CN114790489B - MNP (MNP) marking site of haemophilus influenzae, primer composition, kit and application of MNP marking site - Google Patents
MNP (MNP) marking site of haemophilus influenzae, primer composition, kit and application of MNP marking site Download PDFInfo
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Abstract
The invention discloses an MNP (MNP) marking site of haemophilus influenzae, a primer composition, a kit and application thereof, wherein the MNP marking site refers to a genome region which is screened on the genome of haemophilus influenzae and is separated from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marking 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 haemophilus influenzae 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 property, can be applied to the identification and genetic variation detection of the haemophilus influenzae of a large-scale sample, and has important significance on the scientific research and monitoring of the haemophilus influenzae.
Description
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to an MNP (MNP) marking 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, and is widely lodged in nasal, pharyngeal, ocular and vaginal mucosa of healthy humans, often symbiotic with normal flora, and is an important pathogenic bacterium for community-acquired pneumonia. It can form secondary infections such as bronchitis, sinusitis, otitis media, etc. in influenza and other viral infections, most commonly occurring in adults; can also cause some primary suppurative infections, such as suppurative meningitis, nasopharyngitis, suppurative arthritis, etc., which often occur in children. In developing countries, haemophilus influenzae causes 200 to 300 thousands of pneumonia per year, with most pathogenic people being 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 or not through clinical manifestations. Therefore, the detection and typing of the haemophilus influenzae are rapid and accurate, and the method has important significance for timely diagnosing the etiology and achieving accurate treatment. In addition, haemophilus influenzae is also a common model of pathogenic microorganisms for laboratory research. As a group organism, individuals in the group can be mutated in interaction with hosts and environments. For laboratory studies, such undetectable variations can result in strains of the same name in different laboratories or different times in the same laboratory being virtually different, resulting in irreproducible and incomparable experimental results. Heterogeneity between human hela cell laboratories has resulted in a significant amount of incomparable experimental results and wasted data. Therefore, developing a rapid, accurate and monitorable mutation detection and analysis method for haemophilus influenzae has important significance for scientific research and application of haemophilus influenzae.
Classical haemophilus influenzae detection methods, including isolation and culture, PCR techniques, whole genome and metagenome sequencing, etc., suffer from one or more limitations in terms of duration, complexity of operation, detection throughput, accuracy and sensitivity of detection variation, cost, etc. The targeted molecular marker detection technology integrating the ultra-multiplex PCR amplification and the high-throughput sequencing can enrich target microorganisms in a sample with low microorganism content in a targeted manner, avoids the waste of a large amount of data and the background noise caused by the separation and culture step required by the whole genome sequencing and the metagenome sequencing, and has the advantages of small sample requirement, accurate diagnosis result, data quantity saving and low-frequency variation detection.
The molecular markers detected by the existing targeted detection technology mainly comprise SNP and SSR markers. SSR markers are the most well-accepted markers for polymorphism, but are small in number in microorganisms; the number of SNP markers is huge, the distribution is dense, and the polymorphism of single SNP marker is insufficient to capture the potential allelic diversity in microorganism population.
Therefore, development of a novel molecular marker of a pathogenic microorganism haemophilus influenzae with high polymorphism and a detection technology thereof becomes a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to provide MNP (MNP) marking sites of haemophilus influenzae, a primer composition, a kit and application thereof, which can carry out qualitative identification and mutation detection on haemophilus influenzae 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 of Haemophilus influenzae, the MNP marker locus being a species-specific genomic region screened on the Haemophilus influenzae genome and having a plurality of nucleotide polymorphisms within the species, including the marker locus of MNP-1 to MNP-12 on the L42023 genome.
In the above technical scheme, the marking sites of MNP-1 to MNP-12 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 sequence L42023.
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 12 pairs of primers, the nucleotide sequences of the 12 pairs of primers being shown as SEQ ID NO.1 to SEQ ID NO. 24.
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 MNP marker locus of haemophilus influenzae, 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 said MNP marker locus of haemophilus influenzae or said multiplex PCR primer composition or said detection kit for the detection of haemophilus influenzae for non-diagnostic purposes.
In a fifth aspect of the invention, there is provided the use of said MNP marker locus of haemophilus influenzae or said multiplex PCR primer composition or said detection kit for detecting genetic variations within and between haemophilus influenzae strains.
In a sixth aspect of the invention, there is provided the use of said MNP marker locus of haemophilus influenzae or said multiplex PCR primer composition or said detection kit for constructing a haemophilus influenzae database.
In a seventh aspect of the invention, there is provided the use of said MNP marker locus of haemophilus influenzae or said multiplex PCR primer composition or said detection kit in the accurate and finely divided detection of haemophilus influenzae.
In the above application, firstly, the total DNA of the bacteria of the sample to be tested is obtained; performing a first round of multiplex PCR amplification on the total DNA and the blank control by using the kit, wherein the number of cycles is not higher than 25; purifying the amplified product, and then adding a sample tag and a second generation sequencing joint based on the second-round PCR amplification; quantifying after purifying the second round of amplification products; detecting a plurality of strains by mixing the amplification products of the second round in equal amounts and then performing high throughput sequencing; and comparing the sequencing result with the reference sequence of the haemophilus influenzae to obtain the number of the 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 sequencing sequences of the haemophilus influenzae 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 the identification of the haemophilus influenzae, whether the sample to be detected contains nucleic acid of the haemophilus influenzae or not is judged after quality control according to the number of sequencing sequences of the haemophilus influenzae detected in the sample to be detected and the blank control and the number of MNP sites detected. The quality control scheme and the judging method are characterized in that DNA of the haemophilus influenzae with known copy number is taken as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting the haemophilus influenzae are evaluated, and the quality control scheme and the judging method when the kit detects the haemophilus influenzae are formulated.
When used in the detection of genetic variation in haemophilus influenzae, it includes the detection of genetic variation between and within strains. The detection of genetic variation among strains comprises the steps of obtaining genotype data of 12 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 12 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, the 12 loci of the strains to be compared may be amplified by single PCR, and the amplified products may be subjected to Sanger sequencing to obtain sequences, and the genotypes of each MNP locus of the strains 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 method is used for constructing a haemophilus influenzae DNA fingerprint database, genotype data of the MNP locus of the haemophilus influenzae identified from a sample is input into a database file to form the haemophilus influenzae DNA fingerprint database; and (3) each time different samples are identified, comparing the samples with a DNA fingerprint database of the haemophilus influenzae, and identifying whether the haemophilus influenzae 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 haemophilus influenzae with the main genotype difference at least 1 MNP site is a new mutation type, and recording the new mutation type in the DNA fingerprint database.
When the method is used for haemophilus influenzae typing, the haemophilus influenzae in a sample to be tested is identified, and the genotype of each MNP locus is obtained; collecting genome sequences of the haemophilus influenzae disclosed on the network and constructing a haemophilus influenzae reference sequence library by the constructed haemophilus influenzae DNA fingerprint database; and comparing the genotype of the haemophilus influenzae in the sample to be tested with a 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, so as to realize the fine typing of the haemophilus influenzae.
The invention is initiated in the haemophilus influenzae field, and is not reported in related documents; MNP markers are developed mainly based on reference sequences, and are characterized by large-scale discrimination among MNP sites of other species, polymorphic inside the species of Haemophilus influenzae, conserved on both sides of the sequences, mined according to reported resequencing sequences of the species Haemophilus influenzae; 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 a MNP (MNP) marking site of haemophilus influenzae, a primer composition, a kit and application thereof. The provided 12 MNP loci of the haemophilus influenzae 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, high sensitivity and culture-free detection of the haemophilus influenzae and the requirements of accurate detection of genetic variation among haemophilus influenzae strains are met; meets the requirements of the standard and the sharable fingerprint data construction of the haemophilus influenzae and the requirements of the fine subdivision, and provides technical support for the scientific research and monitoring of the haemophilus influenzae.
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 the MNP-labeling sites of Haemophilus influenzae;
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:
MNP markers suitable for detection of the population organisms are screened as detection targets. 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 MNP labeling is reported in haemophilus influenzae, and corresponding technologies are 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 Haemophilus influenzae, which are genomic regions screened on the Haemophilus influenzae genome that are distinct from other species and have multiple nucleotide polymorphisms within the species, including the marker loci for MNP-1-MNP-12 on the L42023 genome.
Next, the present invention developed a multiplex PCR primer composition for detecting the MNP marker loci of Haemophilus influenzae, which comprises 12 pairs of primers, the nucleotide sequences of which 12 pairs of primers are shown as SEQ ID NO.1 to SEQ ID NO. 24. 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 the haemophilus influenzae.
The kit provided by the invention can accurately and sensitively detect haemophilus influenzae with the concentration of less than 10 copies/reaction.
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 one of the 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 MNP marker loci of Haemophilus influenzae and design of multiplex PCR amplification primers
S1, screening of MNP (MNP) marker locus of haemophilus influenzae
Based on the complete or partial sequences of the genomes of 771 different isolates of haemophilus influenzae disclosed on the net, 12 MNP marker loci are obtained through sequence alignment. For species on which no genomic data is present on the net, genomic sequence information representing a minispecies of the microorganism species to be detected may also be obtained by high throughput sequencing, which may be whole genome or simplified genome sequencing. In order to ensure polymorphism of the selected markers, genomic sequences of at least 10 genetically representative isolates are generally used as reference. The 12 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 haemophilus influenzae 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 haemophilus influenzae;
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
The method comprises the steps of adding a haemophilus influenzae counting standard product with known copy number into human genome DNA to prepare a 1000-copy/reaction simulated template, detecting by using the MNP mark detection method, constructing 4 repeated sequencing libraries, screening a primer combination with uniform amplification and optimal compatibility according to detection conditions of MNP sites in the 4 libraries, and finally screening the primer combination of 12 MNP sites in the table 1.
Example 2 detection of Haemophilus influenzae by MNP loci and primers
Haemophilus influenzae mock samples were prepared at 1 copy/reaction, 10 copy/reaction and 100 copy/reaction using a known copy number haemophilus influenzae count standard, 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 reproducibility, 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 haemophilus influenzae detected in a blank control and a nucleic acid standard of the haemophilus influenzae in 12 repeated experiments, and formulating a threshold value for pollution of a quality control system and detection of 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 7 MNP sites in a 10-copy/reaction sample, and at most 1 MNP site in a 0-copy/reaction sample, 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 Haemophilus influenzae
2. Reproducibility and accuracy assessment of MNP (MNP) marker detection kit for detecting haemophilus influenzae
Based on whether the genotype of the co-detection site is reproducible or not in the two replicates, the reproducibility and accuracy of detection of haemophilus influenzae by the MNP marker detection method are evaluated. Specifically, the data of 12 sets of 100 copies/reaction samples were compared in pairs, and the results are shown in Table 3.
TABLE 3 reproducibility and accuracy assessment of Haemophilus influenzae 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 value judgment for detecting haemophilus influenzae by MNP (MNP) mark detection kit
Sequences aligned to Haemophilus influenzae can be detected in 1 copy/reaction samples, covering at least 1 MNP site. The sequence of H.influenzae 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 3.6 megabases. The measurement and calculation basis is that the number of MNP loci detected by each sample is 12, and the length of one sequencing fragment is 300 bases, so that when the data size is more than 3.6 megabases, most samples can ensure that the number of sequencing fragments covering each locus reaches 1000 times by one experiment, and the accurate analysis of the base sequence of each MNP locus is ensured.
2) Determining whether the contamination is acceptable based on the signal index S of haemophilus influenzae in the test sample and the noise index P of haemophilus influenzae 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 haemophilus influenzae, respectively, in the control.
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 haemophilus influenzae, respectively, 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 SNR of haemophilus influenzae in samples to be tested
As a result, as shown in Table 4, the mean value of the noise index of Haemophilus influenzae in the control was 0.06%, the mean value of the signal index in the 1-copy sample was 0.29%, and the mean value of the signal-to-noise ratio of the 1-copy sample and the control was 4.9, so that the present invention provides that when the signal-to-noise ratio was more than 10-fold, it was judged that the contamination in the detection system was acceptable. The average signal to noise ratio of the 10 copies of the sample and the blank was 55.4, and at least 7 MNP sites were stably detected in the 10 copies/reaction 12 sets of data, accounting for 43.8% of the total sites. Therefore, under the condition of ensuring accuracy, the standard prescribes that the signal-to-noise ratio judgment threshold of the haemophilus influenzae is 30, namely when the signal-to-noise ratio of the haemophilus influenzae in the sample is more than 30 and the site detection rate is more than or equal to 30 percent, the nucleic acid of the haemophilus influenzae is judged to be detected in the sample.
Therefore, the kit provided by the invention can sensitively detect 10 copies/reaction of haemophilus influenzae.
4. Specific evaluation of MNP (MNP) marker detection kit for detecting haemophilus influenzae
The mixed template is prepared by artificially mixing together DNA of haemophilus influenzae, mycobacterium tuberculosis, acinetobacter strain, pertussis baud bacteria, huo Shibao termitis bacteria, chlamydia pneumoniae, mycoplasma pneumoniae, EB 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 according to equimolar amounts, and the method provided by the invention is adopted to detect haemophilus influenzae in the mixed template by taking a sterile water template as a blank control, so that 3 repeated experiments are carried out. After sequence comparison and analysis according to the quality control scheme and the judgment threshold, 12 MNP sites of the haemophilus influenzae 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 Haemophilus influenzae strains
6 haemophilus influenzae strains provided by the disease control and prevention control center of Hubei province are detected by using the kit and the MNP marking site detection method, and samples are sequentially named as S-1 to S-6, wherein S-2 to S-5 are offspring strains of the same strain in different periods. The average coverage of sequencing per sample was more than 1000 fold, and all 12 MNP markers could be detected per strain (table 5). The fingerprint patterns of 6 strains are aligned pairwise, and the results are shown in table 5, wherein the difference between S1 and other strains is at least the difference between the main genotypes of 2 MNP loci; s-2 and S3-S5 also present major genotype differences at 1 MNP site (Table 5), indicating that the same named strains have inter-strain variation.
TABLE 5-6 detection assay for Haemophilus influenzae
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 haemophilus influenzae strains in different laboratories, so that the comparability of research results is ensured, and the kit has important significance for scientific research of haemophilus influenzae. In clinical terms, one can take into account the diagnostic regimen as to whether the site of the difference affects resistance.
Example 4 genetic variation detection of Haemophilus influenzae
As a group organism, the individuals in the inner part of the haemophilus influenzae group are mutated, so that the group is not homozygous any more, and a heterogeneous heterozygous group is formed, and the stability and consistency of the phenotype of the microorganism for the 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.
Absence of chain preferenceGenotypes were judged for authenticity based on the number and proportion of sequenced sequences in table 5. Table 6 lists e calculated based on binom. Inv function under the probability guarantee of α=99.9999% max (n=1) and e max (n.gtoreq.2) is 1.03% and 0.0994%, respectively, and the true hypogenotype is judged only when the number of sequences of the hypogenotype exceeds the critical value. When a plurality of candidate minor alleles exist, multiple correction is carried out on the P value of each candidate allele type, and FDR is carried out<0.5% of candidate alleles are judged to be true minor genotypes. Parameter e related to Table 6 max (n=1) and e max (n.gtoreq.2) 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 Haemophilus influenzae having major genotype differences of S-1 and S-2 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, 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 the two variants of haemophilus influenzae, heterozygous genotype loci can be detected in 24 artificial heterozygous samples, and the applicability of the developed method for detecting MNP markers of haemophilus influenzae in detecting genetic variation of strains is demonstrated.
Example 5 construction of Haemophilus influenzae DNA fingerprint database
All strains or DNA of samples used for constructing the DNA fingerprint database of the haemophilus influenzae are extracted by using a conventional CTAB method, a 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) 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 the haemophilus influenzae 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 use in Haemophilus influenzae Fine subdivision
Firstly, constructing a reference sequence library of haemophilus influenzae, which consists of a published genome sequence of haemophilus influenzae and a constructed DNA fingerprint database of haemophilus influenzae; obtaining MNP fingerprints of haemophilus influenzae in each sample to be tested by using the primer combination and the MNP marker locus detection method described in example 2; comparing the DNA fingerprint of each strain with a constructed reference sequence library, and screening to obtain the strain with the closest genetic distance in the sequence library; 100% identical to the genotype of the existing strain, the existing variant has a main genotype difference at least one MNP site, and the new variant realizes the fine typing of haemophilus influenzae.
The detection results of 6 haemophilus influenzae are shown in table 5, and after the 6 haemophilus influenzae is compared in pairs, the 6 haemophilus influenzae is divided into 3 types according to the difference of main genotypes, and the 3 types are different from strains in a reference sequence library, so that the haemophilus influenzae is a new type. 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.
Sequence listing
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Claims (7)
1. The multiplex PCR primer composition for detecting the haemophilus influenzae 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.
2. A test kit for detecting haemophilus influenzae, 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 to 3 for the detection of non-diagnostic haemophilus influenzae.
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 haemophilus influenzae strains.
6. Use of the primer composition of claim 1 or the detection kit of any one of claims 2-3 for constructing a haemophilus influenzae 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 haemophilus influenzae in a finely divided form.
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| WO2009024844A2 (en) * | 2007-07-13 | 2009-02-26 | Universita' Degli Studi Di Firenze | Method of detecting and serotyping of pathogens in a sample |
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| 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 |
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