CN114214435B

CN114214435B - MNP (MNP) labeling combination of mycoplasma pneumoniae, primer pair combination, kit and application of MNP labeling combination

Info

Publication number: CN114214435B
Application number: CN202111309316.5A
Authority: CN
Inventors: 周俊飞; 高利芬; 方治伟; 肖华峰; 彭海; 陈利红; 李论; 李甜甜; 万人静
Original assignee: Jianghan University
Current assignee: Jianghan University
Priority date: 2021-11-06
Filing date: 2021-11-06
Publication date: 2023-06-16
Anticipated expiration: 2041-11-06
Also published as: CN114214435A

Abstract

The invention belongs to the technical field of molecular biology, and discloses a MNP (MNP) marker combination, a primer pair combination, a kit and application thereof, wherein the MNP marker combination comprises 14 markers of MNP-1-MNP-14, and the specific nucleotide sequence is shown as SEQ ID NO.1-SEQ ID NO. 14; the nucleotide sequence of the primer is shown as SEQ ID NO.15-SEQ ID NO. 42. The MNP marker combination can specifically identify mycoplasma pneumoniae and accurately detect variation; the primers are not interfered with each other, and the multiplex amplification and sequencing technology is integrated, so that the sequence analysis can be carried out on all the marker combinations of multiple samples at one time, the detection advantages of high flux, multiple targets, high sensitivity, high precision and culture free are achieved, the method can be applied to the identification and genetic variation detection of mycoplasma pneumoniae of large-scale samples, and the method has important significance on the scientific research and epidemic prevention monitoring of mycoplasma pneumoniae.

Description

MNP (MNP) labeling combination of mycoplasma pneumoniae, primer pair combination, kit and application of MNP labeling combination

Technical Field

The embodiment of the invention relates to the technical field of biology, in particular to an MNP (MNP) marking combination of mycoplasma pneumoniae, a primer pair combination, a kit and application thereof.

Background

Mycoplasma pneumoniae ((Mycoplasma pneumoniae) is a main pathogen causing mycoplasma pneumoniae, is mainly infected by droplets, and is mainly shown as upper respiratory tract infection, bronchopneumonia, pneumonia and the like after being infected by a human body, and can also affect other systems of the human body, such as liver function injury, brain injury, and other pulmonary complications, serious illness and even life-threatening complications occur, mycoplasma pneumoniae disease incidence can account for 20% -30% of all pneumonia cases worldwide and has a tendency to rise year by year, serum epidemiological studies show that children and young people aged 5-19 are susceptible subjects of mycoplasma pneumonia in the world, but in recent years, mycoplasma pneumoniae can also cause community-acquired pneumonia in old people aged 65 and infants aged 5 years old and less, and once the mycoplasma pneumoniae is developed, symptoms often become more serious.

In addition, mycoplasma pneumoniae is also a common model of pathogenic microorganisms for laboratory research. As a group organism, individuals in the group can be mutated in interaction with hosts and environments. For laboratory studies, such undetectable variations can result in strains of the same name in different laboratories or different times in the same laboratory being virtually different, resulting in irreproducible and incomparable experimental results. Heterogeneity between human hela cell laboratories has resulted in a significant amount of incomparable experimental results and wasted data. Therefore, the development of a rapid, accurate and mutation-monitoring mycoplasma pneumoniae detection and analysis method has important significance for scientific research and application of mycoplasma pneumoniae.

Classical mycoplasma pneumoniae detection methods, including isolation culture, PCR techniques, whole genome and metagenome sequencing, etc., suffer from one or more limitations in terms of duration, complexity of operation, detection throughput, accuracy and sensitivity of detection variation, cost, etc. The targeted molecular marker detection technology integrating the ultra-multiplex PCR amplification and the high-throughput sequencing can enrich target microorganisms in a sample with low microorganism content in a targeted manner, avoids a large amount of data waste and background noise caused by sequencing of a whole genome and a metagenome, and has the advantages of small sample requirement, accurate diagnosis result, data quantity saving and low-frequency variation detection.

The molecular markers detected by the existing targeted detection technology mainly comprise SNP and SSR markers. SSR markers are the most well-accepted markers for polymorphism, but are small in number in microorganisms; the number of SNP markers is huge, the distribution is dense, and the polymorphism of single SNP marker is insufficient to capture the potential allelic diversity in microorganism population. Therefore, development of a novel molecular marker of mycoplasma pneumoniae with high polymorphism and a detection technology thereof become technical problems to be solved urgently.

The invention develops a novel molecular marker-MNP marker specific to species. MNP markers refer to polymorphic markers caused by multiple nucleotides in a region of the genome. MNP markers have the following advantages over SSR markers and SNP markers: (1) The allele type is rich, and single MNP mark has 2 ⁿ The species allelic type is higher than SSR and SNP; (2) The species distinguishing capability is strong, the species identification can be realized by only a small amount of MNP marks, and the detection error rate is reduced. The MNP labeling method for detecting MNP labels based on the combination of super multiplex PCR and a second generation high throughput sequencing technology has the following advantages: (1) The output is a base sequence, a standardized database can be constructed for sharing without parallel experiments; (2) The method has high efficiency, breaks through the limitation of the number of sequencing samples by using the sample DNA bar code, and can type tens of thousands of MNP labels of hundreds of samples at one time; (3) High sensitivity, multiple targets are detected at one time by using multiple PCR, and high false negative and low sensitivity caused by single target amplification failure are avoided; (4) High accuracy, and sequencing the amplified product hundreds of times by using a second-generation high-throughput sequencer.

In view of the advantages and the characteristics, the MNP marking and the detection technology thereof can realize classification and tracing of the multi-allele types of the group organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, genetic variation detection and the like. At present, no report about MNP labeling exists in microorganisms, and corresponding technology and detection standards are lacking. The marker and primer combinations developed in the present invention will also be used to formulate the national standard for pathogen detection (program number 20201830-T-469) which will be released at the end of 2021.

Disclosure of Invention

The embodiment of the invention aims to provide an MNP (MNP) marking combination, a primer pair combination, a kit and application thereof for identifying and mutating mycoplasma pneumoniae, which have the effects of multiple targets, high flux, high sensitivity and fine typing.

In a first aspect of the invention there is provided a combination of MNP markers of Mycoplasma pneumoniae, the combination of MNP markers being a genomic region screened on the Mycoplasma pneumoniae genome that is distinct from other species and has multiple nucleotide polymorphisms within the species, comprising 14 markers of MNP-1 to MNP-14 on a Mycoplasma pneumoniae reference sequence, the specific nucleotide sequences being as shown in SEQ ID NO.1-SEQ ID NO.14, wherein ID NO.15-SEQ ID NO.28 is the upper primer ID NO.29-SEQ ID NO.42 is the lower primer.

In the above technical solution, table 1 of the specification further describes the combination of the MNP-1 to MNP-14, and the start and end positions of the MNP markers marked in table 1 are determined based on the reference sequences corresponding to the same row of MNPs in table 1.

In a second aspect of the present invention, there is provided a multiplex PCR primer pair combination for detecting the MNP tag combination, the multiplex PCR primer pair combination comprising 14 pairs of primers, the nucleotide sequences of the specific primers being shown in SEQ ID NO.15-SEQ ID NO. 42.

In the above technical solution, each MNP labeled combination primer includes an upper primer and a lower primer, which are specifically shown in table 1 of the specification.

In a third aspect of the invention, there is provided a detection kit for detecting said mycoplasma pneumoniae core MNP marker combination, said kit comprising said primer pair combination.

Further, the kit further comprises a multiplex PCR premix.

And the MNP label combination of mycoplasma pneumoniae or the primer pair combination or the application of the detection kit in qualitative detection of mycoplasma pneumoniae of non-diagnostic purpose, and the application in preparing qualitative detection products of mycoplasma pneumoniae.

In a fourth aspect of the invention, there is provided the use of said MNP marker combination of mycoplasma pneumoniae or said multiplex PCR primer pair combination or said detection kit in the identification of mycoplasma pneumoniae, construction of DNA fingerprint databases, and detection of genetic variation.

In the above application, firstly, the total DNA of the bacteria of the sample to be tested is obtained; performing a first round of multiplex PCR amplification on the total DNA and the blank control by using the kit, wherein the number of cycles is not higher than 25; purifying the amplified product, and then adding a sample tag and a second generation sequencing joint based on the second-round PCR amplification; quantifying after purifying the second round of amplification products; detecting a plurality of strains by mixing the amplification products of the second round in equal amounts and then performing high throughput sequencing; and comparing the sequencing result with the reference sequence of the mycoplasma pneumoniae to obtain the number of detection sequences and genotype data of the total DNA. And carrying out data quality control and data analysis on the sequencing data of the total DNA according to the number of the mycoplasma pneumoniae sequencing sequences and the number of the detected MNP markers obtained from the total DNA and the blank control, and obtaining the number of the detected MNP markers, the number of the sequencing sequences covering each MNP marker and the MNP marker genotype data.

When the method is used for mycoplasma pneumoniae identification, whether the sample to be detected contains mycoplasma pneumoniae nucleic acid is judged after quality control according to the number of sequencing sequences of mycoplasma pneumoniae detected in the sample to be detected and a blank control and the number of MNP sites detected. The quality control scheme and the judging method are characterized in that DNA of mycoplasma pneumoniae with known copy numbers is taken as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting mycoplasma pneumoniae are evaluated, and the quality control scheme and the judging method when the kit detects mycoplasma pneumoniae are formulated.

When used for mycoplasma pneumoniae genetic variation detection, it includes inter-and intra-strain genetic variation detection. The detection of genetic variation among strains comprises the steps of obtaining genotype data of 14 MNP markers of each strain to be compared by using the kit and the method. And analyzing whether the main genotypes of the strains to be compared are different from each other on the 14 MNP labels through genotype comparison. If the strain to be compared has a variation in the main genotype of at least one MNP marker, it is determined that there is a genetic variation in both. Alternatively, 14 markers of the strain to be compared may be amplified by single PCR, respectively, and then Sanger sequencing is performed on the amplified products to obtain sequences, and then the genotypes of each MNP marker of the strain to be compared are aligned. If MNP markers of non-identical major genotypes are present, variations are present between the strains to be compared. When detecting genetic variation inside the strain, determining whether the MNP marker of the strain to be detected detects a secondary genotype other than the primary genotype through a statistical model. If the strain to be tested has the subgenotype in at least one MNP mark, judging that the strain to be tested has genetic variation.

When the method is used for constructing a mycoplasma pneumoniae DNA fingerprint database, genotype data of the MNP marker of mycoplasma pneumoniae identified from a sample is input into a database file to form the mycoplasma pneumoniae DNA fingerprint database; and (3) when different samples are identified, comparing the samples with a DNA fingerprint database of the mycoplasma pneumoniae, and identifying whether the mycoplasma pneumoniae in the samples is different from the strains in the database in the main genotype (the genotype supported by more than 50% of sequencing fragments in one MNP mark) in the MNP mark, wherein the mycoplasma pneumoniae with the main genotype difference in at least 1 MNP mark is a new mutation type, and recording the new mutation type in the DNA fingerprint database.

When the method is used for mycoplasma pneumoniae typing, mycoplasma pneumoniae in a sample to be tested is identified, and the genotype of each MNP locus is obtained; collecting genome sequences of mycoplasma pneumoniae disclosed on the net and constructing a mycoplasma pneumoniae DNA fingerprint database to form a mycoplasma pneumoniae reference sequence library; comparing the genotype of mycoplasma pneumoniae in the sample to be detected with a reference sequence library of the mycoplasma pneumoniae, and screening strains which are genetically identical or closest to each other to obtain the typing of the mycoplasma pneumoniae in the sample to be detected. And identifying whether mycoplasma pneumoniae in the sample is an existing type or a new type according to the comparison result with the reference sequence library, and realizing the fine typing of the mycoplasma pneumoniae.

Compared with the prior art, the invention has the following advantages:

the invention provides an MNP (MNP) marking combination of mycoplasma pneumoniae, a primer pair combination, a kit and application thereof. The provided 14 MNP markers of mycoplasma pneumoniae and the primer combination thereof can be used for multiplex PCR amplification, and the amplification products are sequenced by fusing a second generation sequencing platform, so that the requirements of high-throughput, high-efficiency, high-accuracy and high-sensitivity detection of mycoplasma pneumoniae are met, and the requirements of mycoplasma pneumoniae standard and sharable fingerprint data construction are met; the need to accurately detect genetic variation between mycoplasma pneumoniae strains; the requirements of mycoplasma pneumoniae on homozygosity and heterozygosity are identified, and technical support is provided for applications such as mycoplasma pneumoniae detection, DNA fingerprint construction, mycoplasma in-vivo and mycoplasma-mycoplasma genetic variation detection and the like. The invention is initiated in the mycoplasma pneumoniae field, and is not reported in related documents.

Drawings

FIG. 1 is a schematic diagram of MNP marker polymorphism;

FIG. 2 is a flow chart of screening and primer design for Mycoplasma pneumoniae MNP markers;

FIG. 3 is a flow chart of detection of MNP markers;

Detailed Description

In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to specific embodiments that are illustrated in the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or may be prepared by existing methods.

The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:

1. screening a novel MNP marker suitable for high polymorphism of the population organisms as a detection target. MNP markers are mainly developed based on reference sequences, and MNP markers which are large-scale and are distinguished from other species, polymorphic in mycoplasma pneumoniae species and conserved in sequences at two sides can be mined according to reported resequencing data of mycoplasma pneumoniae representative minispecies;

2. MNP marker detection primers suitable for multiplex PCR amplification can be designed through conserved sequences at two sides of MNP markers;

3. and screening a set of MNP (MNPN-specific primers) with maximum polymorphism and high specificity according to the test result of the standard substance, and a primer combination and kit with the best compatibility and a detection and judgment method.

The kit provided by the invention can accurately and sensitively detect 10 copies/reaction of mycoplasma pneumoniae.

In the reproducibility test of the invention, the difference logarithm of MNP marking main genotypes among different libraries and different library construction batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%.

The MNP label and the kit have high specificity in detecting mycoplasma pneumoniae in complex templates.

The MNP marker combination, primer composition, kit and use thereof of mycoplasma pneumoniae of the present application will be described in detail below with reference to examples and experimental data.

Example 1 screening of Mycoplasma pneumoniae MNP marker combinations and design of multiplex PCR amplification primers

S1, screening of MNP (mycoplasma pneumoniae) markers

Based on complete or partial sequences of genomes of 771 mycoplasma pneumoniae different isolates disclosed on the net, 14 MNP markers are obtained through sequence alignment. For species on which no genomic data is present on the net, genomic sequence information representing a minispecies of the microorganism species to be detected may also be obtained by high throughput sequencing, which may be whole genome or simplified genome sequencing. In order to ensure polymorphism of the selected markers, genomic sequences of at least 10 genetically representative isolates are generally used as reference.

The 14 MNP markers screened are shown in table 1:

MNP markers and detection primers starting positions on the reference sequence as described in Table 1

The step S1 specifically includes:

selecting a genome sequence of a representative strain of mycoplasma pneumoniae as a reference genome, and comparing the genome sequence with the reference genome to obtain single nucleotide polymorphism markers of each strain of mycoplasma pneumoniae;

on the reference genome, carrying out window translation by taking 100-300bp as a window and taking 1bp as a step length, and screening to obtain a plurality of candidate MNP (MNP) marker areas, wherein the candidate MNP marker areas contain more than or equal to 2 single nucleotide variation markers, and the single nucleotide polymorphism markers do not exist on sequences of 30bp at both ends;

screening a region with the discrimination DP of more than or equal to 0.2 from the candidate polynucleotide polymorphism marking regions as MNP marks; wherein dp=d/t, t is the log of comparisons when all the minor species in the candidate polynucleotide polymorphism marker region are compared pairwise, and d is the log of samples of at least two single nucleotide polymorphism differences in the candidate polynucleotide polymorphism marker region.

As an optional implementation mode, when screening is performed on the reference genome by taking 100-300bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, so that the comprehensive screening is facilitated.

S2, design of multiplex PCR amplification primer

The MNP marked multiplex PCR amplification primers are designed through primer design software, the primer design follows that the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can be amplified normally in one amplification reaction.

S3, evaluating detection efficiency of primer combination

The detection method of the MNP markers comprises the steps of amplifying all MNP markers at one time through multiplex PCR, sequencing amplified products through second-generation high-throughput sequencing, analyzing sequencing data, and evaluating the compatibility of the primer combination according to the detected markers.

Mycoplasma pneumoniae DNA standard substances (product number: BDS-BW-044, guangzhou Bangding biosciences Co., ltd.) with known copy numbers are added into human genome DNA to prepare 1000 copies/reaction templates, detection is carried out through the MNP mark detection method, 4 repeated sequencing libraries are constructed, the designed primer combinations are screened according to detection results, and finally the 14 MNP marked primer pair combinations which can be detected in the 4 libraries and have the best compatibility are obtained through screening, wherein the specific primer pair combinations are shown in the table 1.

Threshold settings and Performance assessment for MNP markers and primers identification of Mycoplasma pneumoniae described in example 2

In this example, mycoplasma pneumoniae nucleotide standards of known copy numbers were added to human genomic DNA to prepare Mycoplasma pneumoniae-simulated samples of 1 copy/reaction, 10 copy/reaction and 100 copy/reaction. An equal volume of sterile water was set at the same time as a blank. A total of 4 samples, each of which was constructed as 3 replicate libraries per day, were tested continuously for 4 days, i.e., 12 sets of sequencing data were obtained per sample, as shown in table 2. And (3) according to the number of sequencing fragments and the number of markers of the mycoplasma pneumoniae MNP markers detected in the blank control and mycoplasma pneumoniae nucleotide standard in 12 repeated experiments, preparing thresholds for pollution of a quality control system and detection of a target pathogen, and evaluating reproducibility, accuracy and sensitivity of the detection method.

The detection flow of MNP markers is shown in fig. 3.

1. Detection sensitivity and stability analysis for detecting mycoplasma pneumoniae by MNP labeling method

As shown in Table 2, the kit can stably detect more than 6 MNP sites in 10 copies/reaction samples, and can detect at most 1 MNP site in a few samples of 0 copies/reaction, and the kit can clearly distinguish between 10 copies/reaction and 0 copies/reaction samples, and has technical stability and detection sensitivity as low as 10 copies/reaction.

TABLE 2 detection sensitivity and stability analysis of MNP labeling method of Mycoplasma pneumoniae

2. Reproducibility and accuracy assessment of MNP (MNP) marker detection kit for detecting mycoplasma pneumoniae

Based on whether the genotype of the marker is reproducible or not in the two repetitions, the reproducibility and accuracy of detection of mycoplasma pneumoniae by the MNP marker detection method are evaluated. Specifically, the data of 12 groups of 100 copies of the sample were compared in pairs, and the result is shown in Table 3, wherein the number of MNP markers with differences in the main genotypes is 0; according to the principle that the reproducible genotypes are considered to be accurate between 2 repeated experiments, the accuracy a=1- (1-r)/2=0.5+0.5r, r represents the reproducibility, i.e. the ratio of the number of reproducible markers of the main genotype to the number of common markers. In the project reproducibility test, the difference logarithm of MNP marking main genotypes among different libraries and different library construction batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%. Based on this, the kit can accurately and sensitively detect mycoplasma pneumoniae of less than 10 copies/reaction.

TABLE 3 reproducibility and accuracy assessment of Mycoplasma pneumoniae MNP marker detection methods

3. Threshold judgment for detecting mycoplasma pneumoniae by MNP (MNP) marker detection kit

As shown in Table 3, the sequences aligned to Mycoplasma pneumoniae can be detected in 1 copy/reaction sample, and at least 1 MNP marker is covered. The sequence of Mycoplasma pneumoniae was also detected in part of the blank. Because of the extreme sensitivity of MNP marker detection methods, contamination of the data in the detection is prone to false positives. The following quality control scheme is formulated in this example.

The quality control scheme is as follows:

1) The amount of sequencing data is greater than 4.5 megabases. The measurement and calculation basis is that the number of MNP labels detected by each sample is 14, and the length of one sequencing fragment is 300 bases, so that when the data size is more than 4.5 megabases, most samples can ensure that the number of sequencing fragments covering each label reaches 1000 times by one experiment, and the accurate analysis of the base sequence of each MNP label is ensured.

2) Determining whether the contamination is acceptable based on the signal index S of mycoplasma pneumoniae in the test sample and the noise index P of mycoplasma pneumoniae in the blank, wherein:

the noise figure p=nc/Nc for the control, where Nc and Nc represent the number of sequenced fragments and total sequenced fragment number of mycoplasma pneumoniae in the control, respectively.

The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequenced fragments and the total number of sequenced fragments, respectively, of mycoplasma pneumoniae in the test sample.

3) The detection rate of MNP markers in the test sample is calculated and refers to the ratio of the number of detected markers to the total number of designed markers.

As shown in table 4, the average noise figure of mycoplasma pneumoniae in the blank is 0.06%, the average signal figure in 1 copy sample is 0.27%, and the average signal to noise ratio of 1 copy sample and blank is 3.4, so the present invention provides that when the signal to noise ratio is greater than 10 times, it can be determined that contamination in the detection system is acceptable.

As shown in Table 4, the average signal-to-noise ratio of the 10 copies of the sample and the blank was 58.6, and at least 6 MNP markers were stably detected in the 10 copies/reaction 14 sets of data, accounting for 42.9% of the total markers. Therefore, under the condition of ensuring accuracy, the standard prescribes that the signal-to-noise ratio judgment threshold of mycoplasma pneumoniae is 30, namely when the signal-to-noise ratio of mycoplasma pneumoniae in a sample is more than 30 and the mark detection rate is more than or equal to 30%, the nucleotide of mycoplasma pneumoniae is judged to be detected in the sample. Therefore, the kit provided by the invention can sensitively detect 10 copies/reaction of mycoplasma pneumoniae.

TABLE 4 SNR of Mycoplasma pneumoniae in samples to be tested

4. Specific evaluation of MNP marker detection method for detecting mycoplasma pneumoniae

The DNA of Mycoplasma pneumoniae, mycobacterium tuberculosis, acinetobacter strain, pertussis baud bacteria, huo Shibao termitis bacteria, chlamydia pneumoniae, EB virus, haemophilus influenzae, varicella zoster virus, cytomegalovirus, herpes simplex virus, human bocavirus, klebsiella pneumoniae, legionella, moraxella catarrhalis, pseudomonas aeruginosa, rickettsia, staphylococcus aureus, streptococcus pneumoniae and Streptococcus pyogenes are mixed together according to the equimolar amount to prepare a mixed template, and a blank template is used as a control, and the mycoplasma pneumoniae in the mixed template is detected by adopting the method provided by the invention, so that 3 repeated experiments are carried out. Results the sequencing sequences obtained in 3 replicates were aligned to only 14 MNP sites in mycoplasma pneumoniae. After analysis according to the quality control scheme and the judgment threshold, the nucleic acid of mycoplasma pneumoniae is specifically detected in 3 repeated experiments, which shows that the MNP label and the kit detect the high specificity of mycoplasma pneumoniae in complex templates.

Example 3 detection of genetic variation between Mycoplasma pneumoniae strains

The detection method is used for detecting the collected 6 mycoplasma pneumoniae strains by using the kit and MNP mark combination, samples are sequentially named as S1-S6, the average coverage of sequencing of each sample is 1530 times, and all 14 MNP marks can be detected by each strain (table 5). The fingerprints of 6 strains were aligned pairwise, and the results are shown in Table 5, wherein 1 part (S-2) of the fingerprints and 5 parts of Mycoplasma pneumoniae detected together with the same batch all have a partially marked major genotype difference (Table 5), and strain-to-strain variation exists, and the strains may belong to different isolates.

The application of the kit for identifying the genetic variation among strains by detecting MNP markers can be used for ensuring the genetic consistency of mycoplasma pneumoniae strains named in different laboratories, so that the comparability of research results is ensured, and the kit has important significance for scientific research of mycoplasma pneumoniae. Whereas clinically, a diagnostic regimen may be considered for whether the differential markers affect drug resistance.

TABLE 5 6 detection and analysis of Mycoplasma pneumoniae

EXAMPLE 4 detection of genetic variation inside Mycoplasma pneumoniae Strain

As a group organism, partial individuals in the mycoplasma pneumoniae group are mutated, so that the group is no longer homozygous to form a heterogeneous heterozygous group, and the stability and consistency of the phenotype of the microorganism for the test are influenced. Such variants, when detected by molecular markers on a population, appear as alleles outside the main genotype of the marker. When variant individuals have not accumulated, they occupy a very small proportion of the population and exhibit a low frequency of allelic forms. Low frequency alleles tend to mix with technical errors, making the prior art indistinguishable. The present invention detects MNP markers with high polymorphism. Based on the fact that the probability of occurrence of a plurality of errors is lower than that of one error, the technical error rate of MNP markers is significantly lower than that of SNP markers.

The authenticity assessment of the secondary isogenotypes of this example was performed as follows: the allelotype with strand preference (ratio of the number of sequencing sequences covered on the DNA duplex) is first excluded according to the following rule: the strand preference is greater than 10-fold, or the difference from the strand preference of the major allele is greater than 5-fold.

Genotypes without strand preference were judged for authenticity based on the number and proportion of sequenced sequences in table 5. Table 6 lists e calculated based on binom. Inv function under the probability guarantee of α=99.9999% _max (n=1) and e _max (n.gtoreq.2) is 1.03% and 0.0994%, respectively, and the true hypogenotype is judged only when the number of sequences of the hypogenotype exceeds the critical value. When a plurality of candidate minor alleles exist, multiple correction is carried out on the P value of each candidate allele type, and FDR is carried out<0.5% of candidate alleles are judged to be true minor genotypes.

Parameter e related to Table 6 _max (n=1) and e _max (n.gtoreq.2) means that the number of sequences of the wrong allele carrying n SNPs is the highest proportion of the total number of sequences of the marker. e, e _max (n=1) and e _max (n.gtoreq.2) 1.03% and 0.0994%, respectively, are obtained from the frequency of all minor genotypes detected at 930 homozygous MNP markers.

TABLE 6 critical values for determining the hypo-isogenotypes at partial sequencing depth

According to the above parameters, nucleotides of two strains having a difference in genotype were mixed in the following 8 ratios of 1/1000,3/1000,5/1000,7/1000,1/100,3/100,5/100,7/100 to prepare artificial heterozygous samples, each sample was tested 3 times for repetition, and 24 sequencing data were obtained in total. Through the accurate comparison with the MNP marked genotypes of the two strains, the mark with the heterozygous genotype is detected in 24 artificial heterozygous samples, and the applicability of the developed MNP marked detection method for mycoplasma pneumoniae in detecting genetic variation inside strain groups is demonstrated.

EXAMPLE 5 construction of Mycoplasma pneumoniae DNA fingerprint database

All strains or DNA of samples used for constructing a mycoplasma pneumoniae DNA fingerprint database are extracted by using a conventional CTAB method, a commercial kit and other methods, and the quality of the DNA is detected by using agarose gel and an ultraviolet spectrophotometer. If the ratio of the absorbance values of the extracted DNA at 260nm and 230nm is more than 2.0, the ratio of the absorbance values of 260nm and 280nm is between 1.6 and 1.8, the DNA electrophoresis main band is obvious, no obvious degradation and RNA residues exist, the genome DNA reaches the relevant quality requirements, and the subsequent experiments can be carried out.

And (3) carrying out sequence comparison on the sequencing data of the 6 strains to obtain the main genotype of each mark of each strain, thereby forming MNP fingerprint of each strain. The MNP fingerprint of each strain is input into a database file to form an MNP fingerprint database of mycoplasma pneumoniae; and comparing the MNP fingerprint of the mycoplasma pneumoniae detected in the new sample with the constructed MNP fingerprint database, and recording the MNP fingerprint of the sample with the main genotype difference into the constructed MNP fingerprint database. The constructed MNP fingerprint database is based on the gene sequence of the detected strain, is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed and shared and being updated at any time.

Example 6 use in Mycoplasma pneumoniae Fine subdivision

Detecting the 6 mycoplasma pneumoniae strains by using the primer combination and MNP mark combination detection method, so as to obtain MNP fingerprint of each strain; constructing a reference sequence library of mycoplasma pneumoniae, which consists of a published genome sequence of mycoplasma pneumoniae and a constructed MNP fingerprint database of mycoplasma pneumoniae; comparing MNP fingerprint of each strain with a constructed reference sequence library, and screening to obtain the strain with the closest genetic distance to the sequence library; 100% identical to the genotype of the existing strain, is the existing type, has main genotype difference at least one MNP site, is a new variant, and realizes the fine typing of mycoplasma pneumoniae. Detection of 6 samples of Mycoplasma pneumoniae As shown in Table 5, 1 part of Mycoplasma pneumoniae detected was different from the other 5 parts of Mycoplasma pneumoniae detected in 1 MNP-tagged major genotypes, and were judged as different variants. The genotype of the other 5 parts was kept consistent with the NCTC10119 ministrain and should be the progeny strain of NCTC10119 strain. Therefore, the resolution of the method for mycoplasma pneumoniae reaches the level of single base, and the method can realize the fine typing of mycoplasma pneumoniae in a sample.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiment and all such alterations and modifications as fall within the scope of the embodiments of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, the embodiments of the present invention are intended to include such modifications and alterations insofar as they come within the scope of the embodiments of the invention as claimed and the equivalents thereof.

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<210> 5

<211> 135

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

agcaatgaaa gcaatttcac caaactttaa atttgctttt aataagtcct taaagttctt 60

taagttaccg ttaactagtc attccaaggt atagataaag cggtagtgtt caatgaccgc 120

tttgtaatca acatt 135

<210> 6

<211> 146

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

ttttcgtttt tctgctccca ctaaataaat tgagttgggt tttaaaaacc gccaaaaatt 60

tttaatttag tttttgctac tttctcctcc ctccccctca cgagtgaaaa ccccggggcg 120

tgggccttag tgcgcgataa cactgc 146

<210> 7

<211> 150

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

actcttgata aggaaatttt gcattttctc aactttgtgc tttgtaaatt aatctataaa 60

acacaatctt ttcatcactt tgacgctttt taaaacccaa ctcaatttat ttagtggggg 120

caggaaaacg aaaattttgt gttgaaggcc 150

<210> 8

<211> 143

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gaaaacacgg ccggattgaa cggaccgatt aatggcttgt ttatcctgtt agacaccttt 60

gcctatgtga ccccggtgag tgggatgaaa ggggggagta agaatacgga ggcagtgcaa 120

accaagtatc ccgttaagga tga 143

<210> 9

<211> 148

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

tatgtggtgt cggtatccac taaaccgtac ttggtttgca aagcccggaa caataagaat 60

ttcttttgtt tagcgacatc acgaccactt tcagtaagat aatgactacc accatccacg 120

gccattaagt ggatgccgtc tttaccac 148

<210> 10

<211> 150

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

aattattatc tataaatcag gttttcaacc atagattact atggaaaaga aaaataaaaa 60

aaataccgca accgttccta ccaaagaaca attggatggc atgtttaaat ctcttgaaaa 120

agaagagaac tatgatgtct ttgttcaagt 150

<210> 11

<211> 149

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

tggaatttct acaagaatcc tcggtgcttt actgttaacc cacagtgatg actttggact 60

ggtactacca tgaaaagtag caccaattca agtcaaactg tacttatttg acaaaaaagg 120

tgatacaaag acagtagagt tagcacaga 149

<210> 12

<211> 147

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

ggtttaggct tagggcaaat tctccgtgct cccaatacct tcaactcttc ccacgaagtg 60

ccctattctt atgacctcat ctttggtgat gttaacacca ccaattttta cgcccagttt 120

gcgaaaaagc acaacaccca catgtgg 147

<210> 13

<211> 150

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

cttttgttca cctgtttgct gttgtccatc accagatttt gcaactgtta ctgcagtagt 60

actgctagtc atagaactag tctgatcatt acccaacgct ttgcttgtaa acgttgaatg 120

tacattactc tgactcgcag ctagcaacag 150

<210> 14

<211> 143

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

agctgaattt aattaatgga cagcaccaac caaaacctgt tcgcgtcatt gtccaaaaaa 60

ggacccgttc gcaaagaaaa tcaggacttt agtgttgtta ctttcaaccg ctttggtcag 120

ttaatgagcc ttgtttgtga tgg 143

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

ccttgagtga ccagttacaa gtaac 25

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

cagtttttcc tttcactggg agatc 25

<210> 17

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

agctgttcaa acaacttctg gg 22

<210> 18

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

ctgtaaccac cgcccctcta c 21

<210> 19

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

agcaatgaaa gcaatttcac caaac 25

<210> 20

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

ttttcgtttt tctgctccca ctaaa 25

<210> 21

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

actcttgata aggaaatttt gcatttt 27

<210> 22

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

gaaaacacgg ccggattgaa c 21

<210> 23

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

tatgtggtgt cggtatccac taaac 25

<210> 24

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

aattattatc tataaatcag gttttcaacc at 32

<210> 25

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

tggaatttct acaagaatcc tcggt 25

<210> 26

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

ggtttaggct tagggcaaat tctc 24

<210> 27

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

cttttgttca cctgtttgct gttg 24

<210> 28

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

agctgaattt aattaatgga cagca 25

<210> 29

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

gaaaagtcac actggtaccg tgt 23

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

gccaaacccg ctaaggtgaa 20

<210> 31

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

accaaaaaga gtgttgatga gcaat 25

<210> 32

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

ccttagctac tttggtcgtt ttagc 25

<210> 33

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

aatgttgatt acaaagcggt cattg 25

<210> 34

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

gcagtgttat cgcgcactaa g 21

<210> 35

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

ggccttcaac acaaaatttt cgtt 24

<210> 36

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

tcatccttaa cgggatactt ggttt 25

<210> 37

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 37

gtggtaaaga cggcatccac tta 23

<210> 38

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 38

acttgaacaa agacatcata gttctct 27

<210> 39

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 39

tctgtgctaa ctctactgtc tttgt 25

<210> 40

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 40

ccacatgtgg gtgttgtgc 19

<210> 41

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 41

ctgttgctag ctgcgagtca 20

<210> 42

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 42

ccatcacaaa caaggctcat taact 25

Claims

1. The MNP marker fragment combination of mycoplasma pneumoniae is characterized by comprising 14 marker fragments, and the specific nucleotide sequence of the MNP marker fragment combination is shown as SEQ ID NO.1-SEQ ID NO. 14.

2. A multiplex PCR primer pair combination for detecting the mycoplasma pneumoniae MNP-tagged fragment combination according to claim 1, wherein the multiplex PCR primer pair combination comprises 14 pairs of primers, and the specific primer nucleotide sequences are shown in SEQ ID No.15-SEQ ID No. 42.

3. A detection kit for detecting the combination of mycoplasma pneumoniae MNP-tagged fragments according to claim 1, wherein the kit comprises the primer pair combination according to claim 2.

4. The test kit of claim 3, wherein the kit further comprises a multiplex PCR premix.

5. Use of a combination of MNP-labeled fragments of mycoplasma pneumoniae according to claim 1 or a combination of primer pairs according to claim 2 or a detection kit according to any one of claims 3-4 for qualitative detection of mycoplasma pneumoniae of non-diagnostic interest.

6. Use of a combination of MNP-labeled fragments of mycoplasma pneumoniae according to claim 1 or a combination of primer pairs according to claim 2 or a detection kit according to any one of claims 3-4 in the preparation of a qualitative detection product of mycoplasma pneumoniae.

7. Use of a combination of MNP-labeled fragments of mycoplasma pneumoniae according to claim 1 or a combination of primer pairs according to claim 2 or a detection kit according to any one of claims 3-4 for the detection of genetic variations both inside and between mycoplasma pneumoniae strains for non-diagnostic purposes.

8. Use of a combination of MNP-labeled fragments of mycoplasma pneumoniae according to claim 1 or a combination of primer pairs according to claim 2 or a detection kit according to any one of claims 3-4 in the construction of a mycoplasma pneumoniae database.

9. Use of a combination of MNP-labeled fragments of mycoplasma pneumoniae according to claim 1 or a combination of primer pairs according to claim 2 or a detection kit according to any one of claims 3-4 for the detection of mycoplasma pneumoniae of non-diagnostic interest.