isothermal amplification technology, choose the region that six of mycoplasma pneumoniae gene conserved regions are different, design 4 primers, in conjunction with the archaeal dna polymerase with strand displacement function, realize nucleic acid constant-temperature amplification, can fluorescent signal be sent after amplified production is combined with fluorescence dye, read fluorescent signal in real time by detecting instrument, according to amplification curve sentence read result, thus realize the mensuration to mycoplasma pneumoniae in sample.
Test kit sequencing of the present invention operation steps, achieve the detection to mycoplasma pneumoniae in sputum and swab sample, make operating process easy and controlled, add again stable liquid while that testing process adopting stopped pipe to detect and prevent and treat aerocolloidal formation, solve pollution problem, result is more reliable.Test kit of the present invention is because of quick, Gao Min, simple to operate, low cost and other advantages, and applicable Test Field and basic medical unit are applied.
Claims (2)
1. a mycoplasma pneumoniae nucleic acid method for quick, is characterized in that: detection method step is as follows:
(1) sample process, estimate the amount of sputum sample, add the NaOH solution of 4% of 2-4 times of volumes, fully mix, room temperature leaves standstill liquefaction 10 min-20 min, makes it fully liquefy; Swab sample is directly stored in swab conserving liquid;
(2) sample washing, sputum (or the swab washing fluid) sample getting liquefaction is transferred in centrifuge tube, and centrifugal segregation supernatant collection precipitates, with the washing precipitation of sample washings, centrifugal collecting precipitation;
(3) nucleic acid extraction, by the resuspended above-mentioned precipitation of sample lysate, mixing, 100 DEG C of temperature bath cracking 5-15 min, be placed in immediately on ice, then centrifugal, supernatant liquor is sample form DNA;
(4) application of sample reaction, takes out detector tube, marks negative control pipe, detector tube and positive control pipe respectively, adds negative solution successively respectively, supernatant liquor that step (3) obtains and positive solution;
(5) upper machine runs, and puts into Fluorescent reader after being mixed by the detector tube of step (4), is incubated 30-60 min under setting 60 –, 65 DEG C of conditions; Add fluorescence dye in advance in amplification system, can fluorescent signal be sent after amplified production is combined with fluorescence dye, read fluorescent signal in real time by detecting instrument, according to amplification Reading determination result;
(6) result interpretation, if instrument amplification reading is in " S " type or imperfect " S " type, then result interpretation is positive; If instrument amplification reading is "-" type, then result interpretation is negative.
2. a kind of mycoplasma pneumoniae nucleic acid method for quick according to claim 1, it is characterized in that: in described result interpretation step 6, should according to positive control pipe amplified reaction instrument readings in " S " type, negative control pipe amplified reaction instrument readings is "-" type, otherwise should judge that this experiment is invalid.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105403562A (en) * | 2015-12-16 | 2016-03-16 | 合肥泰特信息科技有限责任公司 | Mycoplasma detector |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105403562A (en) * | 2015-12-16 | 2016-03-16 | 合肥泰特信息科技有限责任公司 | Mycoplasma detector |
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Application publication date: 20151021 |