CN111378770A - Kit for detecting mycoplasma pneumoniae - Google Patents
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- CN111378770A CN111378770A CN201811647982.8A CN201811647982A CN111378770A CN 111378770 A CN111378770 A CN 111378770A CN 201811647982 A CN201811647982 A CN 201811647982A CN 111378770 A CN111378770 A CN 111378770A
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Abstract
The invention discloses a detection kit for mycoplasma pneumoniae based on LAMP technology. The LAMP primers are designed according to a specific conserved sequence of the mycoplasma pneumoniae, each group of primers comprises 4 oligonucleotides, and the mycoplasma pneumoniae is subjected to fluorescence identification detection by using a constant temperature amplification instrument through an LAMP reaction system. The invention provides a new technical platform for detecting the mycoplasma pneumoniae, and is suitable for popularization and application in basic units, field monitoring and bedside detection.
Description
Technical Field
The invention belongs to the application of a molecular biology detection method represented by isothermal amplification in the aspect of detection of mycoplasma pneumoniae, and particularly relates to a loop-mediated isothermal amplification detection method and a kit for the mycoplasma pneumoniae.
Background
Mycoplasma pneumoniae (Mpn) is the smallest microorganism that is facultative anaerobic and able to live independently, with a size of 200 nm. Sterile cell walls, which can grow and divide in cell-free medium, contain RNA and DNA, are metabolized to produce energy, and are sensitive to antibiotics. Mycoplasma is a causative agent of various diseases in animals, and 8 types are currently found, of which only Mycoplasma pneumoniae is certainly causative in humans, mainly respiratory diseases. The mulberry-like yeast strain grows well on an agar medium containing 20% of horse serum and yeast, and a typical dome-shaped mulberry-like bacterial colony can be seen under a microscope after primary culture, and turns into a fried egg shape after multiple passages. The pathological changes of mycoplasma pneumonia are mainly interstitial pneumonia, sometimes complicated by bronchopneumonia, and are called primary atypical pneumonia. The mycoplasma pneumoniae is mainly infected by droplets, the incubation period is 2-3 weeks, and the incidence rate is the highest in teenagers. The clinical symptoms are mild, even no symptoms at all, if some, only general respiratory symptoms such as headache, pharyngalgia, fever, cough and the like, but some death reports exist. It can occur all the year round, but it is mostly in autumn and winter.
In recent years, molecular biology methods are continuously applied to rapid detection of respiratory pathogens, laboratory diagnosis methods of respiratory pathogens have been greatly developed, and nucleic acid detection has become a development direction of laboratory diagnosis of respiratory pathogens. Compared with the traditional laboratory detection method, the molecular diagnosis technology has incomparable detection speed, specificity and sensitivity and becomes a new standard of laboratory diagnosis.
The loop-mediated isothermal amplification method has the advantages of high detection sensitivity, intuitive result judgment, no need of expensive equipment such as a fluorescent quantitative PCR instrument and the like, and has wide application prospect.
Loop-mediated isothermal amplification (LAMP) is a novel isothermal nucleic acid amplification technology, has the advantages of rapidness, simplicity, convenience, economy, sensitivity and the like, and is widely applied to the field of rapid nucleic acid detection at present. The principle of LAMP is that 2 pairs of primers (FIP [ F1c + F2], BIP [ B2+ B1c ], F3 and B3) are designed for 6 regions on a target gene, and nucleic acid amplification is carried out under isothermal conditions in a short time (15-90 min) under the action of a strand displacement type DNA polymerase.
Adding a fluorescent substance SYBR GREEN into the reaction liquid, and detecting a fluorescence curve by using an isothermal amplification instrument, wherein the fluorescence curve shows that mycoplasma pneumoniae exists in a sample to be detected (positive), and the non-amplification curve shows that mycoplasma pneumoniae does not exist in the sample to be detected (negative).
Compared with the traditional PCR, LAMP has the characteristics of simple and convenient operation, high sensitivity, strong specificity, simple result judgment, low cost and the like. The LAMP detection sensitivity is at least 2 orders of magnitude higher than that of the common PCR. In addition, only one water bath or thermostat is needed for isothermal amplification, the requirement on equipment is simple, the operation process is short in time consumption and can be completed within 1 hour. The PCR amplification product is developed by fluorescent dyes such as SYBRGREEN and the like, and the detection of the LAMP reaction result can be visually presented by an isothermal amplification instrument, so that the detection method is efficient, simple, convenient, rapid and high-flux.
Therefore, the development of a loop-mediated isothermal amplification kit for mycoplasma pneumoniae is of great significance.
Disclosure of Invention
The invention aims to: providing a method for detection of mycoplasma pneumoniae nucleic acid; it is another object to provide a kit for use in the method.
1. LAMP (loop-mediated isothermal amplification) detection special primer for mycoplasma pneumoniae
According to the specific conserved sequence of the mycoplasma pneumoniae genome, an online Primer design software Primer Explorer V5 is applied, and after a target sequence is uploaded, a plurality of groups of Primer sequences can be obtained preliminarily.
The LAMP primers are screened according to key factors of LAMP primer design, wherein the key factors mainly comprise the stability of the ends of the primers, GC content, the distance between the primers and a secondary structure, and the LAMP primers are finally obtained. Specifically, in order to make it easier to bend F1c and B1c in the reaction, a double-stem loop structure can be formed, and the primers F1c and B1c are higher than the Tm values of the other primers by about 5 ℃. To improve the efficiency of binding of nucleotides to template annealing, the most terminal six bases of each primer have a free energy Δ G of ≦ 4Kcal/mol, F3/B3, a 3 'terminal Δ G of F2/B2 of ≦ 4Kcal/mol, and a Δ G of the 5' terminal of F1c and B1c of ≦ 4 Kcal/mol. The GC content of the primer is set to be in the range of 40% to 60%. For the distance between the primers, the distance from the 5 'end of F2 to the 5' end of B2 should be 120-180 bp, the distance from the 5 'end of F2 to the 3' end of F1c, i.e., the stem ring segment, should be 40-60 bp, and the distance from the 5 'end of F2 to the 3' end of F3 should be 0-20 bp. Finally, special attention should be paid to the fact that no secondary structure can be formed between the primers. The final primers obtained by screening according to the above design principles are as follows.
F3: 5’-TGCTGCTATTCTCAATCCGG-3’；
B3: 5’-GACCCCACAAGGTTGAACC-3’；
FIP:
5’-CGCTCGTACTCGTTAGCAGCAATTAGCAGCTCTTCCCGACA-3’；
BIP:
5’-AACGGTAGCTCCTACCCAAGGAGGTGGAGAAACGGGAAAGC-3’。
2. LAMP (loop-mediated isothermal amplification) detection method for mycoplasma pneumoniae
The LAMP detection method of the mycoplasma pneumoniae mainly comprises the following steps:
[1] nucleic acid extraction: nucleic acid extraction is carried out on a sample to be detected by using a magnetic bead method nucleic acid extraction reagent (nucleic acid extraction and purification reagent) produced by Shanghai Fuxing Changcheng medical science Co., Ltd.
[2] LAMP amplification
And (3) amplifying under the mediation of a special primer by taking the extracted nucleic acid of the object to be detected as a template. Wherein, the LAMP reaction system (20 μ l) comprises: genomic DNA of the test substance 3. mu.L, 20mM Tris-HCl (pH8.8), 10mM (NH)4)2SO4，50mMKCl，2mM MgSO40.1% Tween20, 1M Betaine, 0.4mM dNTPeach, 8U Bst DNA polymerase, SYBR GREEN (1 × DMSO PCR grade), 0.2 mu M F3, 0.2 mu M B3, 0.8 mu M FIP and 0.8 mu M BIP, LAMP amplification conditions can be set to be constant temperature reaction at 58-68 ℃ for 15-60 min, preferably constant temperature reaction at 63 ℃ for 30 min.
[3] Determination of results
And detecting a fluorescence curve by using a constant temperature amplification instrument, wherein the existence of the mycoplasma pneumoniae (positive) in the sample to be detected is represented by the fluorescence curve, and the nonexpansion curve represents the nonexistence of the mycoplasma pneumoniae (negative) in the sample to be detected.
3. LAMP (loop-mediated isothermal amplification) detection kit for mycoplasma pneumoniae
The LAMP detection kit for mycoplasma pneumoniae provided by the invention comprises the special primers for LAMP detection, main reagents and reaction parameters, and has the following advantages:
[1] high specificity
The 4 primers identify 6 specific regions of the target sequence, so that the high specificity of LAMP amplification is ensured, namely LAMP can search out the corresponding target sequence from a gene sample with only one nucleotide difference for amplification;
[2] high efficiency amplification
The sensitivity is about 100 times higher than that of the common PCR;
[3] simple and convenient operation
The result can be judged only by placing the sample to be detected (target nucleic acid) and the detection reagent in a constant temperature amplification instrument at about 63 ℃ for reaction for about 30 min;
[4] visual result
And detecting a fluorescence curve by using a constant temperature amplification instrument to observe a reaction result. The invention can simply, conveniently and rapidly (constant temperature reaction at 63 ℃ for 30 min) detect the mycoplasma pneumoniae in high efficiency and specificity under the isothermal condition. The method does not need complex instruments, provides a new technical platform for the detection of the mycoplasma pneumoniae, is particularly suitable for crowd screening, has wide market prospect and larger economic and social benefits, and is suitable for large-scale popularization and application.
Detailed Description
The present invention is not limited to the following embodiments, but is also applicable to other embodiments and specific operations. The methods used in the following examples are conventional methods unless otherwise specified.
Example 1 primer design for LAMP detection of Mycoplasma pneumoniae
The specific conserved sequence of the mycoplasma pneumoniae genome is obtained by using NCBI database retrieval, and a plurality of groups of primer sequences can be obtained preliminarily by applying online primer design software PrimeExplorer V5 and uploading target sequences. The LAMP primers are screened according to key factors of LAMP primer design, wherein the key factors mainly comprise the stability of the ends of the primers, GC content, the distance between the primers and a secondary structure, and the LAMP primers are finally obtained.
Example 2 establishment of LAMP detection method for Mycoplasma pneumoniae
The primer for LAMP detection of Mycoplasma pneumoniae obtained in example 1 is used for LAMP detection of the nucleic acid extract of the nasopharyngeal swab sample, and the specific operation steps are as follows:
[1] reaction system
Nucleic acid extraction was performed on a sample to be tested using a magnetic bead method nucleic acid extraction reagent (nucleic acid extraction and purification reagent) produced by Shanghai Fuxing Changcheng medical science Co., Ltd, and isothermal amplification was performed under the guidance of the LAMP specific primer obtained in example 1 using the extracted product as a template. Wherein, the LAMP reaction system (20 μ l) comprises: genomic DNA of the test substance 3. mu.L, 20mM Tris. HCl (pH8.8), 10mM (NH)4)2SO4，50mM KCl，2mM MgSO40.1% Tween20, 1M Betaine, 0.4mM dNTPeach, 8U Bst DNA polymerase, SYBR Green (1 × DMSO PCR grade), 0.2. mu. M F3, 0.2. mu. M B3, 0.8. mu.M FIP and 0.8. mu.M BIP.
[2] Determination of results
And detecting a fluorescence curve by using a constant temperature amplification instrument, wherein the existence of the mycoplasma pneumoniae (positive) in the sample to be detected is represented by the fluorescence curve, and the nonexpansion curve represents the nonexistence of the mycoplasma pneumoniae (negative) in the sample to be detected.
Example 3 LAMP detection kit for preparation of Mycoplasma pneumoniae
The LAMP reaction mixture, LAMP reaction primers (4. mu. M F3 and B3; 16. mu.M FIP and BIP), LAMP reaction enzyme (8U/. mu.L Bst DNA polymerase), reaction buffer (40 mM Tris. HCl, pH 8.8; 20mM (NH)4)2SO4；100mM KCl；4mM MgSO4The LAMP detection kit for the mycoplasma pneumoniae is obtained by packaging 0.2% Tween20, 2M Betaine, 0.8mM dNTPeach, SYBR Green (2 × DMSO PCR grade)) and a positive control (plasmid diluent containing a mycoplasma pneumoniae genome specific conserved sequence).
Claims (4)
1. An LAMP detection method and a reaction system for mycoplasma pneumoniae.
2. An LAMP detection method of mycoplasma pneumoniae mainly comprises the following steps: performing LAMP amplification under the guidance of the special primer in claim 1 by using the genome DNA of a to-be-detected object as a template, adding SYBR GREEN into a reaction solution, and detecting a fluorescence curve by using a constant temperature amplification instrument, wherein the existence of the fluorescence curve indicates the existence of mycoplasma pneumoniae in the to-be-detected sample and the result is positive, and the absence of the amplification curve indicates the absence of the mycoplasma pneumoniae in the to-be-detected sample and the result is negative.
3. The detection method according to claim 2, characterized in that: carrying out nucleic acid extraction on a sample to be detected by using a magnetic bead method; the LAMP reaction system comprises: genomic DNA of the test substance 3. mu.L, 20mM Tris-HCl (pH8.8), 10mM (NH)4)2SO4，50mM KCl，2mM MgSO40.1% Tween20, 1M Betaine, 0.4mM dNTPeach, 8U Bst DNApolymerase, SYBR Green (1 × DMSO PCR grade), 0.2. mu. M F3, 0.2. mu. M B3, 0.8. mu.M FIP and 0.8. mu.M BIP.
4. The detection method according to claim 2, characterized in that the LAMP amplification conditions are: the reaction tube is placed at a constant temperature of 58-68 ℃ for reaction for 15-60 min, preferably at a constant temperature of 63 ℃ for reaction for 30 min.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811647982.8A CN111378770A (en) | 2018-12-29 | 2018-12-29 | Kit for detecting mycoplasma pneumoniae |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811647982.8A CN111378770A (en) | 2018-12-29 | 2018-12-29 | Kit for detecting mycoplasma pneumoniae |
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