CN116064740A - Mycoplasma pneumoniae detection kit - Google Patents

Mycoplasma pneumoniae detection kit Download PDF

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Publication number
CN116064740A
CN116064740A CN202111270499.4A CN202111270499A CN116064740A CN 116064740 A CN116064740 A CN 116064740A CN 202111270499 A CN202111270499 A CN 202111270499A CN 116064740 A CN116064740 A CN 116064740A
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mycoplasma pneumoniae
primer
detection
line
lamp
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钟运华
潘秀华
李春华
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Guangzhou Weimi Biological Science & Technology Co ltd
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Guangzhou Weimi Biological Science & Technology Co ltd
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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

The invention discloses a detection kit based on the LAMP technology for mycoplasma pneumoniae. The LAMP primer is designed according to a specific conserved sequence of mycoplasma pneumoniae, and an amplification product obtained by the LAMP technology is used for carrying out fluorescence identification detection on the amplification product, so as to judge whether the sample contains mycoplasma pneumoniae. The invention provides a new technical platform for mycoplasma pneumoniae detection, and is suitable for popularization and application in basic units, on-site monitoring and bedside detection.

Description

Mycoplasma pneumoniae detection kit
Technical Field
The invention belongs to application of a molecular biological detection method combining isothermal amplification and fluorescence lateral chromatography in mycoplasma pneumoniae detection.
Background
Mycoplasma pneumoniae (MPP), commonly referred to simply as mycoplasma pneumonia, is an acute inflammation mainly caused by interstitial lesions of the respiratory tract and lungs by Mycoplasma Pneumoniae (MP), often accompanied by pharyngitis, bronchitis, and pneumonia. The disease has certain self-healing property, but also has extrapulmonary complications such as meningitis, myocarditis, pericarditis, nephritis, immune hemolytic anemia and the like, and the disease can endanger life. At present, mycoplasma pneumoniae has no unified diagnosis standard, and clinical symptoms, physical signs, chest imaging and serological examination results are generally required to be combined for diagnosis.
Loop-mediated isothermal amplification (LAMP) is a novel isothermal nucleic acid amplification technology, has the advantages of rapidness, simplicity, economy, sensitivity and the like, and is widely applied to the field of rapid nucleic acid detection. The LAMP principle is to design 2 pairs of primers [ FIP (F1c+F2), BIP (B2+B1c), F3 and B3] for 6 regions on a target gene, and perform nucleic acid amplification under isothermal conditions in a short time (15-90 min) under the action of a strand displacement type DNA polymerase.
Compared with the traditional PCR, the LAMP has the characteristics of simple operation, high sensitivity, strong specificity, simple result judgment, low cost and the like. LAMP detection sensitivity is at least 2 orders of magnitude higher than that of ordinary PCR. The amplified products are detected by agarose gel electrophoresis, positive products are ladder-shaped strips with different fragment sizes, and the LAMP reaction result is visualized, so that the detection method is efficient, simple, convenient, quick and high-throughput.
Disclosure of Invention
The invention aims at: providing a detection kit for mycoplasma pneumoniae; another object is to provide a detection method for the mycoplasma pneumoniae kit.
A mycoplasma pneumoniae assay kit, the kit comprising: LAMP reaction liquid, positive control liquid, fluorescent detection card and sample diluent;
the LAMP reaction solution comprises 10 XBuffer, 25mmol/LMgSO 4, 25mmol/LdNTPs, 8U/mu LBst DNA polymerase, 5 mu mol/L primers F3 and B3, 20 mu mol/L primers FIP and BIP;
the primer F3 is 5'-acaagccttagccgttat-3',
the primer B3 is 5'-gggctaagcttccatttgg-3',
the primer FIP is 5'-tgccttaaactggttttggtctaatgttgatgccaactataaggaac-3',
the primer BIP is 5'-caccccaatgaggacgatcttctaccacttgttcagcc-3';
the positive control solution is plasmid diluent containing a mycoplasma pneumoniae genome specific conserved sequence.
The mycoplasma pneumoniae detection kit according to the above, wherein the fluorescent detection card comprises a sample pad, a binding pad, a nitrocellulose membrane, and an absorbent pad.
The mycoplasma pneumoniae detection kit as described above, wherein the sample diluent is phosphate buffer.
The mycoplasma pneumoniae detection kit according to the above, wherein the conjugate pad is coated with streptavidin-modified microspheres.
The mycoplasma pneumoniae detection kit is characterized in that the nitrocellulose membrane is provided with two lines, one is a C line, and biotin-BSA is coated to capture redundant streptavidin modified chromogenic microspheres; the other is a T-line, coated with a complementary oligonucleotide to specifically recognize the amplicon by hybridization with the oligonucleotide at the 5' end of the primer.
The detection method of mycoplasma pneumoniae mainly comprises the following steps:
1) Extracting nucleic acid from a sample to be detected by using a magnetic bead method;
2) Performing LAMP amplification under the guidance of the primer of claim 1 by using genomic DNA of an object to be detected as a template;
3) Diluting the amplified product with a sample diluent, dripping 1-2 drops on a sample pad of a fluorescence detection card, and observing whether the C line and the T line on the nitrocellulose membrane generate fluorescence within 15 min.
The method for detecting mycoplasma pneumoniae according to the above, characterized by comprising the steps of: the conditions for LAMP amplification include: the reaction temperature is 60-65 ℃, the amplification time is 60-70min, the concentration ratio of the internal primer to the external primer is 0.2:0.8-0.2:1.6, and the Mg is as follows 2+ The concentration is 4.0-5.0mmoL/L, dNTPs, the concentration is 0.6 mmoL/L, and the concentration of Bst DNA polymerase is 4.8-9.6U.
The method for detecting mycoplasma pneumoniae according to the above, characterized by comprising the steps of: the reaction temperature is preferably 60 ℃, the amplification time is preferably 60min, the concentration ratio of the inner primer to the outer primer is preferably 0.2:0.8, and the Mg 2+ The concentration is preferably 4.0mmol/L, and Bst DNA polymerase is preferably 8.0U.
The method for detecting mycoplasma pneumoniae according to the above, characterized by comprising the steps of: and judging whether the C line and the T line on the fluorescence detection card generate fluorescence or not, and determining that the sample to be detected contains mycoplasma pneumoniae (positive) when the C line and the T line generate fluorescence.
According to the mycoplasma pneumoniae genome specific conserved sequence, a plurality of groups of Primer sequences can be obtained initially after uploading target sequences by applying on-line Primer design software Primer ExplorerV 5.
The LAMP primer is obtained by screening the LAMP primer mainly comprising the stability of the terminal ends of the primer, the GC content, the distance between the primers and the secondary structure according to the key factors of the LAMP primer design. Specifically, in order to make F1c and B1c more easily bent during the reaction, a double stem-loop structure can be formed, and the primers F1c and B1c should have a Tm value of about 5℃higher than those of the other primers. The Tm values were as close as possible to the requirements (60 ℃ C. And 65 ℃ C.). To improve the annealing binding efficiency of the nucleotide with the template, the delta G value of the six bases at the extreme end of each primer is less than or equal to-4 Kcal/mol, the delta G value of the 3 '-end of F3/B3 and F2/B2 is less than or equal to-4 Kcal/mol, and the delta G value of the 5' -ends of F1c and B1c is less than or equal to-4 Kcal/mol. The GC content of the primer is set to be 40% -60%. For the distance between the primers, the distance from the 5 'end of F2 to the 5' end of B2 is 120-180 bp, the distance from the 5 'end of F2 to the 3' end of F1c, namely the stem-loop section is 40-60 bp, and the distance from the 5 'end of F2 to the 3' end of F3 is 0-20 bp. Finally, special attention must be paid to the inability to form secondary structures between the primers. The final primers were obtained by screening according to the above design principle as follows.
F3 5’-acaagccttagccgttat-3’
B3 5’-gggctaagcttccatttgg-3’
FIP 5’-tgccttaaactggttttggtctaatgttgatgccaactataaggaac-3’
BIP 5’-caccccaatgaggacgatcttctaccacttgttcagcc-3’
The detection kit for mycoplasma pneumoniae provided by the invention comprises the special primer, the main reagent and the reaction parameters for LAMP detection, and has the following advantages:
1) The operation is simple and convenient: the result can be judged only by placing the sample to be detected (target nucleic acid) and the detection reagent into a constant-temperature amplification instrument at about 60 ℃ for reaction for about 60 minutes;
2) The result is visual: and (3) utilizing a fluorescence detection card to determine a reaction result by observing whether the C line and the T line generate fluorescence. The invention can efficiently, specifically and intuitively detect mycoplasma pneumoniae under isothermal conditions. The method does not need complex instruments, provides a new technical platform for detecting mycoplasma pneumoniae, is particularly suitable for crowd screening, has wide market prospect and large economic and social benefits, and is suitable for large-scale popularization and application.
Detailed Description
The present invention will now be described with reference to specific embodiments and specific operation procedures on the premise of the technical proposal of the present invention, but the scope of the present invention is not limited to the following examples. The methods used in the examples described below are conventional methods unless otherwise specified.
Example 1 primer design for LAMP detection of Mycoplasma pneumoniae
And searching in NCBI database to obtain a mycoplasma pneumoniae genome specific conserved sequence, and uploading the target sequence by applying online primer design software Primer Explorer V to obtain a plurality of groups of primer sequences preliminarily. The LAMP primer is obtained by screening the LAMP primer mainly comprising the stability of the terminal ends of the primer, the distance between the primers and the secondary structure according to the key factors of the LAMP primer design.
Example 2 preparation of Mycoplasma pneumoniae kit
LAMP reaction solution [ 10 XBuffer (2.5. Mu.L) per 25. Mu.L, 25mmol/LMgSO ] 4 (3.0. Mu.L), 25mmol/LdNTPs (2.5. Mu.L), 8U/. Mu.LBst DNA polymerase (1.0. Mu.L), 5. Mu. Mol-L primers F3 and B3 (1.0. Mu.L each), 20. Mu. Mol/L primers FIP and BIP (1.0. Mu.L each), template 2.0. Mu. L, ddH 2 0 complement to 25. Mu.L]And packaging the kit with positive control solution (plasmid diluent containing mycoplasma pneumoniae genome specific conserved sequence), a fluorescence detection card and a sample diluent together to obtain the mycoplasma pneumoniae detection kit.
Example 3 establishment of Mycoplasma pneumoniae detection method Using known Mycoplasma pneumoniae samples
The primer for LAMP detection of mycoplasma pneumoniae obtained in example 1 is used for LAMP detection of a nasopharyngeal swab sample nucleic acid extract, and the specific operation steps are as follows:
1) Reaction system
Nucleic acid extraction was performed on the sample to be tested using a magnetic bead method nucleic acid extraction reagent (nucleic acid extraction and purification reagent), and isothermal amplification was performed under the guidance of the LAMP-specific primers obtained in example 1 using the extracted product as a template. Wherein the LAMP reaction system (25. Mu.L) comprises: reaction temperature 60 ℃, amplification time 60min, internal and external primer concentration ratio 0.2:0.8, mg 2+ The concentration was 4.0mmol/L and Bst DNA polymerase was 8.0U.
2) Performing LAMP amplification under the guidance of the reaction system of 1) by taking genomic DNA of an object to be detected as a template;
3) Diluting the amplified product with a sample diluent, dripping 1-2 drops on a sample pad of a fluorescence detection card, and observing whether the C line and the T line on the nitrocellulose membrane generate fluorescence within 15 min.
And judging whether the C line and the T line on the fluorescence detection card generate fluorescence or not, and judging samples, wherein the C line and the T line generate fluorescence, so that the samples to be detected contain mycoplasma pneumoniae (positive).
Example 4 Mycoplasma pneumoniae detection kit for detection of unknown samples
The unknown samples were compared with the culture (gold standard) using LAMP amplification product-fluorescence chromatography using the method of example 3.
And (3) diluting the LAMP amplification product by using a sample diluent, dripping 1-2 drops on a sample pad of a fluorescent detection card, and observing the result within 15 minutes.
Detection result
Figure DEST_PATH_IMAGE001
As can be seen from the above table, the sensitivity of the amplified product for fluorescence chromatography was 99/(99+5) =95.2%, and the specificity was 79/(79+4) =96.3%, and the sensitivity and specificity were both high.
Sequence listing
<110> Guangzhou City micro Biotech Co., ltd
<120> Mycoplasma pneumoniae detection kit
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
acaagcctta gccgttat 18
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gggctaagct tccatttgg 19
<210> 3
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
tgccttaaac tggttttggt ctaatgttga tgccaactat aaggaac 47
<210> 4
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
caccccaatg aggacgatct tctaccactt gttcagcc 38

Claims (9)

1. A mycoplasma pneumoniae assay kit, the kit comprising: LAMP reaction liquid, positive control liquid, fluorescent detection card and sample diluent;
the LAMP reaction solution comprises 10 XBuffer, 25mmol/LMgSO 4, 25mmol/LdNTPs, 8U/mu LBst DNA polymerase, 5 mu mol/L primers F3 and B3, 20 mu mol/L primers FIP and BIP;
the primer F3 is 5'-acaagccttagccgttat-3',
the primer B3 is 5'-gggctaagcttccatttgg-3',
the primer FIP is 5'-tgccttaaactggttttggtctaatgttgatgccaactataaggaac-3',
the primer BIP is 5'-caccccaatgaggacgatcttctaccacttgttcagcc-3';
the positive control solution is plasmid diluent containing a mycoplasma pneumoniae genome specific conserved sequence.
2. The mycoplasma pneumoniae detection kit according to claim 1, wherein the fluorescent detection card comprises a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad.
3. The mycoplasma pneumoniae detection kit according to claim 2, wherein the sample diluent is phosphate buffer.
4. The mycoplasma pneumoniae detection kit according to claim 2, wherein the conjugate pad is coated with streptavidin-modified microspheres.
5. The mycoplasma pneumoniae detection kit according to claim 2, wherein the nitrocellulose membrane has two lines, one of which is a C-line, coated with biotin-BSA to capture excess streptavidin-modified chromogenic microspheres; the other is a T-line, coated with a complementary oligonucleotide to specifically recognize the amplicon by hybridization with the oligonucleotide at the 5' end of the primer.
6. The detection method of mycoplasma pneumoniae mainly comprises the following steps: extracting nucleic acid from a sample to be detected by using a magnetic bead method; performing LAMP amplification under the guidance of the primer of claim 1 by using genomic DNA of an object to be detected as a template; and (3) diluting the amplified product with a sample diluent, dripping 1-2 drops on a sample pad of a fluorescent detection card, and observing a result within 15 minutes.
7. The method for detecting mycoplasma pneumoniae according to claim 6, wherein: the conditions for LAMP amplification include: the reaction temperature is 60-65 ℃, the amplification time is 60-70min, the concentration ratio of the internal primer to the external primer is 0.2:0.8-0.2:1.6, and the Mg is as follows 2+ The concentration is 4.0-5.0mmoL/L, dNTPs, the concentration is 0.6 mmoL/L, and the concentration of Bst DNA polymerase is 4.8-9.6U.
8. The method for detecting mycoplasma pneumoniae according to claim 7, wherein the LAMP amplification conditions are: the reaction temperature is preferably 60 ℃, the amplification time is preferably 60min, the concentration ratio of the inner primer to the outer primer is preferably 0.2:0.8, and the Mg 2+ The concentration is preferably 4.0mmol/L, and Bst DNA polymerase is preferably 8.0U.
9. The method for detecting mycoplasma pneumoniae according to claim 6, wherein: and judging whether the C line and the T line on the fluorescence detection card generate fluorescence or not, and determining that the sample to be detected contains mycoplasma pneumoniae when the C line and the T line generate fluorescence.
CN202111270499.4A 2021-11-04 2021-11-04 Mycoplasma pneumoniae detection kit Pending CN116064740A (en)

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