CN114015793B - MNP (MNP) marking site of rickettsia, primer composition, kit and application of MNP marking site - Google Patents

Abstract

The invention discloses MNP (MNP) marking sites of rickettsia, a primer composition, a kit and application thereof, wherein the MNP marking sites refer to genome regions which are screened on the genome of rickettsia and are separated from other species and have a plurality of nucleotide polymorphisms in the species, and the marking sites comprise MNP-1-MNP-15; the primer is shown as SEQ ID NO.1-SEQ ID NO. 30. The MNP marker locus can specifically identify rickettsia and finely distinguish different strains; the primers are not interfered with each other, and the multiplex amplification and sequencing technology is integrated, so that the sequence analysis can be performed on all the marker loci 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 identification and genetic variation detection of rickettsia of a large-scale sample, and the method has important significance on scientific research and epidemic prevention monitoring of the rickettsia.

Description

MNP (MNP) marking site of rickettsia, 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 an MNP (MNP) marking site of rickettsia, a primer composition, a kit and application thereof.

Background

Rickettsia is a genus (Rickettsia) in taxonomy, and a class of strictly intracellular parasitic prokaryotes between bacteria and viruses, close to bacteria, are causative agents of epidemic typhus, endemic typhus, rocky mountain typhus, rickettsia, tsutsugamushi, spot fever, and trench fever. The diseases caused by rickettsia are collectively called rickettsia diseases, and are natural epidemic diseases of people and animals. Rickettsia disease exists throughout the world, has historically been a serious threat to human health, and is a major cause of death in some countries both tropical and subtropical, especially third world countries, where morbidity is still high. Therefore, the rapid and accurate detection of rickettsia is of great importance for early diagnosis and control of diseases. Rickettsia comprises a plurality of species, but the existing Rickettsia classification system is classified based on the modes of an inventor and the like, and comprises Prikettsia (Rickettsia prowazekii), morse Rickettsia (Rickettsia mooseri) and Kang Shili g Rickettsia (Rickettsia Conorii) and the like, and with the development of molecular biology technology, a more scientific classification system based on genetic materials is urgently needed.

Existing rickettsia detection and typing methods, including isolation culture, PCR techniques, whole genome and metagenome sequencing, and the like, have one or more limitations in terms of duration, operational complexity, detection throughput, accuracy and sensitivity of detection variation, cost, and the like. 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, low-frequency mutation detection and accurate typing. 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, the development of a novel molecular marker with high polymorphism of rickettsia and a detection technology thereof is a technical problem to be solved urgently.

Disclosure of Invention

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

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

in a first aspect of the invention there is provided a MNP marker locus for Rickettsia, the MNP marker locus being a region of the genome screened on the Rickettsia genome that is distinct from other species and has multiple nucleotide polymorphisms between internal species, including the marker locus for MNP-1 to MNP-15 on the Rickettsia reference sequence.

In the above technical scheme, the marking sites of MNP-1 to MNP-15 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 reference sequences corresponding to the same row of MNPs in the table 1.

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 15 pairs of primers, the specific primer sequences being shown in SEQ ID NO.1-SEQ ID NO. 30.

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 invention, there is provided a detection kit for detecting the MNP marker locus of rickettsia, 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 rickettsia or the multiplex PCR primer composition or the detection kit for the identification of rickettsia for non-diagnostic purposes.

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

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

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

In the application, the specific operation steps are as follows:

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 rickettsia 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 rickettsia sequencing sequences and the number of the detected MNP sites obtained in the total DNA and the blank control, 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 Rickettsia body identification, quality control is carried out according to the number of sequencing sequences of Rickettsia bodies detected in a sample to be detected and a blank control and the number of MNP sites detected, and whether the sample to be detected contains Rickettsia body nucleic acid or not is judged. The quality control scheme and the judging method are characterized in that DNA of Rickettsia with known copy number is taken as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting Rickettsia are evaluated, and the quality control scheme and the judging method when the kit detects Rickettsia are formulated.

When used in rickettsia genetic variation assays, they include inter-and intra-strain genetic variation assays. The detection of genetic variation among strains comprises the steps of obtaining genotype data of 15 MNP sites of each strain to be compared by using the kit and the method. By genotype comparison, the strains to be compared are analyzed for differences in major genotypes at the 15 MNP sites. 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, 15 loci of the strain to be compared may be amplified by single PCR, and then Sanger sequencing is performed on the amplified products to obtain sequences, and then the genotypes of each MNP locus of the strain to be compared are 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 method is used for constructing a Rickettsia DNA fingerprint database, genotype data of the MNP locus of Rickettsia identified from a sample is input into a database file to form the Rickettsia DNA fingerprint database; each time a different sample is identified, it is identified whether rickettsia in the sample differs from strains in the database in that the MNP locus has a major genotype (with more than 50% of the genotypes supported by the sequencing fragments at one MNP locus), and rickettsia having a major genotype difference at least 1 MNP locus is a new variant type, by comparison with the DNA fingerprint database of rickettsia, and is included in the DNA fingerprint database.

When the method is used for the typing of rickettsia, the rickettsia in a sample to be tested is identified, and the genotype of each MNP locus is obtained; collecting genome sequences of Rickettsia bodies disclosed on the network and a constructed Rickettsia body DNA fingerprint database to construct a Rickettsia body reference sequence library; and comparing the genotype of the rickettsia in the sample to be detected with a reference sequence library of the rickettsia. And identifying whether the rickettsia in the sample is an existing strain type or a new variant strain type according to the comparison result with the reference sequence library, and realizing the fine typing of the rickettsia.

The invention belongs to the initiative in the Rickettsia field, and is not reported in related documents; MNP markers are developed mainly based on reference sequences, and MNP sites which are large-scale and are distinguished from other species, polymorphic in the rickettsia species and conserved in sequence at two sides can be mined according to reported resequencing data of rickettsia representing small 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 a set of MNP locus with the largest polymorphism and high specificity and a primer combination with the best compatibility can be screened out according to the test result of the standard substance.

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 rickettsia, a primer composition, a kit and application thereof. The provided 15 MNP loci of the rickettsia 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 the rickettsia are met, and the requirements of the rickettsia standard and sharable fingerprint data construction are met; the need to accurately detect genetic variation between rickettsia strains; the requirement of homozygous and heterozygous Rickettsia is identified, and technical support is provided for scientific research, scientific monitoring and prevention and control of Rickettsia.

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 for MNP marker loci of Rickettsia;

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:

the invention develops a novel molecular marker-MNP marker which is suitable for detecting group organisms and is specific to species. MNP markers refer to polymorphic markers caused by multiple nucleotides in a region of the genome. Compared to SSR markers and SNP markers, MNP markers have the followingAdvantages are: (1) The alleles are abundant, and 2 are arranged on single MNP locus ⁿ Species alleles 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 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. Thus, the present invention developed MNP marker loci for Rickettsia that are genomic regions screened on Rickettsia genome that are distinct from other species and have multiple nucleotide polymorphisms inside the species, including MNP-1-MNP-15 marker loci with AJ235269 as reference genome.

Next, the present invention has developed a multiplex PCR primer composition for detecting MNP marker loci of Rickettsia, characterized in that the multiplex PCR primer composition comprises 15 pairs of primers, the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID NO.1 to SEQ ID NO. 30. The primers do not collide with each other, and efficient amplification can be performed by multiplex PCR.

The multiplex PCR primer composition can be used as a detection kit for detecting the MNP marker locus of the rickettsia.

The kit of the invention can accurately and sensitively detect rickettsia down to 10 copies/reaction.

The MNP markers and the kits of the invention detect high specificity of target microorganisms in complex templates.

The MNP marker locus, primer composition, kit and use thereof of one of rickettsia of the present application will be described in detail below in connection with examples, comparative examples and experimental data.

Example 1 selection of MNP marker loci of Rickettsia and design of multiplex PCR amplification primers

S1, screening of MNP (MNP) marker loci of rickettsia

Based on complete or partial sequences of genomes of 133 different isolates of rickettsia disclosed on the net, 15 MNP marker loci are obtained by 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 15 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 of a representative strain of the rickettsia 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 rickettsia;

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.

In this embodiment, the primers used to identify the MNP marker sites are shown in table 1.

S3, evaluating detection efficiency of primer combination

The detection method of the MNP marker is that all MNP loci are amplified at one time through multiplex PCR, amplification products are sequenced through second-generation high-throughput sequencing, sequencing data are analyzed, and compatibility of the primer combination is evaluated according to the detected loci.

The primer combination was used to screen a primer combination of 15 MNP sites according to the present invention as described in Table 1 by using Kang Shili g of a quality control substance (cargo number: VIP (VC) 042) of a purchased copy number, adding to human genome DNA, preparing a 1000 copy/reaction template, and screening a primer combination of optimal compatibility with uniformity of amplification according to detection of MNP sites in 4 libraries.

Threshold settings and Performance assessment for MNP site and primer identification of Rickettsia by example 2

In this example, kang Shili g of a quality control of hyposomal DNA with a known copy number was added to human genomic DNA to prepare 1 copy/reaction, 10 copy/reaction and 100 copy/reaction of Rickettsia-induced samples. 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 according to the number of sequencing fragments and the number of sites of MNP sites of Rickettsia detected in blank control and Rickettsia simulated samples in 12 repeated experiments, evaluating the reproducibility, accuracy and sensitivity of the detection method, and formulating a quality control system pollution and a threshold value for detecting a target pathogen. The detection flow of MNP markers is shown in fig. 3.

1. Sensitivity and stability assessment of MNP (MNP) marked detection kit for detecting rickettsia

As shown in Table 2, the kit can stably detect 1-2 MNP sites in a 1-copy/reaction sample, 7 MNP sites in a 10-copy/reaction sample, and at most 1 MNP site in a 0-copy/reaction sample, and can clearly distinguish between a 10-copy/reaction sample and a 0-copy/reaction sample, and has technical stability and detection sensitivity as low as 10-copy/reaction.

TABLE 2 detection sensitivity and stability analysis of MNP labeling method of Rickettsia

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

Based on whether the genotype of the co-detected site is reproducible in the two replicates, the reproducibility and accuracy of detection of rickettsia by the MNP marker detection method is assessed. Specifically, the data of 12 sets of 100 copies/reaction samples were compared in pairs, respectively, and the results are shown in Table 3.

TABLE 3 reproducibility and accuracy assessment of MNP marker detection method of Rickettsia

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 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%. Thus, the kit of the present invention is capable of accurately detecting rickettsia as low as 10 copies/reaction.

3. Threshold value judgment for detecting rickettsia by MNP (MNP) mark detection kit

As shown in Table 2, the sequence aligned to Rickettsia was detected in 1 copy/reaction sample, covering at least 1 MNP site. The sequence of rickettsia 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 4.5 megabases. The measurement and calculation basis is that the number of MNP loci detected by each sample is 15, 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 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 rickettsia signal index S in the test sample and the rickettsia noise index P in the blank, wherein:

the blank noise index p=nc/Nc, where Nc and Nc represent the number of sequenced fragments and total sequenced fragment number of rickettsia, respectively, in the blank.

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

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.

TABLE 4 Signal to noise ratio of Rickettsia in samples to be tested

As a result, as shown in Table 4, the average value of the noise index of Rickettsia in the control was 0.06%, the average value of the signal index in the 1-copy sample was 0.29%, and the average value of the signal-to-noise ratio of the 1-copy sample and the control was 5.23, so that the present invention provides that when the signal-to-noise ratio is more than 10 times, it can be judged that the contamination in the detection system is acceptable.

The average signal to noise ratio of the 10 copies of the sample and the blank was 52.1, and at least 7 MNP sites were stably detected in the 10 copies/reaction 15 sets of data, accounting for 43.8% of the total sites. Therefore, under the condition of ensuring accuracy, the standard prescribes that the rickettsia positive judgment standard is: when the signal to noise ratio of the rickettsia in the sample is greater than 30 and the site detection rate is greater than or equal to 30%, determining that the nucleic acid of the rickettsia is detected in the sample.

Therefore, the kit provided by the invention can accurately and sensitively detect the rickettsia with the concentration as low as 10 copy/reaction.

4. Specific evaluation of MNP marker detection method for detecting rickettsia

Kang Shili g of Mycobacterium tuberculosis, acinetobacter strain, pertussis baud bacteria, huo Shibao t bacteria, chlamydia pneumoniae, mycoplasma 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 artificially mixed together according to equimolar amounts to prepare a mixed template, and the Rickettsia in the mixed template is detected by using a blank template as a control by adopting the method provided by the invention, so that 3 repeated experiments are performed. After sequence comparison and analysis according to the quality control scheme and the judgment threshold, 15 MNP sites of the rickettsia 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 Rickettsia strains

6 rickettsia strains stored in the same laboratory are detected by using the kit and the MNP marker locus detection method, samples are sequentially named as S1-S6, the average coverage of sequencing of each sample is 1534 times, and all 15 MNP markers can be detected by each strain (Table 5). The results of pairwise alignment of fingerprints of 6 strains are shown in Table 5, and 1 part (S-2) and 5 parts of rickettsia detected together with the same batch all have major genotype differences of 2 MNP sites (Table 5), and there are inter-strain variations, possibly belonging to different isolates.

TABLE 5 detection analysis of 6 Rickettsia

As can be seen from Table 5, the application of the kit of the invention in identifying genetic variation among strains by detecting MNP markers can be used for ensuring the genetic consistency of the same named rickettsia strains in different laboratories, thereby ensuring the comparability of research results, and having great significance for scientific research of rickettsia. 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 inside Rickettsia Strain

As a group organism, the individuals in the Rickettsia 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 test are influenced. Such variants, when detected by molecular marker detection on the population, appear as alleles outside the major genotype of the locus. 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 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 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) 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, nucleic acids 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 genotypes of MNP loci of the two strains, loci with heterozygous genotypes are detected in 24 artificial heterozygous samples, and the applicability of the developed MNP marker detection method for mycoplasma pneumoniae in detecting genetic variation inside strain groups is demonstrated.

Example 5 construction of Rickettsia DNA fingerprint database

All strains used for constructing the database of the DNA fingerprint of the Cryptosporidium or the DNA of samples are extracted by using the conventional CTAB method, the commercial kit and the like, 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) comparing the sequencing data of the 6 strains with the reference genotype, and obtaining the main genotype of each site of each strain to form the MNP fingerprint of each strain. And recording the obtained MNP fingerprint of each strain into a database file to form a Rickettsia body DNA 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. And comparing the MNP fingerprint of the strain obtained by each detection with an MNP fingerprint database constructed based on the existing genome data, and inputting the MNP fingerprint of the strain with the main genotype difference into the constructed MNP fingerprint database to achieve real-time updating and co-construction sharing of the database.

Example 6 use in Rickettsia Fine refinement

Constructing a reference sequence library consisting of the disclosed genome sequence of rickettsia and a rickettsia MNP fingerprint database; detecting Rickettsia in a sample to be detected by using the primer combination and MNP marking site detection method, and obtaining MNP fingerprint of each strain; comparing the DNA fingerprint of each strain with a constructed reference sequence library, and identifying the DNA fingerprint as a very similar strain of the existing strain with the same reference sequence, wherein the DNA fingerprint of each strain has main genotype difference at more than one MNP locus and is identified as a new variant strain, so that the fine typing of the P.

Detection of 6 rickettsia strains as shown in table 5, among the 6 rickettsia detected, S1 and other 5 were different in major genotypes at 2 or more MNP sites, and 5 strains consistent in genotype were consistent with Kang Shili g of the rickettsia Malish 7 strain, suggesting that they are very similar strains. The S1 strain and the Malish 7 strain were also most similar, but were identified as new variants by the presence of a difference in 2 MNP sites. Therefore, the resolution of the MNP locus and the kit to the rickettsia reaches the level of single base, and the fine typing of the rickettsia in the sample can be realized.

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> 9

gaccggaaac tattgcctag acttt 25

<210> 10

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

gatttgcagc gcgatgagtg tatag 25

<210> 11

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gtagctcagt tggttagagc gtc 23

<210> 12

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

agaatgtatc acttggtttt gaaatatca 29

<210> 13

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

agacaaaggc agtgaatata gaaca 25

<210> 14

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

tgatgacatc acctaataca ctgtt 25

<210> 15

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

tgttggaatc ttgttgtata gactaga 27

<210> 16

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

actgaatata aaaccttcat tgctatgga 29

<210> 17

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

tagcaagtta tattgtgaca ccacg 25

<210> 18

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

tatcggtgcc ataatattag ccatc 25

<210> 19

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

ataacaatct aatgaacaga gttgtga 27

<210> 20

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

tttctttgta atctttcaac aatactctag 30

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

accagaatgc atactcatca tgact 25

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

agattatatg cttggaccta atgct 25

<210> 23

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

aacaatacaa agagcaacag cagtt 25

<210> 24

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

acgtaattta gaatagaggt tgcgg 25

<210> 25

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

actccttatt ttgatccatg aaatattgc 29

<210> 26

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

acacaagaag aaatttatca atgaaattgt 30

<210> 27

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

tgtttgtgta aaagttttca atatgct 27

<210> 28

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

agatatgtca ctttcatatt gtatacttct 30

<210> 29

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

atattaatgt taaattttca aatgtgtccg 30

<210> 30

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

aagctactct ttttagtatt gcttgt 26

Claims (7)

1. A multiplex PCR primer composition for detecting a rickettsia MNP marker locus, wherein the MNP marker locus is a genomic region screened on the rickettsia genome that is distinct from other species and has multiple nucleotide polymorphisms within the species, comprising the marker locus of MNP-1 to MNP-15 of an AJ235269 reference genome, the multiplex PCR primer composition comprising 15 pairs of primers, the nucleotide sequences of the 15 pairs of primers being shown in SEQ ID No.1 to SEQ ID No. 30.

2. A detection kit for detecting the rickettsia MNP marker locus of claim 1, wherein the kit comprises 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 a primer composition according to claim 1 or a detection kit according to any one of claims 2-3 for the identification of rickettsia 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 rickettsia strains.

6. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2-3 for the construction of a rickettsia database.

7. Use of the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 in the detection of rickettsia finely divided type.