CN114836550B

CN114836550B - MNP (MNP) marking site of klebsiella pneumoniae, primer composition, kit and application of MNP marking site

Info

Publication number: CN114836550B
Application number: CN202111329161.1A
Authority: CN
Inventors: 李论; 方治伟; 李甜甜; 肖华锋; 彭海; 陈利红; 高利芬; 周俊飞
Original assignee: Jianghan University
Current assignee: Jianghan University
Priority date: 2021-11-10
Filing date: 2021-11-10
Publication date: 2023-06-16
Anticipated expiration: 2041-11-10
Also published as: CN114836550A

Abstract

The invention discloses MNP (MNP) marking sites of klebsiella pneumoniae, a primer composition, a kit and application thereof, wherein the MNP marking sites refer to genome regions which are screened on a genome of the klebsiella pneumoniae and are separated from other species and have a plurality of nucleotide polymorphisms in the species, and the MNP marking sites comprise MNP-1-MNP-9; the primer is shown as SEQ ID NO. 1-SEQ ID NO. 18. The MNP marker locus can specifically identify klebsiella pneumoniae and finely distinguish different subtypes; the primers are not interfered with each other, and the sequence analysis can be carried out on all the marking sites of multiple samples at one time by integrating multiple amplification and sequencing technologies, so that the method has the advantages of high flux, multiple targets, high sensitivity and culture-free detection, can be applied to identification and genetic variation detection of the Klebsiella pneumoniae of a large-scale sample, and has important significance on scientific research and degradation monitoring of the Klebsiella pneumoniae.

Description

MNP (MNP) marking site of klebsiella pneumoniae, primer composition, kit and application of MNP marking site

Technical Field

The embodiment of the invention relates to the technical field of biology, in particular to MNP (MNP) marking sites of klebsiella pneumoniae, a primer composition, a kit and application thereof.

Background

Klebsiella pneumoniae (Klebsiella Pneumoniae) is a common bacterium in nature, belongs to conditional pathogenic bacteria, can cause diseases such as pneumonia, wound infection and urinary tract, and has high incidence rate in diabetics, oncology patients, infants and patients who use antibiotics for a long time. In addition, klebsiella pneumoniae is also one of the main microorganisms responsible for animal contamination without specific pathogens. When the immune function of the body of experimental animals such as big and small mice is reduced or in a stress state, the quality of the experimental animals is often influenced and animal experiments are interfered due to the infection of klebsiella pneumoniae, even animal death is caused, so that the experimental data and economy are lost, and the teaching and scientific research are seriously influenced. Klebsiella pneumoniae is also a common model pathogen 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 quick, accurate and mutation-monitoring detection and analysis method for klebsiella pneumoniae has important significance for scientific research and application of klebsiella pneumoniae.

Classical klebsiella pneumoniae detection methods, including isolation culture, PCR techniques, whole genome and metagenome sequencing, etc., suffer from one or more limitations in terms of duration, operational complexity, 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 the whole genome separation culture step and metagenome sequencing, and has the advantages of no culture, less sample requirement, accurate diagnosis result, data 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 with high polymorphism of pathogenic microorganism klebsiella pneumoniae and detection technology thereof become a technical problem to be solved urgently.

Disclosure of Invention

The invention aims to provide MNP (MNP) marking sites of klebsiella pneumoniae, a primer composition, a kit and application thereof, which can carry out qualitative identification and mutation detection on the klebsiella pneumoniae and have the effects of multiple targets, high flux, high sensitivity and fine typing.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect of the invention there is provided a MNP marker locus for klebsiella pneumoniae, the MNP marker locus being a species specific genomic region screened on the klebsiella pneumoniae genome and having a plurality of nucleotide polymorphisms within the species, comprising the marker locus for MNP-1 to MNP-9 on the CP003200 genome.

In the above technical scheme, the marking sites of MNP-1 to MNP-9 are specifically shown in the specification table 1, and the starting and ending positions of the MNP marks marked in the table 1 are determined based on the CP003200 sequence.

In a second aspect of the present invention, there is provided a multiplex PCR primer composition for detecting the MNP marker loci, the multiplex PCR primer composition comprising 9 pairs of primers, the nucleotide sequences of the 9 pairs of primers being shown as SEQ ID NO.1 to SEQ ID NO. 18.

In the above technical solution, the primers of each MNP marker locus include an upper primer and a lower primer, and are specifically shown in table 1 of the specification.

In a third aspect of the present invention, there is provided a detection kit for detecting the Klebsiella pneumoniae MNP marker locus, the kit comprising the primer composition.

Further, the kit further comprises a multiplex PCR premix.

In a fourth aspect of the invention, there is provided the use of the MNP marker locus of klebsiella pneumoniae or the multiplex PCR primer composition or the detection kit in qualitative detection of klebsiella pneumoniae for non-diagnostic purposes.

In a fifth aspect of the invention, there is provided the use of the MNP marker locus of klebsiella pneumoniae or the multiplex PCR primer composition or the detection kit for detecting genetic variations within and among klebsiella pneumoniae strains.

In a sixth aspect of the invention, there is provided the use of the MNP marker locus of klebsiella pneumoniae or the multiplex PCR primer composition or the detection kit in constructing a klebsiella pneumoniae database.

In a seventh aspect of the invention, there is provided the use of the MNP marker locus of klebsiella pneumoniae or the multiplex PCR primer composition or the detection kit in the detection of fine typing of klebsiella pneumoniae.

The application steps of the qualitative detection of the klebsiella pneumoniae, the detection of genetic variation inside and among the klebsiella pneumoniae strains, the construction of a klebsiella pneumoniae database and the fine typing detection application of the klebsiella pneumoniae include: firstly, obtaining total bacterial DNA of a sample to be detected; 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 Klebsiella 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 sequence sequences of the Klebsiella pneumoniae obtained from the total DNA and the blank control and the number of the detected MNP sites, and obtaining the number of the detected MNP sites, the number of the sequencing sequences covering each MNP site and the genotype data of the MNP sites.

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

When used in klebsiella pneumoniae genetic variation detection, it includes inter-strain and intra-strain genetic variation detection. The detection of genetic variation among strains comprises the steps of obtaining genotype data of each strain to be compared at 9 MNP sites by using the kit and the method. And analyzing whether the main genotypes of the strains to be compared are different at the 9 MNP loci through genotype comparison. If the strain to be compared has a variation in the main genotype of at least one MNP site, it is determined that there is a genetic variation in both. Alternatively, the 9 sites of the strain to be compared may be amplified by single PCR, respectively, and then Sanger sequencing may be performed on the amplified products to obtain sequences, and then the genotypes of each MNP site of the strain to be compared may be aligned. If there are MNP sites of inconsistent main genotypes, there are variations between the strains to be compared. When detecting genetic variation inside the strain, determining whether the secondary genotype other than the primary genotype is detected at the MNP locus of the strain to be detected through a statistical model. If the strain to be tested has the subgenotype at least one MNP site, judging that the strain to be tested has genetic variation.

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

When the method is used for the typing of klebsiella pneumoniae, the klebsiella pneumoniae in a sample to be tested is identified, and the genotype of each MNP locus is obtained; collecting genome sequences of Klebsiella pneumoniae disclosed on the network and constructing a Klebsiella pneumoniae DNA fingerprint database to form a Klebsiella pneumoniae reference sequence library; comparing the genotype of the klebsiella pneumoniae in the sample to be detected with a reference sequence library of the klebsiella pneumoniae. And identifying whether the klebsiella 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 klebsiella pneumoniae.

The invention belongs to the first creation in the field of klebsiella pneumoniae, and is not reported in related literature; MNP markers are mainly developed based on reference sequences, and are characterized in that MNP sites which are different from other species, polymorphic in the interior of the Klebsiella pneumoniae species and conserved in sequence at two sides are excavated on a large scale according to reported resequencing sequences of the Klebsiella pneumoniae representative species; MNP site detection primers suitable for multiplex PCR amplification can be designed through conserved sequences at two sides of the MNP site; and then according to the test result of the standard substance, a set of primer combination with the largest polymorphism, high specificity MNP locus and the best compatibility and a detection kit are screened.

One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:

the invention provides MNP (MNP) marking sites of klebsiella pneumoniae, a primer composition, a kit and application thereof. The 9 MNP loci of the klebsiella pneumoniae and the primer combination thereof can be used for multiplex PCR amplification, and the amplification products are sequenced by fusing with a second generation sequencing platform, so that the requirements of high-throughput, high-efficiency, high-accuracy and high-sensitivity detection on the klebsiella pneumoniae are met, and the requirements of accurate detection on genetic variation among klebsiella pneumoniae strains are met; meets the requirement of identifying the degeneration of Klebsiella pneumoniae bacterial group; meets the requirements of the standard and the shared fingerprint data construction of the klebsiella pneumoniae and provides technical support for the scientific research and the degradation monitoring of the klebsiella pneumoniae.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

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

FIG. 2 is a flow chart of screening and primer design of Klebsiella pneumoniae MNP marker loci;

FIG. 3 is a flow chart of detection of MNP marker loci.

Detailed Description

The advantages and various effects of the embodiments of the present invention will be more clearly apparent from the following detailed description and examples. Those skilled in the art will appreciate that these specific implementations and examples are provided to illustrate, but not limit, examples of the present invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Thus, 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 embodiments of the invention belong. In case of conflict, the present specification will control.

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:

screening MNP markers suitable for detection of population organisms as detectionA target. 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 alleles are abundant, and 2 are arranged on single MNP locus ⁿ Species alleles, higher than SSR and SNP, are suitable for detection of microorganisms, a typical population organism; (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 labeling fuses the ultra-multiplex PCR and the second-generation high-throughput sequencing technology, and has the following advantages: (1) The output is a base sequence, and 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 sites 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 is lacking. The development, screening and application of MNP labeling method has better application foundation in plants.

Thus, the present invention developed MNP marker loci for Klebsiella pneumoniae that are genomic regions screened on the Klebsiella pneumoniae genome that are distinct from other species and have multiple nucleotide polymorphisms within the species, including the marker loci for MNP-1 to MNP-9 on the CP003200 genome.

Next, the present invention developed a multiplex PCR primer composition for detecting the Klebsiella pneumoniae MNP marker locus, comprising 9 pairs of primers, the nucleotide sequences of the 9 pairs of primers being shown as SEQ ID NO.1 to SEQ ID NO. 18. The primers do not collide with each other, and efficient amplification can be performed through multiplex PCR;

the multiplex PCR primer composition can be used for a detection kit for detecting the MNP labeling site of klebsiella pneumoniae.

The kit provided by the invention can sensitively detect 1 copy/response Klebsiella 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 markers and the kits of the invention have high specificity in detecting target microorganisms in complex templates.

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

Example 1 screening of Klebsiella pneumoniae MNP marker loci and design of multiplex PCR amplification primers

S1, screening MNP (MNP) marker locus of klebsiella pneumoniae

Based on complete or partial sequences of genomes of 529 klebsiella pneumoniae different isolates disclosed on the net, 9 MNP (MNP) marker loci are obtained through sequence comparison. 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 9 MNP marker loci screened are shown in table 1:

TABLE 1 MNP marker loci and detection primers starting position on the reference sequence

The step S1 specifically includes:

selecting a genome sequence representing subtype of the klebsiella pneumoniae as a reference genome, and comparing the genome sequence with the reference genome to obtain single nucleic acid polymorphic sites of each strain of the klebsiella pneumoniae;

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

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

As an optional implementation mode, when screening is performed on the reference genome by taking 100-300 bp 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 multiplex PCR amplification primers of the MNP locus 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

DNA of a klebsiella pneumoniae standard product (commodity number JC 10072) with known copy number is added into human genome DNA to prepare a 1000-copy/reaction simulation template, detection is carried out through the MNP mark detection method, 4 repeated sequencing libraries are constructed, a primer combination with uniform amplification and optimal compatibility is screened according to the detection condition of MNP sites in the 4 libraries, and finally the primer combination of 9 klebsiella pneumoniae specific MNP sites and the MNP sites with optimal compatibility is screened out.

Example 2 detection of Klebsiella pneumoniae by MNP site and primer

1. Detection of MNP markers

Samples of 1 copy/reaction, 10 copy/reaction and 100 copy/reaction of Klebsiella pneumoniae were prepared using DNA from a known copy number Klebsiella pneumoniae count standard and added to human genomic DNA. 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. The detection flow of MNP markers is shown in fig. 3. And evaluating the repeatability, accuracy and sensitivity of the detection method according to the number of sequencing fragments and the number of sites of the MNP sites of the klebsiella pneumoniae detected in a blank control sample and a klebsiella pneumoniae simulation sample in 12 repeated experiments, and formulating a quality control system pollution and a threshold value for detecting a target pathogen.

1. Sensitivity and stability assessment for detecting haemophilus influenzae by MNP (MNP) marker detection kit

As shown in Table 2, the kit can stably detect more than 3 MNP sites in a 1-copy/reaction sample, and can detect at most 1 MNP site in a 0-copy/reaction sample, and the kit can clearly distinguish between a 1-copy/reaction sample and a 0-copy/reaction sample, and has technical stability and detection sensitivity as low as 1-copy/reaction.

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

2. Reproducibility and accuracy assessment for detecting klebsiella pneumoniae by MNP (MNP) mark detection kit

Based on whether the genotype of the co-detected locus can be reproduced or not in the two repetitions, the reproducibility and the accuracy of detecting klebsiella pneumoniae by the MNP marker detection method are evaluated. Specifically, the data of 12 sets of 100 copies of the sample were compared in pairs, and the results are shown in Table 3.

TABLE 3 reproducibility and accuracy evaluation of Klebsiella pneumoniae MNP marker detection method

As can be seen from Table 3, the number of MNP sites having a difference in the main genotypes was 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, and r represents the reproducibility, namely the ratio of the reproducible site number of the main genotype to the common site number. 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%.

3. Threshold judgment for detecting klebsiella pneumoniae by MNP (MNP) mark detection kit

Sequences aligned to klebsiella pneumoniae can be detected in 1 copy/reaction sample, covering at least 3 MNP sites. The sequence of Klebsiella pneumoniae was also detected in a partial blank. Because of the extreme sensitivity of MNP marker detection methods, contamination of the data in the detection is prone to false positives. Therefore, the quality control scheme is formulated in this example, and is specifically as follows:

1) The amount of sequencing data is greater than 3 megabases. The measurement and calculation basis is that the number of MNP loci detected by each sample is 9, and the length of one sequencing fragment is 300 bases, so that when the data size is more than 3 megabases, most samples can ensure that the number of sequencing fragments covering each locus reaches 1000 times by one experiment, and ensure the accurate analysis of the base sequence of each MNP locus.

2) Determining whether the contamination is acceptable based on the signal index S of klebsiella pneumoniae in the test sample and the noise index P of klebsiella 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 klebsiella 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 of klebsiella pneumoniae in the test sample, respectively.

3) Calculating the detection rate of MNP marking sites in a test sample, wherein the detection rate refers to the ratio of the number of detected sites to the number of total designed sites. The results are shown in Table 4;

TABLE 4 SNR of Klebsiella pneumoniae in sample to be tested

As can be seen from Table 4, the average noise index of Klebsiella pneumoniae in the control is 0.02%, the average signal index in 1copy sample is 0.28%, and the average signal-to-noise ratio of 1copy sample and control is 11.38, so that the present invention provides that when the signal-to-noise ratio is greater than 12, it can be determined that the contamination in the detection system is acceptable. At least 3 sites can be detected in the 1copy sample, accounting for 33.3% of the total sites; therefore, under the condition of ensuring the accuracy, the standard prescribes that the signal-to-noise ratio judgment threshold of the klebsiella pneumoniae is 12, namely when the signal-to-noise ratio of the klebsiella pneumoniae in a sample is more than 12 and the site detection rate is more than or equal to 30%, the nucleic acid of the klebsiella pneumoniae is judged to be detected in the sample.

Therefore, the kit provided by the invention can sensitively detect 1 copy/reaction Klebsiella pneumoniae.

4. Specific evaluation for detecting klebsiella pneumoniae by MNP (MNP) mark detection kit

The DNA of Klebsiella pneumoniae, mycobacterium tuberculosis, acinetobacter strain, pertussis baud bacterium, huo Shibao termyces, chlamydia pneumoniae, mycoplasma pneumoniae, EB virus, haemophilus influenzae, varicella zoster virus, cytomegalovirus, herpes simplex virus, human bocavirus, klebsiella pneumoniae strain, moraxella catarrhalis, pseudomonas aeruginosa, rickettsia, staphylococcus aureus, streptococcus pneumoniae and Streptococcus pyogenes are artificially mixed together according to an equimolar amount to prepare a mixed template, and the Klebsiella pneumoniae in the mixed template is detected by adopting the method provided by the invention by using the sterile water template as a blank control, so that 3 repeated experiments are performed. After sequence comparison and analysis according to the quality control scheme and the judgment threshold, 9 MNP sites of the klebsiella pneumoniae can be specifically detected in 3 repeated experiments, which shows that the MNP markers and the kit detect the high specificity of target microorganisms in complex templates.

Example 3 detection of genetic variation between klebsiella pneumoniae strains

6 Klebsiella pneumoniae strains provided by the Hubei province disease control prevention control center are detected by using the kit and the MNP marking site detection method, samples are sequentially named as S1-S6, and 5 parts of the samples are offspring strains propagated in different periods of the same strain. The average fold coverage for sequencing of each MNP site was 3103 fold, and all 9 MNP markers could be detected for 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 Klebsiella pneumoniae detected together in the same batch all have a partial site major genotype difference (Table 5), and the strain-to-strain variation exists and may belong to different isolates.

TABLE 5 detection and analysis of 6 Klebsiella pneumoniae

As can be seen from Table 5, 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 the same named Klebsiella pneumoniae strains in different laboratories, so that the comparability of research results is ensured, and the kit has important significance for scientific research of Klebsiella pneumoniae. In clinical terms, one can take into account the diagnostic regimen as to whether the site of the difference affects resistance.

Example 4 detection of genetic variation in Klebsiella pneumoniae

Detection of genetic variation of klebsiella pneumoniae, including inter-and intra-strain variation. Since Klebsiella pneumoniae is parasitic in a host, that is, genetic variation of Klebsiella pneumoniae between and within the host is detected. The variation among hosts is detected by comparing the main genotypes, the obtained fingerprint of klebsiella pneumoniae is compared pairwise, 100% reproducibility and accuracy of the main genotypes are identified based on MNP labeling, and the main genotype difference of two strains with one locus can be detected.

And it is the variation inside the klebsiella pneumoniae host that is difficult to detect. As a group organism, klebsiella pneumoniae is mutated in a host or in a group, and when the group is detected by molecular marker, it is expressed as an allele outside the main genotype of the site. 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 invention distinguishes true minor genotypes from error genotypes caused by technical errors through a statistical model. Specifically:

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 6. 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 number of sequences sequenced in each locus of the hypo-isogenotype is determined to be true only when the number of sequences in the hypo-isogenotype exceeds the thresholdTrue minor isogenotypes. 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) refers to the highest proportion of the total sequence of the locus of the sequence of the wrong allele carrying n SNPs. 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 sites.

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

According to the above parameters, DNAs of 2 Klebsiella pneumoniae strains having a difference in genotype of Table 5 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 to obtain 24 sequencing data in total. Through the accurate comparison of the genotypes of MNP loci of two klebsiella pneumoniae strains, heterozygous genotype loci can be detected in 24 artificial heterozygous samples, and the applicability of the developed method for detecting MNP markers of klebsiella pneumoniae in detecting genetic variation of the strains is demonstrated.

Example 5 construction of klebsiella pneumoniae DNA fingerprint database

All strains used for constructing a klebsiella pneumoniae DNA fingerprint database or DNA of samples 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 site of each strain, forming MNP fingerprint of each strain, and recording a database file to form a Klebsiella pneumoniae DNA fingerprint database. The constructed MNP fingerprint database is based on the gene sequences of the detected strains and is therefore compatible with all high throughput sequencing data. The MNP fingerprint of the strain obtained by each detection is compared with the constructed MNP fingerprint database, and the MNP fingerprint database constructed by the MNP fingerprint of the strain with the main genotype difference is realized, so that the co-construction sharing and the random updating of the database are realized.

Example 6 application in the Fine typing of Klebsiella pneumoniae

The MNP fingerprint of each strain was obtained using the primer combination and MNP marker locus detection method described in example 2. The DNA fingerprint of each strain is compared with the constructed Klebsiella pneumoniae reference sequence library in pairs, and the DNA fingerprint of each strain is the same as the existing sequence library, is an existing variant, has main genotype difference at least one MNP site, is defined as a new variant, and realizes the fine typing of the Klebsiella pneumoniae.

The detection results of 6 haemophilus influenzae are shown in table 5, and after the 6 haemophilus influenzae are compared in pairs, the 6 haemophilus influenzae is divided into 2 types according to the difference of main genotypes, and the 2 types have genotype differences with strains in a reference sequence library, so that the strains are new variant strains. Therefore, the resolution of the method for haemophilus influenzae reaches the level of single base, and the method can realize the fine typing of haemophilus influenzae 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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<213> Artificial sequence (Artificial Sequence)

<400> 2

cgttataaac tattcagatc cagcaca 27

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ccctccgctt cacctttca 19

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

aaacatttcc gcttgaggat attgc 25

<210> 5

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

attaacgtca ttattatgcc agcgc 25

<210> 6

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

accaaaacta aacgaggtaa aatatgt 27

<210> 7

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tgaagctgtt gagcatcatc ag 22

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

ggcgacgctg gataacaa 18

<210> 9

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

cctggccgat ctgcagaa 18

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

gagccttcgc ctcgttgata g 21

<210> 11

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gctttgacga agactaaacg tgac 24

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

ggcctggatc ttacgcca 18

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

acgaccattg aggcggtc 18

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

gtccagcgac tgcagctc 18

<210> 15

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

gaaatggcgc tatgactcca aatc 24

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

tcgatggtcg gcaggaac 18

<210> 17

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ccctttgcta agcaggtttg tc 22

<210> 18

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

cttgccagag tttgagggat ttac 24

Claims

1. The multiplex PCR primer composition for detecting the klebsiella pneumoniae is characterized by comprising 9 pairs of primers, wherein the nucleotide sequences of the 9 pairs of primers are shown as SEQ ID NO. 1-SEQ ID NO. 18.

2. A test kit for detecting klebsiella pneumoniae, comprising the primer composition of claim 1.

3. The test kit of claim 2, wherein the kit further comprises a multiplex PCR premix.

4. Use of the primer composition of claim 1 or the detection kit of any one of claims 2-3 in qualitative detection of klebsiella pneumoniae for non-diagnostic purposes.

5. Use of the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 for detecting genetic variation within and among klebsiella pneumoniae strains.

6. Use of the primer composition of claim 1 or the detection kit of any one of claims 2-3 for constructing a klebsiella pneumoniae database.

7. Use of the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 in the fine typing detection of klebsiella pneumoniae.