CN114790489A - MNP (MNP) marker site of haemophilus influenzae, primer composition, kit and application of MNP marker site - Google Patents

MNP (MNP) marker site of haemophilus influenzae, primer composition, kit and application of MNP marker site Download PDF

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CN114790489A
CN114790489A CN202111323161.0A CN202111323161A CN114790489A CN 114790489 A CN114790489 A CN 114790489A CN 202111323161 A CN202111323161 A CN 202111323161A CN 114790489 A CN114790489 A CN 114790489A
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李论
高利芬
方治伟
肖华锋
陈利红
李甜甜
周俊飞
彭海
万人静
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Abstract

The invention discloses an MNP marker site of haemophilus influenzae, a primer composition, a kit and application thereof, wherein the MNP marker site refers to a genome region which is screened on the genome of haemophilus influenzae, is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marker sites of MNP-1-MNP-12; the primer is shown as SEQ ID NO. 1-SEQ ID NO. 24. The MNP marker locus can specifically identify the haemophilus influenzae and finely distinguish different subtypes; the primers are not interfered with each other, and the multiplex amplification and sequencing technology is integrated, so that sequence analysis can be performed on all marked sites of multiple samples at one time, the primers have the advantages of high throughput, multiple targets, high sensitivity and no culture, can be applied to identification and genetic variation detection of haemophilus influenzae of large-scale samples, and have important significance on scientific research and monitoring of haemophilus influenzae.

Description

MNP (MNP marker locus) of haemophilus influenzae, primer composition, kit and application of MNP marker locus and primer composition
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to an MNP (MNP) marker site of haemophilus influenzae, a primer composition, a kit and application thereof.
Background
Haemophilus influenzae (Haemophilus influenzae) is the most common bacterium of Haemophilus which is pathogenic to humans, inhabits the nasal, pharyngeal, ocular and vaginal mucosa of healthy human bodies more widely, is often symbiotic with normal flora, and is an important pathogenic bacterium of community-acquired pneumonia. It can form secondary infection such as bronchitis, sinusitis, otitis media, etc. in influenza and other viral infections, which often occur in adults; some primary pyogenic infections, such as pyogenic meningitis, nasopharyngitis, pyogenic arthritis, etc., are also caused, and are often seen in children. In developing countries, haemophilus influenzae causes 200 to 300 million pneumonia per year, and most of the causative agents are haemophilus influenzae type b. The clinical symptoms of respiratory tract infection caused by Haemophilus influenzae and other pathogens are very similar, so that it is difficult to accurately judge whether a patient is infected with Haemophilus influenzae through clinical manifestations. Therefore, the rapid and accurate detection and typing of the haemophilus influenzae have important significance for timely diagnosing the cause of the disease and accurately treating the disease. In addition, haemophilus influenzae is also a common model pathogenic microorganism for laboratory studies. As a group organism, the individual in the group can generate variation in the interaction with a host and the environment. For laboratory studies, this inconspicuous variation can lead to the fact that the identically named strains are not actually identical in different laboratories or at different times in the same laboratory, leading to irreproducible and incomparable experimental results. Heterogeneity between human Hella cell laboratories has resulted in a large number of incomparable experimental results and wasted data. Therefore, the development of a rapid and accurate Haemophilus influenzae detection and analysis method capable of monitoring variation is of great significance to scientific research and application of Haemophilus influenzae.
The classical haemophilus influenzae detection method comprises isolation culture, PCR technology, whole genome and metagenome sequencing and the like, and has one or more limitations in the aspects of time length, operation complexity, detection flux, accuracy and sensitivity of detection variation, cost and the like. The targeted molecular marker detection technology combining the ultra-multiplex PCR amplification and the high-throughput sequencing can be used for enriching target microorganisms in a sample with low microorganism content in a targeted manner, avoids a separation culture step required by whole genome sequencing and a large amount of data waste and background noise caused by metagenome sequencing, and has the advantages of less 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 (single nucleotide polymorphism) markers and SSR (simple sequence repeat) markers. SSR markers are generally accepted as the most polymorphic markers, but are few in microorganisms; SNP markers are large in number, densely distributed, and polymorphic, single SNP markers are insufficient to capture the potential allelic diversity in a microbial population.
Therefore, the development of novel molecular markers with high polymorphism of pathogenic microorganism haemophilus influenzae and detection technology thereof become a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to provide an MNP marker locus of haemophilus influenzae, a primer composition, a kit and application thereof, which can perform qualitative identification and variation detection on haemophilus influenzae and have the effects of multiple targets, high flux, high sensitivity and fine typing.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided an MNP marker site of Haemophilus influenzae, which is a genomic region specific to a species selected on the genome of Haemophilus influenzae and having a plurality of nucleotide polymorphisms within the species, including marker sites of MNP-1 to MNP-12 on the L42023 genome.
In the above technical solution, the labeling sites of MNP-1 to MNP-12 are specifically shown in table 1 of the specification, and the starting and ending positions of the MNP label marked in table 1 are determined based on the L42023 sequence.
In a second aspect of the invention, a multiplex PCR primer composition for detecting the MNP marker locus is provided, and the multiplex PCR primer composition comprises 12 pairs of primers, wherein the nucleotide sequences of the 12 pairs of primers are shown as SEQ ID NO. 1-SEQ ID NO. 24.
In the above technical scheme, the primer of each MNP marker site 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, a detection kit for detecting the MNP marker site of the haemophilus influenzae is provided, and the kit comprises the primer composition.
Further, the kit further comprises a multiplex PCR premix.
In the fourth aspect of the invention, the MNP marker site of the haemophilus influenzae, the multiplex PCR primer composition or the detection kit is provided for application in the detection of non-diagnostic haemophilus influenzae.
In the fifth aspect of the invention, the application of the MNP marker locus of the haemophilus influenzae, the multiplex PCR primer composition or the detection kit in the detection of the genetic variation in and among haemophilus influenzae strains is provided.
In the sixth aspect of the invention, the application of the MNP marker site of the haemophilus influenzae, the multiplex PCR primer composition or the detection kit in the construction of a haemophilus influenzae database is provided.
In the seventh aspect of the invention, the MNP marker site of the haemophilus influenzae or the multiplex PCR primer composition or the detection kit is provided for the application in the fine typing detection of haemophilus influenzae.
In the application, firstly, the total bacterial DNA of a sample to be detected is obtained; carrying out first round of multiplex PCR amplification on the total DNA and a blank control by using the kit disclosed by the invention, wherein the cycle number is not higher than 25; after purifying the amplification product, adding a sample label based on the second round of PCR amplification and a second-generation sequencing adaptor; purifying and quantifying the second round amplification product; when a plurality of strains are detected, performing high-throughput sequencing by equivalently mixing second round amplification products; and aligning the sequencing result to the reference sequence of the haemophilus influenzae to obtain the number of the detected sequences in the total DNA and genotype data. And performing data quality control and data analysis on the sequencing data of the total DNA according to the obtained quantity of the Haemophilus influenzae sequencing sequences and the detected MNP sites on the total DNA and the blank control to obtain the detected MNP site quantity, the sequencing sequence quantity covering each MNP site and the MNP site genotype data.
When the method is used for identifying the haemophilus influenzae, whether the sample to be detected contains nucleic acid of the haemophilus influenzae or not is judged after quality control is carried out according to the number of sequencing sequences of the haemophilus influenzae detected in the sample to be detected and a blank control and the number of MNP sites detected. The quality control scheme and the determination method are characterized in that DNA of Haemophilus influenzae with known copy number is used as a detection sample, the sensitivity, the accuracy and the specificity of the kit for detecting the Haemophilus influenzae are evaluated, and the quality control scheme and the determination method are formulated when the kit is used for detecting the Haemophilus influenzae.
When used for detecting genetic variation of Haemophilus influenzae, the method comprises the detection of genetic variation among strains and in strains. The detection of genetic variation among strains comprises the steps of obtaining genotype data of each strain to be compared at 12 MNP sites by using the kit and the method. And analyzing whether the main genotypes of the strains to be compared on the 12 MNP sites are different or not by genotype comparison. If the strains to be compared have variation in the major genotype of at least one MNP site, the strains are judged to have genetic variation. Alternatively, the 12 sites of the strains to be compared can be amplified by single PCR, and the amplified products can be subjected to Sanger sequencing, and after the sequences are obtained, the genotypes of each MNP site of the strains to be compared are compared. If there are MNP sites with inconsistent master genotypes, there are variations between the strains to be compared. When the genetic variation in the strain is detected, whether a minor genotype other than the major genotype is detected at the MNP site of the strain to be detected is determined by a statistical model. And if the to-be-detected strain has a minor genotype at least one MNP site, judging that the genetic variation exists in the to-be-detected strain.
When the method is used for constructing a Haemophilus influenzae DNA fingerprint database, the genotype data of the MNP site of the Haemophilus influenzae identified from a sample is recorded into a database file to form the Haemophilus influenzae DNA fingerprint database; and when different samples are identified, comparing the different samples with the DNA fingerprint database of the Haemophilus influenzae to identify whether the Haemophilus influenzae in the samples has the difference of the major genotypes (more than 50 percent of the genotypes supported by the sequencing fragments at one MNP site) at the MNP sites with the strains in the database, wherein the Haemophilus influenzae with the difference of the major genotypes at least 1 MNP site is a new variant type, and recording the new variant type into the DNA fingerprint database.
When the method is used for typing haemophilus influenzae, identifying haemophilus influenzae in a sample to be detected to obtain the genotype of each MNP site; collecting the genome sequence of the haemophilus influenzae disclosed on the Internet and the constructed haemophilus influenzae DNA fingerprint database to construct a haemophilus influenzae reference sequence database; and comparing the genotype of the haemophilus influenzae in the sample to be detected with the reference sequence library of the haemophilus influenzae. And identifying whether the haemophilus influenzae in the sample is an existing strain type or a new variant strain type according to the comparison result with the reference sequence library, thereby realizing the fine typing of the haemophilus influenzae.
The invention belongs to the field of haemophilus influenzae, is pioneered, and has no related literature report; MNP markers are mainly developed based on reference sequences, and are distinguished from MNP sites of other species, internal polymorphism of Haemophilus influenzae species and conserved sequences on two sides of the MNP sites on a large scale according to reported re-sequencing data of small species represented by Haemophilus influenzae; MNP locus detection primers suitable for multiplex PCR amplification can be designed through conserved sequences on two sides of the MNP locus; and then according to the test result of the standard product, a set of MNP sites with the largest polymorphism and high specificity, a primer combination with the best compatibility and a detection kit are screened.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the invention provides an MNP marker locus of haemophilus influenzae, a primer composition, a kit and application thereof. The provided 12 MNP sites of the haemophilus influenzae and the primer combination thereof can carry out multiple PCR amplification, and are fused with a second-generation sequencing platform to carry out sequencing on an amplification product, thereby meeting the detection requirements of carrying out high throughput, high efficiency, high accuracy, high sensitivity and culture-free on the haemophilus influenzae and the requirement of accurately detecting the genetic variation among haemophilus influenzae strains; the method meets the demand of shareable fingerprint data construction and the demand of fine typing of the Haemophilus influenzae standard, and provides technical support for scientific research and monitoring of the Haemophilus influenzae.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic diagram of MNP marker polymorphism;
FIG. 2 is a flow chart of the screening and primer design of the Haemophilus influenzae MNP marker locus;
FIG. 3 is a flow chart of detection of MNP marker sites.
Detailed Description
The embodiments of the present invention will be specifically explained below with reference to specific embodiments and examples, and the advantages and various effects of the embodiments of the present invention will be more clearly presented thereby. It should be understood by those skilled in the art that the detailed description and examples are intended to illustrate, but not limit, the embodiments of the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, 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. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or can be prepared by an existing method.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
screening MNP markers suitable for detecting the population organisms as detection targets. MNP markers refer to polymorphic markers caused by multiple nucleotides over a region of the genome. Compared with SSR markers and SNP markers, MNP markers have the following advantages: (1) abundant alleles, 2 at a single MNP locus n Species alleles, higher than SSR and SNP, are suitable for detection of microorganisms, a typical population of organisms; (2) the species distinguishing capability is strong, species identification can be realized only by a small amount of MNP marks, and the detection error rate is reduced. MNP labeling method for detecting MNP label and fusion of super-multiplex PCR and second generation high-molecular-weight PCRThe flux sequencing technology 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 quantity of sequencing samples by using the DNA barcodes of the samples, and can type tens of thousands of MNP sites of hundreds of samples at one time; (3) the sensitivity is high, multiple targets are detected at one time by utilizing multiple PCR, and high false negative and low sensitivity caused by amplification failure of a single target are avoided; (4) high accuracy, using the second generation high throughput sequencer to sequence the amplification product hundreds of times.
In view of the advantages and the characteristics, the MNP marker and the detection technology MNP marking method thereof can realize the classification and the tracing of the multi-allelic genotypes of the population organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, detection of genetic variation and the like. At present, no report about MNP labeling exists in haemophilus influenzae, and corresponding technology is lacked. The development, screening and application of the MNP marking method have better application foundation in plants.
Therefore, the present invention has developed MNP marker sites for Haemophilus influenzae, which are genomic regions screened from the genome of Haemophilus influenzae that are differentiated from other species and have a plurality of nucleotide polymorphisms within the species, including marker sites for MNP-1 to MNP-12 on the L42023 genome.
Then, the invention develops a multiplex PCR primer composition for detecting the MNP marker locus of the haemophilus influenzae, wherein the multiplex PCR primer composition comprises 12 pairs of primers, and the nucleotide sequences of the 12 pairs of primers are shown as SEQ ID NO. 1-SEQ ID NO. 24. The primers are not conflicted with each other, and can be efficiently amplified through multiple PCR;
the multiplex PCR primer composition can be used for a detection kit for detecting the MNP marker locus of the haemophilus influenzae.
The kit provided by the invention can accurately and sensitively detect less than 10 copies/reaction of haemophilus influenzae.
In the reproducibility test of the invention, the logarithm of difference of the MNP labeling main gene type among different libraries and different library establishing batches of each sample is 0, the reproducibility r is 100%, and the accuracy a is 100%.
The MNP marker and the kit of the present invention have high specificity in detecting a target microorganism in a complex template.
The MNP marker site, primer composition, kit and application of Haemophilus influenzae of the present application will be described in detail below with reference to examples, comparative examples and experimental data.
Example 1 screening of Haemophilus influenzae MNP marker loci and design of multiplex PCR amplification primers
S1, screening of Haemophilus influenzae MNP marker locus
Based on the complete or partial genome sequences of 771 different isolates of Haemophilus influenzae disclosed in the network, 12 MNP marker sites are obtained by sequence comparison. For species without genomic data on the net, the genomic sequence information of the representative microspecies of the microbial species to be detected can also be obtained by high-throughput sequencing, wherein the high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure polymorphism of the selected marker, the genomic sequence of at least 10 genetically representative isolates is generally used as a reference. The 12 MNP marker sites screened are shown in table 1:
TABLE 1-the MNP marker sites and the starting positions of the detection primers on the reference sequence
Figure BDA0003338358780000061
The step S1 specifically includes:
selecting a genome sequence of a representative subtype of the haemophilus influenzae as a reference genome, and performing sequence comparison on the genome sequence and the reference genome to obtain single nucleic acid polymorphic sites of each strain of the haemophilus influenzae;
on the reference genome, window translation is carried out by taking 100-300 bp as a window and 1bp as a step length, and a plurality of candidate MNP site regions are obtained by screening, wherein the candidate MNP site regions 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 two ends;
screening a region with the division DP more than or equal to 0.2 in the candidate polynucleotide polymorphic site region as an MNP marker site; wherein, DP ═ d/t, t is the comparison logarithm of all the minor species in the region of the candidate polynucleotide polymorphic site when compared pairwise, d is the sample logarithm of at least two single nucleic acid polymorphisms that differ in the region of the candidate polynucleotide polymorphic site.
As an optional implementation mode, when the reference genome is screened by taking 100-300 bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, which is beneficial to comprehensive screening.
S2 design of multiplex PCR amplification primers
The multiplex PCR amplification primers of the MNP sites are designed through primer design software, the design of the primers 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, evaluation of detection efficiency of primer combination
Adding a haemophilus influenzae counting standard substance with a known copy number into human genome DNA to prepare a 1000-copy/reaction simulation template, detecting by the MNP marker detection method, constructing 4 repeated sequencing libraries, screening primer combinations with uniform amplification and optimal compatibility according to the detection condition of MNP sites in the 4 libraries, and finally screening out the primer compositions of 12 MNP sites in the table 1.
Example 2 detection of Haemophilus influenzae sites and primers by MNP sites
Using a haemophilus influenzae counting standard with a known copy number, 1 copy/reaction, 10 copies/reaction and 100 copies/reaction haemophilus influenzae mock samples were prepared by adding to human genomic DNA. An equal volume of sterile water was also set as a blank. A total of 4 samples were obtained, each sample was constructed into 3 duplicate libraries each day, and the assay was continued for 4 days, i.e. 12 sets of sequencing data were obtained for each sample, as shown in table 2. The detection procedure for MNP markers is shown in FIG. 3. According to the sequencing fragment number and the site number of MNP sites of the haemophilus influenzae detected in a blank control and a haemophilus influenzae nucleic acid standard in 12 repeated experiments, the repeatability, the accuracy and the sensitivity of the detection method are evaluated, and the threshold values of pollution of a quality control system and detection of target pathogens are set.
1. Sensitivity and stability evaluation of MNP (MNP) labeled detection kit for detecting haemophilus influenzae
As shown in Table 2, the kit can stably detect more than 7 MNP sites in a sample of 10 copies/reaction, and can detect 1 MNP site at most in a sample of 0 copy/reaction, can obviously distinguish the samples of 10 copies/reaction and 0 copy/reaction, and has technical stability and detection sensitivity as low as 10 copies/reaction.
TABLE 2 detection sensitivity, stability analysis of MNP labeling method for Haemophilus influenzae
Figure BDA0003338358780000071
Figure BDA0003338358780000081
2. Evaluation of reproducibility and accuracy of MNP (MNP) marker detection kit for detecting haemophilus influenzae
Based on whether the genotype of the co-detected site can be reproduced in the two repetitions, the reproducibility and accuracy of the MNP marker detection method for detecting the haemophilus influenzae are evaluated. Specifically, two-by-two comparisons were made for each of 12 sets of data for 100 copies/reaction samples, and the results are shown in Table 3.
TABLE 3 evaluation of reproducibility and accuracy of Haemophilus influenzae MNP marker detection method
Figure BDA0003338358780000082
As can be seen from Table 3, the number of MNP sites with differences in major genotypes is 0; the accuracy rate a is 1- (1-r)/2 is 0.5+0.5r, and r represents the reproducibility rate, i.e., the ratio of the number of sites where the major genotype is reproducible to the number of common sites, which is considered to be the principle of accuracy among 2 repeated experiments. In the reproducibility test of the invention, the logarithm of difference of the MNP marked main genotype among different libraries and different library establishing batches of each sample is 0, the reproducibility r is 100%, and the accuracy a is 100%.
3. Threshold judgment of Haemophilus influenzae detection by MNP marker detection kit
Sequences aligned to Haemophilus influenzae could be detected in 1 copy/reaction sample, covering at least 1 MNP site. The sequence of Haemophilus influenzae was also detected in the partial blank. Due to the extreme sensitivity of MNP marker detection methods, contamination of the data during detection is prone to the generation of false positives. Therefore, in this example, a quality control scheme is established, specifically as follows:
1) the amount of sequencing data was greater than 3.6 megabases. The measurement and calculation are based on the fact that the number of MNP sites detected by each sample is 12, and the length of a sequencing fragment is 300 bases, so that when the data volume is more than 3.6 million bases, most samples can ensure that the number of the sequencing fragments covering each site reaches 1000 times through one-time experiment, and the accurate analysis of the base sequence of each MNP site is ensured.
2) And judging whether the contamination is acceptable according to the signal index S of the haemophilus influenzae in the test sample and the noise index P of the haemophilus influenzae in the blank control, wherein:
the blank noise index P is Nc/Nc, wherein Nc and Nc represent the number of sequencing fragments of Haemophilus influenzae and the total number of sequencing fragments in the blank, respectively.
The signal index S of the test sample is Nt/Nt, where Nt and Nt represent the number of sequenced fragments and the total number of sequenced fragments of haemophilus influenzae, respectively, in the test sample.
3) And calculating the detection rate of the MNP marker locus in the test sample, which is the ratio of the number of the detected locus to the number of the total design locus.
TABLE 4 Signal to noise ratio of Haemophilus influenzae in samples tested
Figure BDA0003338358780000091
As shown in Table 4, the average value of the noise index of Haemophilus influenzae in the blank was 0.06%, while the average value of the signal index in the sample of 1 copy was 0.29%, and the average value of the signal-to-noise ratio of the sample of 1 copy and the blank was 4.9, and therefore, the present invention provides that when the signal-to-noise ratio is more than 10 times, contamination in the detection system can be judged to be acceptable. The average of the signal-to-noise ratios of the 10 copies of the sample and blank was 55.4, and at least 7 MNP sites were stably detected in the 10 copies/reaction of 12 sets of data, accounting for 43.8% of the total sites. Therefore, in the case of ensuring accuracy, the standard stipulates that the signal-to-noise ratio determination threshold value of haemophilus influenzae is 30, that is, when the signal-to-noise ratio of haemophilus influenzae in a sample is greater than 30 and the site detection rate is greater than or equal to 30%, the detection of nucleic acid of haemophilus influenzae in the sample is determined.
Therefore, the kit provided by the invention can sensitively detect 10 copies/response of the haemophilus influenzae.
4. Specific evaluation of MNP (MNP) marker detection kit for detecting haemophilus influenzae
Artificially mixing DNAs of haemophilus influenzae, mycobacterium tuberculosis, acinetobacter strains, bordetella pertussis, bordetella hollandii, chlamydia pneumoniae, mycoplasma pneumoniae, epstein barr virus, varicella zoster virus, cytomegalovirus, herpes simplex virus, human bocavirus, klebsiella pneumoniae, legionella, moraxella catarrhalis, pseudomonas aeruginosa, rickettsia, staphylococcus aureus, streptococcus pneumoniae and streptococcus pyogenes together according to equimolar amount to prepare a mixed template, and detecting the haemophilus influenzae in the mixed template by using a sterile water template as a blank control for 3 repeated experiments. After sequence comparison and analysis according to the quality control scheme and the judgment threshold, the 12 MNP sites of the haemophilus influenzae can be specifically detected in 3 repeated experiments, which shows that the MNP marker and the kit detect the high specificity of the target microorganism in a complex template.
Example 3 detection of genetic variation among Haemophilus influenzae strains
6 haemophilus influenzae strains provided by the Hubei province disease control and prevention control center are detected by using the kit and an MNP marker locus detection method, samples are sequentially named as S-1-S-6, wherein S-2-S-5 are filial strains of the same strain in different periods. The mean fold coverage of sequencing was over 1000 fold for each sample and all 12 MNP markers were detected per strain (table 5). The fingerprints of 6 strains are compared pairwise, and the result is shown in table 5, wherein the difference between S1 and other strains is at least different in the major genotypes of 2 MNP sites; s-2 and S3-S5 also exhibited major genotype differences at 1 MNP site (Table 5), indicating that the same named strains exhibited inter-strain variation.
TABLE 5-6 detection assays for Haemophilus influenzae
Figure BDA0003338358780000101
As can be seen from Table 5, the application of the kit in identifying genetic variation among strains by detecting MNP markers can be used for ensuring the genetic consistency of the same named Haemophilus influenzae strains in different laboratories, thereby ensuring the comparability of research results, which has important significance for scientific research on Haemophilus influenzae. Clinically, diagnostic protocols can be weighed against whether differential sites affect drug resistance.
Example 4 detection of genetic variation in Haemophilus influenzae
As a group organism, a part of individuals in the Haemophilus influenzae group are mutated, so that the group is not homozygous any more, and a heterogeneous heterozygous group is formed, thereby influencing the stability and consistency of the phenotype of the microorganism for testing in particular. The variant shows an allelic form outside the main genotype of the locus when the population is subjected to molecular marker detection. When the variant individuals have not accumulated, they represent a very small fraction of the population and are characterized by a low frequency of alleles. Low frequency alleles tend to be confused with technical errors, making prior art techniques difficult to distinguish. The present invention detects highly polymorphic MNP markers. The technical error rate of MNP labeling is significantly lower than that of SNP labeling, based on the probability of multiple errors occurring simultaneously being lower than the probability of one error occurring.
The authenticity assessment of the sub-allelic genotypes of this example was performed as follows: alleles with strand bias (ratio of the number of sequencing sequences overlaid on the DNA double strand) were first excluded according to the following rule: the strand preference is greater than 10 fold, or the difference in strand preference from the dominant allele is greater than 5 fold.
Genotypes without strand preference were judged for authenticity based on the number and ratio of sequenced sequences in table 5. Inv function calculation under a 99.9999% probability guarantee, e max (n-1) and e max (n.gtoreq.2) 1.03% and 0.0994%, respectively, the number of sequenced sequences of the sub-allelic gene in each locus is a critical value, and only when the number of sequenced sequences of the sub-allelic gene exceeds the critical value, the true sub-allelic gene is determined. When multiple candidate sub-alleles are present, multiple corrections are made to the P-value for each candidate allele, FDR<0.5% of the candidate alleles were judged to be true sub-allelic genotypes. Table 6 relates to the parameter e max (n-1) and e max (n.gtoreq.2) means that the highest proportion of the number of sequencing sequences carrying the wrong allele of n SNPs to the total number of sequencing sequences at that site. e.g. of the type max (n-1) and e max (n.gtoreq.2) 1.03% and 0.0994%, respectively, were obtained from the frequency of all minor alleles detected at 930 homozygous MNP sites.
TABLE 6 critical value for determination of sub-allelic genotypes at partial sequencing depth
Figure BDA0003338358780000111
Figure BDA0003338358780000121
The DNAs of Haemophilus influenzae S-1 and S-2 having major genotype differences in 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, and each sample was examined 3 times to obtain a total of 24 sequencing data, according to the above parameters. By accurately comparing the gene types of the MNP sites of the two variants of the haemophilus influenzae, the heterozygous gene type sites can be detected in 24 artificial heterozygous samples, thereby demonstrating the applicability of the developed MNP marker detection method of the haemophilus influenzae in detecting the genetic variation of the strains.
Example 5 construction of Haemophilus influenzae DNA fingerprint database
Extracting DNA of all strains or samples for constructing a Haemophilus influenzae DNA fingerprint database by using a conventional CTAB method, a commercial kit and other methods, and detecting the quality of the DNA by using agarose gel and an ultraviolet spectrophotometer. If the ratio of the absorbance values of the extracted DNA at 260nm to 230nm is more than 2.0, the ratio of the absorbance values of 260nm to 280nm is between 1.6 and 1.8, the main band of the DNA electrophoresis is obvious, and obvious degradation and RNA residue do not exist, the genome DNA reaches the relevant quality requirement, and subsequent experiments can be carried out.
And (3) carrying out sequence comparison on the sequencing data of the 6 strains to obtain the major genotype of each site of each strain, forming an MNP fingerprint of each strain, and recording a database file to form a DNA fingerprint database of the haemophilus influenzae. 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 spectrum of the strains obtained by each detection is compared with the established MNP fingerprint database, and the MNP fingerprint database established by the MNP fingerprint spectrum of the strains with different main genotypes realizes the co-establishment sharing and the updating of the database at any time.
Example 6 application in Fine typing of Haemophilus influenzae
Firstly, constructing a reference sequence database of the haemophilus influenzae, wherein the reference sequence database consists of a disclosed genome sequence of the haemophilus influenzae and a constructed DNA fingerprint database of the haemophilus influenzae; obtaining an MNP fingerprint of the haemophilus influenzae in each sample to be detected by using the primer combination and the MNP marker locus detection method in the embodiment 2; comparing the DNA fingerprint of each strain with a constructed reference sequence library, and screening to obtain the strain with the genetic distance closest to that in the sequence library; is 100% identical to the genotype of the existing strain, is an existing variant, has a major genotype difference in at least one MNP site, is a new variant, and realizes the fine typing of the Haemophilus influenzae.
The detection results of 6 haemophilus influenzae are shown in table 5, after the 6 haemophilus influenzae detected are pairwise compared, the 6 haemophilus influenzae are divided into 3 types according to the difference of major genotypes, and the 3 types are different from the strains in the reference sequence library and are new types. Therefore, the resolution of the method for the haemophilus influenzae reaches the level of single base, and the fine typing of the haemophilus influenzae in a sample can be realized.
Finally, it should also be 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. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments 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 in the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, if such modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to encompass such modifications and variations.
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Claims (8)

1. An MNP marker site of Haemophilus influenzae, which is a genomic region that is screened from the genome of Haemophilus influenzae and is distinguished from other species and has a plurality of nucleotide polymorphisms within the species, and comprises marker sites of MNP-1 to MNP-12 on the L42023 genome.
2. The multiplex PCR primer composition for detecting the MNP marker locus of the haemophilus influenzae according to claim 1, which is characterized by comprising 12 pairs of primers, wherein the nucleotide sequences of the 12 pairs of primers are shown as SEQ ID No. 1-SEQ ID No. 24.
3. A test kit for detecting the MNP marker site of Haemophilus influenzae according to claim 1, wherein the kit comprises the primer composition according to claim 2.
4. The test kit of claim 3, wherein the kit further comprises a multiplex PCR premix.
5. Use of a MNP marker site of haemophilus influenzae according to claim 1 or a primer composition according to claim 2 or a test kit according to any one of claims 3 to 4 in the detection of haemophilus influenzae for non-diagnostic purposes.
6. Use of a MNP marker site of haemophilus influenzae according to claim 1 or a primer composition according to claim 2 or a test kit according to any one of claims 3 to 4 for the detection of genetic variations within and between strains of haemophilus influenzae.
7. Use of the MNP marker site of haemophilus influenzae according to claim 1 or the primer composition according to claim 2 or the detection kit according to any one of claims 3-4 for the construction of a haemophilus influenzae database.
8. Use of the MNP marker site of haemophilus influenzae according to claim 1 or the primer composition according to claim 2 or the test kit according to any one of claims 3-4 in the fine typing test of haemophilus influenzae.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009024844A2 (en) * 2007-07-13 2009-02-26 Universita' Degli Studi Di Firenze Method of detecting and serotyping of pathogens in a sample
WO2012103353A2 (en) * 2011-01-26 2012-08-02 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Selective detection of haemophilus influenzae
CN102653794A (en) * 2012-05-14 2012-09-05 江苏大学 Multiple touchdown PCR (polymerase chain reaction) detection kit of haemophilus influenzae
CN102660645A (en) * 2012-05-07 2012-09-12 镇江和创生物科技有限公司 Primer and probe combination and kit for specifically detecting streptococcus pneumoniae and haemophilus influenzae
CN113195737A (en) * 2018-02-20 2021-07-30 奥斯瓦道·克鲁兹基金会 Oligonucleotide, oligonucleotide set, method and kit for simultaneously detecting neisseria meningitidis, streptococcus pneumoniae and haemophilus influenzae

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009024844A2 (en) * 2007-07-13 2009-02-26 Universita' Degli Studi Di Firenze Method of detecting and serotyping of pathogens in a sample
WO2012103353A2 (en) * 2011-01-26 2012-08-02 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Selective detection of haemophilus influenzae
CN102660645A (en) * 2012-05-07 2012-09-12 镇江和创生物科技有限公司 Primer and probe combination and kit for specifically detecting streptococcus pneumoniae and haemophilus influenzae
CN102653794A (en) * 2012-05-14 2012-09-05 江苏大学 Multiple touchdown PCR (polymerase chain reaction) detection kit of haemophilus influenzae
CN113195737A (en) * 2018-02-20 2021-07-30 奥斯瓦道·克鲁兹基金会 Oligonucleotide, oligonucleotide set, method and kit for simultaneously detecting neisseria meningitidis, streptococcus pneumoniae and haemophilus influenzae

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PICKERING J等: "A PCR-High-Resolution melt assay for rapid differentiation of nontypeable haemophilus influenzae and haemophilus haemolyticus", J CLIN MICROBIOL, vol. 52, no. 2, pages 663 - 667 *
万成松,余清: "流感嗜血杆菌的基因组学研究进展", 生命科学研究, no. 2, pages 143 - 147 *

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