CN112831578B - Primer group, kit and method for detecting mycoplasma pneumoniae - Google Patents
Primer group, kit and method for detecting mycoplasma pneumoniae Download PDFInfo
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Abstract
The invention discloses a primer group, a kit and a method for detecting Mycoplasma Pneumoniae (MP), wherein the primer group comprises an MP outer primer pair, an MP inner primer pair and an MP ring primer pair, the sequences of the MP outer primer pair are shown as SEQ ID NO. 1 and SEQ ID NO. 2, the sequences of the MP inner primer pair are shown as SEQ ID NO. 3 and SEQ ID NO. 4, and the sequences of the MP ring primer pair are shown as SEQ ID NO. 5 and SEQ ID NO. 6. The primer group, the kit and the method provided by the invention have the advantages of high sensitivity (up to 100 copies/reaction), accurate detection, high detection speed and simple detection process.
Description
Technical Field
The invention belongs to the fields of molecular biology, in-vitro diagnosis and detection, and particularly relates to a primer group, a kit and a method for detecting mycoplasma pneumoniae.
Background
The mycoplasma pneumoniae is a main pathogen of community-acquired pneumonia, is a main epidemic pathogen in winter and spring seasons around the world, has high infectious diseases, is particularly susceptible to infants, has high infection rate in Chinese people, and has an increasing trend in recent years. Infection with mycoplasma pneumoniae can cause pneumonia and even attack extrapulmonary organs, which in severe cases can lead to death. Respiratory pathogens are of various types, mainly including viruses, bacteria, mycoplasma pneumoniae and chlamydia pneumoniae, and also belong to protozoa, fungi and the like. The first choice treatment drugs and treatment schemes of different pathogenic bacteria are different, and for antibiotics, the first choice effective antibiotics are completely different due to pathogen difference, but the disease signs caused by different pathogens have great similarity and are difficult to distinguish, and the accurate and rapid distinguishing of the pathogen types becomes the key for effectively treating diseases and controlling epidemic spread so as to strive for using correct drugs as early as possible and avoid the abuse of ineffective antibiotics.
At present, in domestic mycoplasma pneumoniae clinical diagnosis, immune method products represented by 'indirect fluorescence method respiratory tract nine joint inspection antibody detection' and 'gold-labeled chromatography 2-5 joint inspection antigen detection' are mainly used, and other technical products are provided. From the viewpoint of technical principle, the methods can be classified into a culture method, an antigen immunoassay method, a serum-specific antibody immunoassay method, and a molecular diagnostic method represented by Real-time fluorescent quantitative PCR (Real-time fluorescent PCR, fluorescent quantitative PCR, real-time quantitative PCR, qPCR, etc.). However, these methods all have obvious technical defects and application restriction factors, for example, the real-time fluorescence quantitative PCR technology faces a series of problems of complex nucleic acid extraction and detection operations, high requirements on instrument platforms and operators, high cost and limited use scenes.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a primer group, a kit and a method used based on the LAMP constant temperature amplification technology, and innovatively develops a one-step method sample nucleic acid extraction technology without purification, so that complicated manual operation steps are omitted; a primer group with optimal target specificity is designed, an optimized reaction formula is developed, and the detection efficiency is improved; the freeze-drying formula is developed and optimized, and the kit capable of being stored in a refrigerated manner or transported at normal temperature is prepared, so that the application scenes of the product are greatly widened.
The core technical method used by the invention is loop-mediated isothermal amplification (LAMP), which is a novel isothermal nucleic acid amplification method developed in recent years and belongs to one of molecular diagnosis methods. The LAMP technology has the advantages of strong specificity, short reaction time, high sample tolerance, convenience in operation, low hardware cost and the like. The method is a patent technology of Japan Rongand research company, and the patents include CN100393875C, CN1222614C, CN100422323C and the like.
The LAMP method designs 6 specific primers (including upstream and downstream outer primers F3 and B3, upstream and downstream inner primers FIP and BIP, and upstream and downstream loop primers LF and LB) aiming at 8 regions of a target sequence, utilizes a Bst DNA polymerase with strand displacement activity, and carries out heat preservation for less than 60min under constant temperature condition, thus completing nucleic acid amplification reaction, generating macroscopic reaction by-product-white magnesium pyrophosphate precipitate, and also carrying out more accurate fluorescence quantification through an embedded dye. The present inventors creatively used this technique, designed a primer set with strong specificity and high sensitivity, and searched for various conditions in the reaction system.
The invention is realized by the following technical scheme:
the first aspect of the invention relates to a primer group for detecting mycoplasma pneumoniae, which has the characteristics of strong specificity (no detection activity on other pathogens), high sensitivity and good universality (universality on various subtypes in species). The specific sequence of the primer set is shown in SEQ ID NO 1 to SEQ ID NO 6 in Table 1.
TABLE 1 primer sequence Listing
The design process of the primer is as follows:
firstly, homology analysis is carried out on MP genome, a highly conserved region is identified, and LAMP detection primers are designed by using high-quality and high-efficiency LAMP primer design software LAMP designer 1.16. In the invention, the primer group capable of amplifying the specific base sequence of the MP genome is a part of the nucleic acid sequence of the 710397-710673 bp site of the MP genome of GenBank LR214945.1 or a part of a complementary strand thereof. Wherein the MP genome consensus specific base sequence is a base sequence unique to the MP genome only and not included in the genome of other microorganisms.
And (3) primer specificity analysis: and respectively carrying out theoretical analysis on the specificity of the primer groups, wherein the method comprises the steps of comparing the sequence between the primers F3 and B3 with the MP genome in a GenBank database to determine a detection region, carrying out Blast comparison on non-MP in public database resources of the detection region sequence, and detecting the matching degree of the primer region and the genome sequence. If the matching degree is higher, the specificity is worse; if the primers can not be compared with the strains of the non-detection strains at the same time, the specificity is good. The result shows that primer matching required by LAMP amplification cannot be formed when no primer is aligned to a non-MP sequence in each primer group, and the specificity of each primer group is better.
Primer universality analysis: and respectively carrying out theoretical analysis on the universality of the primer groups, wherein the method comprises the steps of comparing the sequence between the primers F3 and B3 with the genome of the MP in a GenBank database to determine a detection region, then comparing a plurality of MPs of the detection region sequence in public database resources to detect the matching degree of the primer region and the genome sequence, and if the primer regions are completely matched, the universality is good. As a result, it was found that the primer regions of the respective primer sets completely matched the plural genomes of the above MP, indicating that the respective primer sets are excellent in versatility.
The second aspect of the present invention relates to a kit for detecting mycoplasma pneumoniae, which comprises a reaction system, wherein the reaction system comprises the primer set according to the first aspect.
Preferably, the reaction system further comprises dNTP, bst DNA polymerase, tris-HCl, KCl, (NH 4) 2 SO4, triton X-100, betaine, mg 2+ And a fluorescent dye.
More preferably, the reaction system comprises:
0.125-5mmol/L dNTP；
0.15-2U/. Mu.L of Bst DNA polymerase;
10-100mmol/L Tris-HCl with pH 7-9;
0-100mmol/L KCl；
0-100mmol/L(NH 4 ) 2 SO 4 ；
0-1％Triton X-100；
0-1.5mol/L betaine;
2-9mmol/L Mg 2+ ；
0.15-0.3 mu mol/L MP outer primer pair;
0.8-2.0 mu mol/L MP inner primer pair; and
0.15-0.6 mu mol/L MP loop primer pair.
Said Mg 2+ May be conventional in the art, preferably MgSO 4 Or MgCl 2 。
The fluorescent dye may be any one of SYBR Green, evaGreen, SYTO-9 and SYTO-82.
In one embodiment of the present invention, the reaction system comprises:
1.0mmol/L dNTP；
0.5U/. Mu.L of Bst DNA polymerase;
20mmol/L Tris-HCl(pH 8.8)；
10mmol/L KCl；
10mmol/L(NH 4 ) 2 SO 4 ；
0.1％Triton X-100；
100 × diluted SYBR Green (NEB);
0.5mol/L betaine;
6mmol/L MgSO 4 ；
0.2 mu mol/L MP outer primer pair;
1.6 mu mol/L MP inner primer pair;
0.4. Mu. Mol/L MP loop primer pair.
The kit is preferably in the form of a lyophilized powder.
Preferably, the protective agent for preparing the freeze-dried powder is 5-20% of sucrose or trehalose, and preferably 10% of trehalose.
As a preferred embodiment, the freeze-drying process of the freeze-dried powder is as follows:
subpackaging 15-30 uL of the liquid reagent mixture into a reaction tube, then placing the reaction tube into an ultralow temperature freeze dryer, firstly reducing the temperature to below-40 ℃, vacuumizing the reaction tube for 20-30 hours, sublimating and removing water until small dry powder is formed at the bottom of the tube, covering the tube in a vacuum state to obtain a freeze-dried dry powder reagent tube, taking out the freeze-dried dry powder reagent tube, and refrigerating the freeze-dried dry powder reagent tube at the temperature of 2-8 ℃. The reagent mixture in the form of freeze-dried powder is the core component of the kit.
In a third aspect the present invention relates to a method of detecting mycoplasma pneumoniae, said method comprising the steps of:
(1) Extracting total DNA in a sample to be detected;
(2) Taking the total DNA extracted in the step (1) as a template, and carrying out LAMP amplification reaction by using the kit in the second aspect to obtain an amplification product;
(3) Detecting a result;
the detection in the step (3) is any one of fluorescence detection, visual color development detection, turbidity detection and electrophoresis detection.
Preferably, the amplification time of the LAMP amplification reaction is 20min, and the temperature is 60-65 ℃.
As a preferred embodiment, the specific steps for extracting the pathogen nucleic acid of the sample to be detected are as follows:
a. taking a pharyngeal swab/a nasal swab/a nasopharyngeal swab, sampling, immersing into a sample preservation solution optimized in a formula, rinsing for 5-10 min, and thermally cracking the leachate at 80-100 ℃ for 5-20 min;
b. standing at room temperature for 1-2 minutes and cooling;
c. instantly throwing by a low-speed centrifuge or throwing by hands, collecting the liquid to the bottom of the tube for later use or storing the liquid at 2-8 ℃ for the day for use, and storing the liquid at the temperature below-20 ℃ for a long time to be used as a template for subsequent LAMP amplification for later use.
In a fourth aspect the present application relates to the use of a primer set according to the first aspect or a kit according to the second aspect in the manufacture of a product for use in the diagnosis of mycoplasma pneumoniae, said product being suitable for use in a method according to the third aspect.
In the experimental process, the MP genome DNA for accurately determining the nucleic acid concentration or the synthetic plasmids matched with each primer group for accurately determining the nucleic acid concentration are used as detection templates for experimental tests. The synthetic plasmid synthesis and quantification method matched with each primer group comprises the following steps: customizing and synthesizing DNA between each primer group F3 and B3, constructing on a plasmid vector pUC57, transferring into escherichia coli DH5 alpha, expressing and amplifying plasmids, extracting and purifying the plasmid DNA, then adopting a nucleic acid quantitative instrument (OD 260/OD280 method) to accurately measure the concentration of nucleic acid, calculating the copy number concentration according to the molecular weight of the plasmids, and gradually diluting into a standard substance of 10^9 copies/uL-10 ^ 1copies/uL. The preparation and quantitative method of the genome DNA comprises the following steps: purchasing MP FH strain (15531-TTR) of ATCC, culturing according to the instructions, extracting and purifying pathogen genome DNA, preparing a gradient dilution, simultaneously testing the gradient dilution of the genome DNA and the quantified plasmid gradient standard by using a QPCR method, preparing a standard curve by using the concentration of the plasmid gradient standard and the starting peak Ct data, substituting the standard curve into the Ct data of the gradient dilution of the genome DNA, and calculating the concentration of the genome DNA in a reverse mode.
The plasmid vectors pUC57, E.coli DH 5. Alpha. Are conventional in the art.
As a preferred embodiment, the LAMP amplification detection step is:
and (2) directly adding the extracted nucleic acid of the sample to be detected into a detection tube as a template of LAMP, uniformly mixing the nucleic acid with the reagent in the form of freeze-dried powder to form an amplification reaction system, placing the amplification reaction system into a reaction hole of a constant-temperature nucleic acid amplification analyzer (such as a real-time fluorescence detector), performing amplification at a constant temperature of 60-65 ℃ for 20min, selecting a proper fluorescence channel to monitor the change of a fluorescence signal in the amplification process, and recording a real-time fluorescence curve. And after the amplification reaction is finished, judging the positive and negative results of the detection items according to the predetermined change rate of the fluorescence intensity signal and the fluorescence intensity signal threshold value, and displaying the results on a screen interface in real time.
In the method, fluorescent dyes including but not limited to SYBR Green, evaGreen, SYTO-9, SYTO-82 and the like can be used in a reaction system, a fluorescent PCR instrument (such as ABI 7500) or other constant-temperature fluorescent detectors are used for detection, and according to the result of the amplification curve judgment, the result shows that the S-shaped amplification curve is positive and the result shows that the S-shaped amplification curve is negative.
In the method of the present invention, the LAMP amplification detection method includes, but is not limited to, the above-mentioned fluorescence detection, and the following visual color detection, turbidity detection, electrophoresis detection, and the like.
The visual color development detection is to add a color development reagent including but not limited to calcein or Hydroxy Naphthol Blue (HNB) into the reaction tube. When calcein or SYBR Green is used as a color developing agent, if the color is orange after reaction, the sample to be detected is negative; if the color is green after the reaction, the sample to be tested is positive. When hydroxyl naphthol blue is used as a color developing agent, if the color after reaction is violet, the sample to be detected is negative; if the color after the reaction is sky blue, the sample to be detected is positive. The color development detection can be carried out in real time or at the end point by a detection instrument besides observing the reaction result by naked eyes, and the sample to be detected is negative by reasonably setting the threshold value of the negative reaction when the reaction result of the sample to be detected is lower than or equal to the threshold value; and when the reaction result of the sample to be detected is greater than the threshold value, determining that the sample to be detected is positive. The detection instrument comprises but is not limited to a fluorescence spectrophotometer, a fluorescence quantitative PCR instrument, a constant temperature amplification micro-fluidic chip nucleic acid analyzer, an isothermal amplification fluorescence detection system and the like.
The turbidity detection is carried out by observing with naked eyes or detecting turbidity by a turbidity meter, and if the detection tube is obviously turbid, the sample to be detected is positive and contains MP; if no turbidity is found, the sample to be tested is negative. Or the bottom of the reaction tube can be observed by naked eyes after centrifugation to see whether the sediment exists, if so, the sample to be detected is positive; if no precipitate is formed at the bottom of the reaction tube, the sample to be detected is negative.
The electrophoresis detection is preferably a gel electrophoresis detection method, and may be agarose gel or polyacrylamide gel. In the electrophoresis detection result, if the electrophoresis image shows a characteristic step-shaped strip, the sample to be detected is positive; and if the electrophoretogram does not present a characteristic step-shaped strip, the sample to be detected is negative.
The positive progress effects of the invention are:
the simple sample nucleic acid extraction method gets rid of the high requirement that a large nucleic acid extractor is needed for molecular diagnosis samples, avoids the disadvantage that the traditional manual extraction kit is long and time-consuming, and greatly enhances the convenience of use of customers;
the constant temperature amplification technology can be used on a small instrument with low price, so that the high requirement and high cost in the aspect of molecular diagnosis hardware are greatly reduced;
the optimized primer sequence has the advantages of high sensitivity, accurate detection, intraspecific compatibility of various subtypes (universality) and interspecific exclusion (specificity);
the reaction speed is high, the consumed time is shortened to 20 minutes (the most common real-time fluorescent quantitative PCR technology generally consumes 40-60 min), and the detection accuracy and detection speed are greatly improved;
the product kit can be stored in a refrigerated storage mode (2-8 ℃) and transported at normal temperature, overcomes the defects that molecular diagnostic reagents generally need to be stored in a refrigerated storage mode (minus 20 ℃) and transported in a cold chain (minus 20 ℃ or 2-8 ℃), and is particularly suitable for low-grade medical detection units with poor medical conditions.
In conclusion, the method has the characteristics of high speed, high efficiency, simple and convenient operation, high specificity, high sensitivity, simple and convenient identification, suitability for field detection and the like, and can be used for quickly detecting the DNA of the mycoplasma pneumoniae.
Drawings
FIG. 1 is a graph showing the sensitivity & amplification time of LAMP amplification of Mycoplasma pneumoniae.
FIG. 2 is a photograph of the LAMP amplification system lyophilized powder.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the invention thereto.
Example 1: MP sequence amplification by LAMP method
(1) An amplification reaction system:
1.0mmol/L dNTP；
0.2 mu mol/L MP outer primer pair;
1.6 mu mol/L MP inner primer pair;
0.4 mu mol/L MP loop primer pair;
0.5U/. Mu.L of Bst DNA polymerase;
20mmol/L Tris-HCl(pH 8.8)；
10mmol/L KCl；
10mmol/L(NH 4 ) 2 SO 4 ；
0.1％Triton X-100；
6mmol/L MgSO 4 ；
0.5mol/L betaine;
SYBR Green at 100X dilution.
(2) Amplification conditions: placing the mixture in a constant temperature nucleic acid amplification analyzer, and amplifying for 20 minutes at 65 ℃.
Example 2: LAMP reaction sensitivity detection
Currently, the mainstream molecular diagnostic products in the market are mostly QPCR, and although the sensitivity performance is generally considered to be better (100-500 copies/reaction), the method is limited by the defects of needing an expensive temperature-variable fluorescence quantitative PCR instrument, low temperature rise and fall speed of PCR circulation and the like, and the popularization is always limited. In contrast, the characteristics of the isothermal LAMP technology just compensate the defects of the QPCR technology, but the sensitivity of LAMP products on the market is not uniform in literature reports, so that the bottleneck in the popularization and use of LAMP is whether the primer sequence is optimized and the reaction system is optimized, thereby ensuring that LAMP has high detection sensitivity.
As shown in FIG. 1 and Table 2, the S-shaped curve in the graph is the fluorescence curve of LAMP amplification experiment, the S-shaped curve means the increase of fluorescence signals, the essential principle is that more fluorescent dyes such as SYBR Green are embedded into a double-chain product along with the increase of amplification products, and compared with the original free state, the fluorescent dyes generate stronger luminescence performance and the fluorescence signals are enhanced. Table 2 summarizes the data of the peak-off time in FIG. 1, i.e., how many minutes the LAMP reaction took, the fluorescent signal curve started to enter the rising stage of the sigmoid curve (NoCt indicates that the LAMP reaction did not peak, i.e., no amplification product was generated).
As shown in FIG. 1 and Table 2, the detection sensitivity of the primer set selected by the invention can reach 100 copies/reaction, which is superior to the conventional sensitivity performance (500-1000 copies/reaction) of the LAMP product on the market reported in the literature and is equivalent to the conventional sensitivity performance of the QPCR product on the market. A good sensitivity table ensures clinical compliance in clinical sample testing and avoids false negative conclusions in testing samples with low levels of pathogens (true positive patient samples, failing to detect positive results, being misdiagnosed as negative, i.e., not infected) (see example 9 below).
Table 2: summary of sensitivity & amplification time for Mycoplasma pneumoniae LAMP amplification
| copies/reactions | Peak time (mins) | |
| | NoCt | |
| 10^1 | |
|
| 10^2 | 15.07 | |
| 10^3 | 9.19 | |
| 10^4 | 9.17 | |
| 10^5 | 8.23 |
Example 3: reaction time-consuming assay
As shown in the figure 1, the table 2 and the table 3, the LAMP amplification reaction can be completed within 20 minutes by the primer group selected by the invention, which is greatly superior to the time consumption of 30-60 minutes of the LAMP/mainstream QPCR products on the market reported in the literature.
Table 3: comparison of time to Peak for different MP primer sets
Example 4 storage stability and reconstitution efficiency test of dried reaction System
As shown in the figure 2, the table 6 and the table 7, the LAMP freeze-drying process developed by the invention has good dry powder form, can be stably stored under various temperature conditions, can at least meet the requirements of no obvious loss of performance for 7 days at 37 ℃, 180 days at 2-8 ℃ and 180 days at-20 ℃, does not influence the detection sensitivity and the reaction speed of products, and realizes refrigeration storage and normal-temperature transportation.
Example 5: primer screening process
MP primer design As described above, in the experimental stage of the present invention, LAMP design 1.16 software is used to design 3 sets of primers, i.e., MP to MP-3 in Table 1, and 2 sets of LAMP primer sets, i.e., MP-4 to MP-5 in Table 1, reported in the literature are synthesized.
And detecting the same genomic DNA gradient dilution in the same reaction system formula by using each primer group, and screening by performance performances in the aspects of sensitivity and reaction time. As shown in Table 3, the primer group MP-primer group 1 selected by the invention has the best performance, the sensitivity can reach 100 copies/reaction, the reaction time is within 20min, and 100 copies/reaction and reactions above can all generate peaks normally. The sensitivity of the MP-primer group 4 can also reach 100 copies/reaction, but the reaction takes longer, the sensitivity and the reaction time of the MP-primer group 2 and the MP-primer group 5 are not similar to those of the MP-primer group 1 and the MP-primer group 3, although the sensitivity and the reaction time are similar to those of the MP-primer group 1, the negative contrast peak is probably caused by primer polymers, and the false positive result is easily caused, so the false positive result is eliminated.
Example 6: enzyme dosage screening process
The enzyme is a core material of LAMP reaction, the dosage is too small, the reaction efficiency is low, the dosage is too much, and the cost is increased. Taking the preferred primer set of MP as an example, the appropriate amount of the selected enzyme is optimized according to literature reports and recommendations of enzyme suppliers.
Adding different amounts of enzyme into the same reaction system, detecting the same genomic DNA gradient dilution, and screening by performance expression in the aspects of sensitivity and reaction time. The invention selects the enzyme dosage, namely the final concentration of 0.5U/uL concentration, the performance is best, the sensitivity can reach 100 copies/reaction, the reaction time is within 20min, and all positive reactions can generate peaks normally (as shown in table 4). The lower dosage is 0.125U/uL and 0.25U/uL, and the sensitivity and the reaction time are influenced; higher dosages, 1U/uL and 2U/uL, also have similar performance, but additionally increase the cost.
Table 4: bst enzyme concentration screening
Example 7: dNTP concentration screening Process
dNTP is a basic unit of DNA composition, is a substrate of LAMP reaction, has too low concentration, low reaction efficiency and too high concentration in a reaction system, and can increase the cost and even form pairing with a template/primer to inhibit the reaction. Taking the MP preferred primer set as an example, the appropriate concentration of dNTPs is screened optimally according to literature reports and recommendations of enzyme suppliers.
dNTP with different dosage is added into the same reaction system, the same genomic DNA gradient dilution is detected, and screening is carried out through performance performances in two aspects of sensitivity and reaction time. The invention selects dNTP concentration, namely the final concentration is 1mM, the performance is optimal, the sensitivity can reach 100 copies/reaction, the reaction time is within 20min, and all positive reactions can normally generate peaks (as shown in Table 5). Lower amounts of 0.25mM and 0.5mM, sensitivity and reaction time are affected; 2mM also has similar performance, but the cost is additionally increased; the 4mM sensitivity and reaction time-consuming do not perform well, probably because, as described earlier, dNTPs form a pair with the template/primer, inhibiting the reaction.
Table 5: dNTP concentration screening
Example 8 Freeze-drying protectant screening procedure
In the experimental stage of the invention, according to literature reports, freeze-drying protective agents of various molecular reaction reagents are tested, as shown in the following table, sucrose and trehalose are finally selected as optimized freeze-drying protective agents through detection on aspects such as the inhibition effect of LAMP, the titer after freeze-drying, the dry powder form, the storage stability and the like (as shown in tables 6 and 7). And the use concentration was optimized, preferably 10%, the concentration optimization screening procedure was the same as in table 7 below, and the data is not shown.
Table 6: screening of lyophilisates
Table 7: data summary table of the storage stability of the LAMP amplification system lyophilized powder.
Example 9 clinical sample alignment
By using the product of the invention, the respiratory tract clinical samples of patients who have MP negative and positive confirmed by using the commercial NMPA certified products on the market are detected, and the negative and positive coincidence rate is basically consistent. The test result of only one sample identified as positive by the comparison system is negative by using the product of the invention (as shown in Table 8), but the test result of the sample is also negative by using a QPCR method reported in the literature (as shown in Table 9), and the possible reason is that DNA is degraded in the process of storing clinical samples and freezing and thawing, so that the two methods cannot be successfully detected.
Table 8 comparison of MP sample detection results by lamp method with comparison system
Table 9: comparison of MP sample detection results with reference to comparison System by QPCR method described in literature
SEQUENCE LISTING
<110> Shanghai ao Pu biomedical corporation
<120> primer set, kit and method for detecting mycoplasma pneumoniae
<130> P20014349C
<160> 30
<170> PatentIn version 3.5
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<211> 21
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ccacaagaag aacaggacga 20
<210> 12
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> LB_MP-2
<400> 12
agcgattgac cgcatctt 18
<210> 13
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> F3_MP-3
<400> 13
gtactgtgaa ccacatgagt t 21
<210> 14
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> B3_MP-3
<400> 14
tagttcacat tgtggtactg c 21
<210> 15
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> FIP_MP-3
<400> 15
gggttggcag tccactttca ctagcttaac tcagcaaccc 40
<210> 16
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> BIP_MP-3
<400> 16
tgtggacaag tacactggtg ccacccaaac taaggtcagt aat 43
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> LF_MP-3
<400> 17
cgtaagctga ttgttccttg tg 22
<210> 18
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> LB_MP-3
<400> 18
ctcacctagt taacgttgac ca 22
<210> 19
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> F3_MP-4
<400> 19
caccctcggg ggcagtcag 19
<210> 20
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> B3_MP-4
<400> 20
ctgattgtcc ctgctggtc 19
<210> 21
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> FIP_MP-4
<400> 21
ttcagagctg gaggttggct tttgatgatt acaggcggtt cg 42
<210> 22
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> BIP_MP-4
<400> 22
gggcggggtg aaggaatgat atttctcgtg aacttggtgt ggt 43
<210> 23
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> LF_MP-4
<400> 23
aggcttcaag tggacaaagt ga 22
<210> 24
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> LB_MP-4
<400> 24
gtcgagggtt cttcgaggcg aac 23
<210> 25
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> F3_MP-5
<400> 25
atgcagtacc accccgac 18
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> B3_MP-5
<400> 26
ccctcaccaa agacactgtt 20
<210> 27
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> FIP_MP-5
<400> 27
cctcgtacgc ctcattgacc tcataagggt gagggtgaga ct 42
<210> 28
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> BIP_MP-5
<400> 28
taagtgacac cgagaagcgt ggacccagtt tcatggaagc c 41
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> LF_MP-5
<400> 29
cgctttggcc atgaagggtt 20
<210> 30
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> LB_MP-5
<400> 30
acttctcttc gttttgtttt tggac 25
Claims (12)
1. The primer group for detecting mycoplasma pneumoniae is characterized by comprising an MP outer primer pair, an MP inner primer pair and an MP ring primer pair, wherein the sequences of the MP outer primer pair are shown as SEQ ID NO. 1 and SEQ ID NO. 2, the sequences of the MP inner primer pair are shown as SEQ ID NO. 3 and SEQ ID NO. 4, and the sequences of the MP ring primer pair are shown as SEQ ID NO. 5 and SEQ ID NO. 6.
2. A kit for detecting mycoplasma pneumoniae, comprising a reaction system, wherein the reaction system comprises the primer set of claim 1.
3. The kit of claim 2, wherein the reaction system further comprises dNTP, bst DNA polymerase, tris-HCl, KCl, (NH) 4 ) 2 SO 4 Triton X-100, betaine, mg 2+ And a fluorescent dye.
4. The kit of claim 3, wherein the reaction system comprises:
0.125-5 mmol/L dNTP；
bst DNA polymerase 0.15-2U/. Mu.L;
10-100mmol/L Tris-HCl with pH 7-9;
10-100 mmol/L KCl；
10-100 mmol/L (NH 4 ) 2 SO 4 ；
0.1-1% Triton X-100；
0.5-1.5 mol/L betaine;
2-9 mmol/L Mg 2+ ；
0.15-0.3 mu mol/L MP outer primer pair;
0.8-2.0 mu mol/L MP inner primer pair; and
0.15-0.6 mu mol/L MP loop primer pair.
5. The kit of claim 4, wherein said Mg is 2+ From MgSO 4 Or MgCl 2 。
6. The kit of claim 4, wherein the fluorescent dye is any one of SYBR Green, evaGreen, SYTO-9, and SYTO-82.
7. The kit of claim 6, wherein the reaction system comprises:
1.0 mmol/L dNTP；
0.5 U/. Mu.L Bst DNA polymerase;
20mmol/L Tris-HCl pH 8.8;
10mmol/L KCl；
10mmol/L (NH 4 ) 2 SO 4 ；
0.1% Triton X-100；
100 × diluted SYBR Green;
0.5mol/L betaine;
6 mmol/L MgSO 4 ；
0.2 Mu mol/L MP outer primer pair;
1.6 Mu mol/L MP inner primer pair; and
0.4 Mu mol/L MP loop primer pair.
8. The kit of any one of claims 2 to 7, wherein the kit is in the form of a lyophilized powder.
9. The kit of claim 8, wherein the protective agent for preparing the lyophilized powder is sucrose or trehalose with a mass volume ratio of 5% to 20%.
10. The kit of claim 9, wherein the protectant is 10% trehalose by mass/volume.
11. The kit of claim 8, wherein the lyophilization process of the lyophilized powder is: and (3) subpackaging the reaction system into reaction tubes in 15-30uL, placing the reaction tubes into an ultralow temperature freeze dryer, reducing the temperature to below-40 ℃, vacuumizing, subliming after 20-30 hours, removing water until small dry powder is formed at the bottom of the tubes, covering the tubes in a vacuum state to obtain freeze-dried dry powder reagent tubes, taking out the reagent tubes, and refrigerating and storing at 2-8 ℃.
12. Use of the primer set according to claim 1 or the kit according to any one of claims 2 to 11 in the preparation of a product for the diagnosis of mycoplasma pneumoniae.
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