CN114277164B - MNP (MNP-associated protein) marking combination, primer pair combination, kit and application of MNP marking combination and primer pair combination - Google Patents
MNP (MNP-associated protein) marking combination, primer pair combination, kit and application of MNP marking combination and primer pair combination Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology, and discloses a MNP (MNP) marker combination, a primer pair combination and a kit of streptococcus pneumoniae and application thereof, wherein the MNP marker combination comprises 15 markers, and a specific nucleotide sequence is shown as SEQ ID NO.1-SEQ ID NO. 15; the primer pair combination comprises 15 pairs of primers, and the specific primer nucleotide sequence is shown as SEQ ID NO.16-SEQ ID NO. 45. The MNP marker combination can specifically identify streptococcus pneumoniae and finely distinguish different strains; the primers are not interfered with each other, and the multiplex amplification and sequencing technology is integrated, so that sequence analysis can be carried out on all the mark combinations of multiple samples at one time; the kit has the advantages of high flux, multiple targets, high sensitivity, high accuracy and culture-free detection, can be applied to detection of large-scale samples, and has important significance for scientific research and monitoring of streptococcus pneumoniae.
Description
Technical Field
The embodiment of the invention relates to the field of biotechnology, in particular to MNP (MNP) marker combination, primer pair combination, kit and application thereof of streptococcus pneumoniae.
Background
Streptococcus pneumoniae (Streptococcus pneumonia) is a human pathogenic gram-positive bacterium, mainly causes human lobar pneumonia, and is one of important pathogenic microorganisms for infectious pneumonia; can also invade other parts of the body to cause secondary thoracomembrane inflammation, otitis media, mastoiditis, endocarditis, suppurative meningitis and the like. Can be transmitted by spray, and is common in winter and early spring, and is often parallel to respiratory tract virus infection. Therefore, rapid and accurate diagnosis of Streptococcus pneumoniae infectious diseases is clinically significant.
In addition, streptococcus pneumoniae is also a common model of pathogenic microorganisms for laboratory research. As a group organism, it can be mutated in the interaction with the host and the environment. For laboratory studies, such undetectable variations may result in strains that are not identical in nature from laboratory to laboratory or from different times in the same laboratory, resulting in irreproducible and incomparable experimental results. Heterogeneity between human hela cell laboratories has resulted in a significant amount of incomparable experimental results and wasted data. Therefore, the development of a rapid and accurate streptococcus pneumoniae detection and analysis method has important significance for clinical diagnosis, disease control monitoring and scientific research of streptococcus pneumoniae.
The classical culture method is a gold standard for detecting streptococcus pneumoniae, but is long in time consumption and complex in operation, and can not meet the requirements of large samples and rapid detection. In recent years, molecular detection technology has been rapidly developed, and fluorescent multiplex PCR technology is one of the most commonly used molecular methods for detecting microorganisms, and is also one of the commonly used detection technologies in current microorganism detection countries and industry standards. However, the number of targets detected by the detection device is limited, variation cannot be detected, and detection of a few markers is easy to detect failure. Metagenome sequencing is another technology for detecting streptococcus pneumoniae, but metagenome sequencing often comprises a large amount of host sequencing data, so that a large amount of data is wasted and background noise is generated, and particularly when detecting a sample of low-abundance bacteria, ultra-deep sequencing is required, and mutation detection and variant distinction are required, so that ultra-high sequencing cost is caused.
The ultra-multiplex PCR amplification combined with the high-throughput sequencing technology can enrich target microorganisms in samples with low microorganism content in a targeted manner, avoids massive data waste and background noise caused by whole genome-dependent pathogen isolation and culture steps and metagenome sequencing, can achieve deep sequencing at low cost, and can be used for detecting base variation through deep sequencing to distinguish the species to the finest taxonomic level, and has the advantages of small sample requirement, high sensitivity, high accuracy and fine 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 found in small numbers 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 of high-polymorphism streptococcus pneumoniae and a high-throughput, accurate and sensitive detection technology thereof becomes a technical problem to be solved urgently.
The invention provides MNP (MNP) labeling combination of streptococcus pneumoniae, a primer composition, a detection method and application. Wherein, MNP marker is a novel molecular marker, which refers to a polymorphic marker caused by multiple nucleotide variations in a region on a genome.
In view of the advantages and characteristics, MNP markers and detection technology MNP marking methods thereof have application potential in the aspects of streptococcus pneumoniae detection, mutation monitoring, fingerprint database construction and the like. The development, screening and application of MNP labeling method have better application foundation in plants. The invention is initiated in the field of streptococcus pneumoniae and is not reported in related documents.
Disclosure of Invention
The invention aims to provide a MNP (MNP) marking combination, a primer pair combination, a kit and application thereof, which can be used for qualitatively identifying and detecting mutation of streptococcus pneumoniae and have the effects of multiple targets, high throughput, high sensitivity and fine typing.
In order to achieve the above effects, the present invention adopts the following technical scheme:
in a first aspect of the present invention there is provided a combination of MNP markers for streptococcus pneumoniae, the combination of MNP markers being a genomic region screened on the genome of streptococcus pneumoniae that is distinct from other species and has a plurality of nucleotide polymorphisms within the streptococcus pneumoniae species, comprising 15 markers for MNP-1 to MNP-15 on the genomic sequence AE007317 of streptococcus pneumoniae as reference nucleotide sequence.
In the technical scheme, the labeled nucleotide sequences of MNP-1 to MNP-15 are specifically shown as SEQ ID NO.1 to SEQ ID NO.15, wherein ID NO.16 to SEQ ID NO.30 are upper primers ID NO.31 to SEQ ID NO.45 are lower primers. Description table 1 further illustrates that the start and end positions of the MNP markers noted in table 1 are determined based on the reference sequence AE 007317.
In a second aspect of the invention, there is provided a multiplex PCR primer pair combination for detecting said MNP marker combination, said multiplex PCR primer pair combination comprising 15 pairs of primers, the specific primer nucleotide sequences being shown in SEQ ID NO.16-SEQ ID NO. 45.
In the above technical solution, each MNP-labeled primer includes an upper primer and a lower primer, and is specifically shown in table 1.
In a third aspect of the invention there is provided a detection kit for detecting the MNP marker combination of streptococcus pneumoniae, the kit comprising the primer pair combination.
Further, the kit further comprises a multiplex PCR premix.
In a fourth aspect of the invention, there is provided the use of the combination of core MNP markers of Streptococcus pneumoniae or the combination of primer pairs or the detection kit in qualitative detection of Streptococcus pneumoniae of non-eruption purpose, in the preparation of a product for qualitative detection of Streptococcus pneumoniae.
In a fifth aspect of the invention, there is provided the use of said MNP marker combination of streptococcus pneumoniae or said multiplex PCR primer pair combination or said detection kit in the construction of DNA fingerprint databases of streptococcus pneumoniae, genetic variation detection and typing.
In the application, the primer combination is firstly utilized to carry out a first round of multiplex PCR amplification on the DNA of the streptococcus pneumoniae strain to be detected, and the cycle number 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 measurement; comparing the sequencing result with the reference sequence of the streptococcus pneumoniae to obtain genotype data of the strain to be detected on the 15 MNP labels.
When the method is used for qualitative identification of streptococcus pneumoniae, whether the sample to be detected contains nucleic acid of the streptococcus pneumoniae is judged after quality control according to the number of sequencing sequences of the streptococcus pneumoniae detected in the sample to be detected and a blank control and the number of MNP marks detected. The quality control scheme and the judging method are characterized in that DNA of streptococcus pneumoniae with known copy numbers is taken as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting the streptococcus pneumoniae are evaluated, and the quality control scheme and the judging method when the kit detects the streptococcus pneumoniae are formulated.
When the DNA fingerprint database of streptococcus pneumoniae is used, the genotype data of all streptococcus pneumoniae obtained from a reference sequence and the genotype data of the MNP mark of a new strain obtained by actual measurement are input into a database file, namely the DNA fingerprint database of streptococcus pneumoniae is formed. Therefore, the DNA fingerprint database can be continuously enriched by utilizing the primer combination.
When the method is used for typing the streptococcus pneumoniae, the genotype of the strain to be tested in the MNP marked genotype is obtained and compared with a reference sequence library consisting of a reference sequence of the public streptococcus pneumoniae and a constructed DNA fingerprint database, and the streptococcus pneumoniae in a sample is identified as an existing strain or a new variant strain.
When used in the detection of genetic variation in Streptococcus pneumoniae, it includes the detection of genetic variation between strains and within strains. The detection of genetic variation among strains comprises the steps of amplifying and sequencing genome DNA of streptococcus pneumoniae to be detected by utilizing the MNP primer combination, and obtaining genotype data of each strain marked by MNP. And analyzing whether the main genotypes of the strains to be detected on the MNP markers are different or not through pairwise comparison. If the difference exists, the mutation exists in the strain to be detected. Alternatively, 15 markers of the strain to be tested may be amplified by single PCR, respectively, and then Sanger sequencing is performed on the amplified products to obtain sequences, and genotypes are aligned pairwise. If there is a mark with inconsistent genotypes, it indicates that there is variation among the strains to be tested. When detecting genetic variation inside the strain, determining whether the MNP marker of the strain to be detected detects a secondary genotype other than the primary genotype through a statistical model. If the strain to be tested has the subgenotype in at least one MNP mark, judging that the strain to be tested has genetic variation.
Compared with the prior art, the invention has the following advantages:
the invention provides a MNP (MNP-associated protein) marker combination, a primer pair combination, a kit and application of the MNP marker combination and the primer pair combination.
Compared with the existing marking and marking detection technology, the technical scheme provided by the invention has the following advantages:
compared with conventional SSR markers and SNP markers, MNP markers have the following advantages: (1) The allelic gene type is rich, and single MNP mark has 2 n The allelotype is higher than that of the traditional common SSR and SNP, and can meet the detection of the multi-allelotype widely existing in microorganisms; (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. MNP markers are mainly developed based on reference sequences, and MNP markers which are distinguished from other species on a large scale, polymorphic in the species and conserved in sequence at two sides can be mined according to the reported resequencing data of the streptococcus pneumoniae representative strain; by conserved sequences on both sides of MNP markers, the design is applicable to multiplePCR amplified MNP marks detection primer; and then according to the test result of the standard substance, a set of MNP marked primer combination with the largest polymorphism and the widest coverage and the best compatibility can be screened out.
The MNP label combination is subjected to super-multiplex PCR amplification through the primer pair combination, and the amplification product is subjected to sequence analysis by a second-generation high-throughput sequencing technology, so that the method has the following detection advantages: (1) And (3) finely dividing streptococcus pneumoniae by utilizing multiple PCR to detect a plurality of streptococcus pneumoniae specific targets at one time, detecting the multiple targets by adopting a second generation sequencing technology, and finely dividing the streptococcus pneumoniae according to sequence characteristics 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 detect tens of thousands of MNP marks 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) And the amplification product is sequenced hundreds of times by using a second-generation high-throughput sequencer with high accuracy, so that the accurate genotype is obtained. (5) Culture-free, enrichment of targets based on PCR technology is carried out without isolation and culture of pathogenic bacteria.
In view of the advantages and characteristics, the MNP labeling combination, the primer pair combination and the kit have the characteristics of multiple targets, high flux, high efficiency, high accuracy and high sensitivity for identifying the streptococcus pneumoniae, and meet the requirements for identifying the streptococcus pneumoniae in a large number of samples; meets the requirement of monitoring genetic variation among streptococcus pneumoniae strains and inside strains; meeting the requirement of constructing a sharable DNA fingerprint database of streptococcus pneumoniae standard; meets the requirement of accurate typing of streptococcus pneumoniae. Therefore, the 15 MNP labeling combinations, the primer pair combinations and the kit provided by the invention can provide technical support for scientific research and epidemic strain monitoring of streptococcus pneumoniae.
Drawings
FIG. 1 is a schematic diagram of MNP marker polymorphism;
FIG. 2 is a flow chart of screening and primer design for Streptococcus pneumoniae MNP marker combinations;
FIG. 3 is a flow chart of detection of MNP marker combinations;
FIG. 4 is a graph showing the relationship between the copy number of Streptococcus pneumoniae and the number of effective sequencing in a sample to be tested.
Detailed Description
In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to specific embodiments that are illustrated in the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or may be prepared by existing methods.
Example 1 screening of Streptococcus pneumoniae MNP marker combinations and design of multiplex PCR amplification primers
Screening of S1 Streptococcus pneumoniae MNP marker combinations
Based on complete or partial sequences of genomes of 538 different streptococcus pneumoniae isolates disclosed on the net, 15 MNP markers are obtained through sequence alignment. For species on which no genomic data is present on the net, genomic sequence information of the strain representative 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 markers screened are shown in table 1:
MNP markers and detection primers starting positions on the reference sequence as described in Table 1
The step S1 specifically includes:
selecting a genome sequence of a representative strain of streptococcus pneumoniae as a reference genome, and comparing the genome sequence with the reference genome to obtain single nucleotide polymorphism markers of each strain of streptococcus pneumoniae;
on the reference genome, carrying out window translation by taking 100-300bp as a window and taking 1bp as a step length, and screening to obtain a plurality of candidate MNP (MNP) marker areas, wherein the candidate MNP marker areas contain more than or equal to 2 single nucleotide variation markers, and the single nucleotide polymorphism markers do not exist on sequences of 30bp at both ends;
screening a region with the discrimination DP of more than or equal to 0.2 from the candidate polynucleotide polymorphism marking regions as MNP marks; wherein dp=d/t, t is the log of comparisons when all the small species are compared pairwise in the candidate polynucleotide polymorphic marker region, and d is the log of samples of differences in at least two single nucleotide polymorphisms in the candidate polynucleotide polymorphic marker region.
As an optional implementation mode, when screening is performed on the reference genome by taking 100-300bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, so that the comprehensive screening is facilitated.
S2, design of multiplex PCR amplification primer
The MNP marked multiplex PCR amplification primers are designed through primer design software, the primer design follows that the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can be amplified normally in one amplification reaction.
In this embodiment, the primers used to identify the MNP marker combinations are as shown in table 1.
S3, evaluating detection efficiency of primer combination
The detection method of the MNP markers comprises the steps of amplifying all MNP markers at one time through multiplex PCR, sequencing amplified products through second-generation high-throughput sequencing, analyzing sequencing data, and evaluating the compatibility of the primer combination according to the detected markers.
A simulated sample of Streptococcus pneumoniae ATCC6303 strain with known copy number is prepared by using DNA of Streptococcus pneumoniae ATCC6303 strain provided by Hubei province disease control center, namely Streptococcus pneumoniae with known copy number is added into human genome DNA (2 ng/reaction), a 1000-copy/reaction template is prepared, and the primer combination is used for detection by the MNP marker detection method, so that 4 repeated sequencing libraries are constructed. According to the detection results in 4 libraries, the 15 MNP markers with high species specificity and high intraspecies distinction degree provided by the invention are finally screened out, and the detection primer pair combination with high amplification efficiency and good compatibility is obtained.
Performance assessment and threshold setting for identification of Streptococcus pneumoniae by MNP markers and kits described in example 2
Streptococcus pneumoniae DNA of example 1 was added to human genomic DNA to prepare 1 copy/reaction, 10 copy/reaction and 100 copy/reaction of Streptococcus pneumoniae-simulated samples, and an equal volume of sterile water was set as a blank control for a total of 4 samples. Each sample was constructed with 4 replicate libraries per day and run continuously for 3 days, i.e., 12 sets of sequencing data were obtained per sample, as shown in table 2. And evaluating the reproducibility, accuracy and sensitivity of the MNP mark and the kit to the identification of the streptococcus pneumoniae according to the number of sequencing fragments and the number of MNP marks of the MNP mark detected in a blank control and a streptococcus pneumoniae nucleotide standard substance in 12 repeated experiments, and formulating a quality control system pollution and threshold values of target pathogen detection.
The detection flow of MNP markers is shown in fig. 3.
1. Detection sensitivity and stability analysis for detecting streptococcus pneumoniae by MNP labeling method
As shown in Table 2, the kit can stably detect more than 7 MNP markers in 10 copies/reaction samples, and can detect 2 MNP markers at most in a few samples of 0 copies/reaction, and the kit can clearly distinguish between 10 copies/reaction and 0 copies/reaction samples, and has technical stability and detection sensitivity as low as 10 copies/reaction.
TABLE 2 primer compatibility and detection sensitivity analysis of MNP labeling method of Streptococcus pneumoniae
2. MNP marker detection method for qualitatively detecting streptococcus pneumoniae and evaluating repeatability and accuracy
Sequencing results of 4 groups of sequencing data are obtained for 3 templates of 100 copies/reaction of streptococcus pneumoniae, and are compared in pairs, and the results are shown in table 3, wherein the MNP markers with the main genotypes different are all 0; according to the principle that the reproducible genotypes are considered to be accurate between 2 repeated experiments, the accuracy a=1- (1-r)/2=0.5+0.5r, r represents the reproducibility, i.e. the ratio of the number of reproducible markers of the main genotype to the number of common markers. In the project reproducibility test, the difference logarithm of MNP marking main genotypes among different libraries and different sequencing batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%.
TABLE 3 accuracy analysis of Streptococcus pneumoniae MNP markers
3. Threshold setting for detecting streptococcus pneumoniae by MNP (MNaphthyl) marker detection method
Because of the extremely sensitive MNP marker detection method, the data contamination in the detection is liable to cause false positive generation, and therefore quality control of the generated sequencing data is required, and the number of sequenced fragments of streptococcus pneumoniae MNP markers, internal standard DNA markers and detection markers detected in each sample are analyzed after quality control.
The quality control scheme adopted by the invention is as follows:
1) The amount of sequencing data is greater than 4.5 megabases. The measurement and calculation basis is that the number of MNP labels detected by each sample is 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 label reaches 1000 times by one experiment, and the accurate analysis of the base sequence of each MNP label is ensured.
2) Determining whether the contamination is acceptable based on the signal index S of streptococcus pneumoniae in the test sample and the noise index P of streptococcus pneumoniae in the blank, wherein:
the noise figure p=nc/Nc for the control, where Nc and Nc represent the number of sequenced fragments and total sequenced fragment number of streptococcus pneumoniae in the control, respectively.
The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequenced fragments and total number of sequenced fragments, respectively, of streptococcus pneumoniae in the test sample.
3) The detection rate of MNP markers in the test sample is calculated and refers to the ratio of the number of detected markers to the total number of designed markers.
As shown in Table 4, the average signal-to-noise ratio of Streptococcus pneumoniae at 1 copy of the sample and the blank was 4.3, and therefore the present invention provides that contamination in the assay system can be judged to be acceptable when the signal-to-noise ratio is greater than 10-fold.
As shown in Table 4, the average signal to noise ratio of the 10 copies of the sample and the blank was 54.8, and the minimum value was 41.4, and at least 7 MNP markers were stably detected in the 10 copies/reaction 12 sets of data, 46.7% of the total markers. Therefore, under the condition of ensuring the accuracy, the standard prescribes that the signal-to-noise ratio judgment threshold value of the streptococcus pneumoniae is 41, namely when the signal-to-noise ratio of the streptococcus pneumoniae in the sample is more than 41 and the mark detection rate is more than or equal to 40%, the nucleotide of the streptococcus pneumoniae is judged to be detected in the sample. Therefore, the kit provided by the invention can sensitively detect 10 copies/reaction of streptococcus pneumoniae.
Table 44 S.pneumoniae signal-to-noise ratios in 12 assays for each of the 44 samples
4. Specific evaluation of MNP marker detection method for detecting Streptococcus pneumoniae
The DNA of Streptococcus pneumoniae, mycobacterium tuberculosis, acinetobacter strain, pertussis baud bacteria, huo Shibao termyces, chlamydia pneumoniae, mycoplasma pneumoniae, EB virus, haemophilus influenzae, varicella zostera virus, cytomegalovirus, herpes simplex virus, human bocavirus, klebsiella pneumoniae, legionella, moraxella catarrhalis, pseudomonas aeruginosa, rickettsia, staphylococcus aureus and Streptococcus pyogenes are artificially mixed together according to the equimolar amount to prepare a mixed template, and the Streptococcus pneumoniae in the mixed template is detected by adopting the method provided by the invention by using a blank template as a control, so that 3 repeated experiments are carried out. Results the sequencing obtained in 3 replicates all detected 15 MNP markers of Streptococcus pneumoniae. After analysis according to the quality control scheme and the decision threshold, the nucleic acid of the streptococcus pneumoniae was determined to be positive in 3 repeated experiments, indicating that the MNP markers and the kit detect high specificity of streptococcus pneumoniae in complex templates.
In summary, the MNP labeling combination, the primer pair combination and the kit of the streptococcus pneumoniae provided by the invention can detect the streptococcus pneumoniae with high sensitivity, high accuracy and specificity.
EXAMPLE 3 construction of Streptococcus pneumoniae DNA fingerprint database
All strains or DNA of samples used for constructing a streptococcus pneumoniae DNA fingerprint database are extracted by using a conventional CTAB method, a commercial kit and other methods, and the quality of the DNA is detected by using agarose gel and an ultraviolet spectrophotometer. If the ratio of the absorbance values of the extracted DNA at 260nm and 230nm is more than 2.0, the ratio of the absorbance values of 260nm and 280nm is between 1.6 and 1.8, the DNA electrophoresis main band is obvious, no obvious degradation and RNA residues exist, the genome DNA reaches the relevant quality requirements, and the subsequent experiments can be carried out.
The primer combination and MNP mark combination detection method is utilized to detect 6 sub-generation strains which are preserved in different periods of 1 streptococcus pneumoniae strains provided by the Hubei province disease control prevention control center, samples are sequentially named as S-1 to S-6, the average coverage of sequencing of each sample is 2032 times, and all 15 MNP marks can be detected by each strain (table 5). Sequence comparison is carried out on the sequencing data of the 6 strains to obtain the main genotype of each mark of each strain, so as to form the MNP fingerprint of each strain. Inputting MNP fingerprint of strains with main genotypes different into a database, and constructing an MNP fingerprint database of streptococcus pneumoniae; comparing the MNP fingerprint of the streptococcus pneumoniae detected in the new sample with the constructed MNP fingerprint database, and recording the MNP fingerprint of the sample with the main genotype difference into the constructed MNP fingerprint database. The constructed MNP fingerprint database is based on the gene sequence of the detected strain, is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed and shared and being updated at any time.
Table 5 6 detection and analysis of Streptococcus pneumoniae strains
EXAMPLE 4 use in Streptococcus pneumoniae Fine refinement
Obtaining MNP finger print patterns of each strain by using the primer combination and MNP mark combination detection method; comparing the obtained MNP fingerprint of each strain with the published genome sequence of streptococcus pneumoniae and the constructed MNP fingerprint database of streptococcus pneumoniae; the strain was determined to be a very similar strain to the existing strain in that the genotype of the strain was 100%, and a new mutant strain was determined to be a strain having a major genotype difference in one or more MNP markers. Detection and typing of 6 Streptococcus pneumoniae strains As shown in Table 5, the detected 6 Streptococcus pneumoniae S-4 and other 5 strains were judged to be 2 strains, differing in major genotypes at 1 MNP marker. The genotypes of 5 strains with the same genotype and NCTC7465 strain remained the same, and could be offspring strains of the strains. The strains in the S-4 and reference sequence libraries were not identical in the major genotypes of the 2 MNP markers, and were judged as new variants. Therefore, the resolution ratio of the method to the streptococcus pneumoniae reaches the level of single base, and the method can realize the fine typing of the streptococcus pneumoniae in the sample.
EXAMPLE 5 detection of genetic variation between strains of Streptococcus pneumoniae
Genetic variation detection of streptococcus pneumoniae, including inter-and intra-strain variation. Because the pneumococci are parasitic in the host, i.e., the genetic variation of Streptococcus pneumoniae is detected between and within the host. The variation among hosts is detected by comparing the main genotypes, the obtained fingerprint spectrums of the streptococcus pneumoniae are compared in pairs, 100% reproducibility and accuracy of the main genotypes are identified based on an MNP labeling method, and the difference of the main genotypes of two strains with one label can be detected.
As shown in Table 5, 5 of the 6 progeny strains had consistent genotypes and no inter-strain genetic variation; whereas S-4 numbered strains and other strains have inter-strain genetic variation.
Therefore, 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 strains in different laboratories, so that the comparability of research results is ensured, and the kit has important significance for scientific research of streptococcus pneumoniae.
EXAMPLE 6 detection of genetic variation within strains of Streptococcus pneumoniae
As a group organism, streptococcus pneumoniae is mutated in a host or in a group, and when the group is detected by molecular marker, it is expressed as an allele outside the main genotype of the marker. When variant individuals have not accumulated, they occupy a very small portion of the population, and are characterized by low frequency alleles, which are difficult to detect in the prior art. Low frequency alleles tend to mix with technical errors, making the prior art indistinguishable. The present invention detects MNP markers with high polymorphism. Based on the fact that the probability of occurrence of a plurality of errors is lower than that of one error, the technical error rate of MNP markers is significantly lower than that of SNP markers. The invention distinguishes true minor genotypes from error genotypes caused by technical errors through a statistical model. Specifically:
the authenticity assessment of the secondary isogenotypes of this example was performed as follows: the allelotype with strand preference (ratio of the number of sequencing sequences covered on the DNA duplex) is first excluded according to the following rule: the strand preference is greater than 10-fold, or the strand preference differs from the major allele by greater than 5-fold.
The statistical model of genotype reuse without strand preference is true. The principle of the statistical model is to assume that the candidate minor allele is the product of the major allele due to PCR or sequencing errors. Under this assumption, the number of sequencing supported by the candidate minor allele (c) corresponds to a binomial distribution based on the marker sequencing depth and the amplicon sequencing error rate e (n), where n refers to the number of SNPs that differ between the candidate minor allele and the major genotype. If the number of supported sequencing sequences of the candidate allele exceeds a threshold value, the assumption that the allele is a major allele due to PCR or sequencing errors is overridden, and the true minor allele is judged. When there are multiple candidate minor alleles, multiple corrections are made for the P value of each candidate allele, with FDR <0.5% of the candidate alleles being considered true minor genotypes.
The statistical model relates to the parameter amplicon sequencing error rate e (n) which refers to the highest proportion of the number of sequences of the wrong allele carrying n SNPs to the total number of sequences of the marker. The present example obtains e in the statistical model by calculating the frequency of the detected minor genotypes in the MNP markers expected to be homozygous, i.e. the error rate of these genotypes max (n=1) and e max (n≥2)。
The calculation method is as follows:
the difference base number between the ith secondary genotype and the main genotype is ni, the number of the supported sequencing sequences is ci, and the error rate ei is the ratio of the number of sequencing sequences ci of the secondary genotype to the total number of sequencing sequences N of the marker, namely ei=ci/N;
calculating e at different sequencing depths max (n). This example summarizes the error rates of all the minor genotypes of all 930 homozygous MNP markers. When sequencing depth>＝1000X,e max (n=1) and e max (n is more than or equal to 2) is 1.03 percent and 0.0994 percent respectively.
In order to detect low frequency variation in a population of microorganisms, the sequencing depth needs to be up to 1000X or more. Then e is calculated under the probability guarantee of alpha=99.9999% based on BINOM.INV function max (n=1) and e max When (n.gtoreq.2) was 1.03% and 0.0994%, respectively, the number of sequences sequenced in the hypoisogenotypes was critical value in each marker, and the true hypoisogenotypes were judged only when the number of sequences in the hypoisogenotypes exceeded the critical value (Table 4). When multiple candidate minor alleles exist, multiple corrections are made to the P value of each candidate allele, FDR<0.5% of candidate alleles are judged to be true minor genotypes.
TABLE 4 critical values for determining the hypo-isogenotypes at partial sequencing depth
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According to the above parameters, DNAs of S-1 and S-4 strains having inter-strain variation 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 with the genotypes of MNP markers of two modified streptococcus pneumoniae, heterozygous genotype markers can be detected in 24 artificial heterozygous samples, and the applicability of the developed detection method for MNP markers of streptococcus pneumoniae in detecting genetic variation of strains is demonstrated.
Finally, it is also intended that the term "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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Sequence listing
<110> Jiang Handa science
<120> MNP labeling combination of Streptococcus pneumoniae, primer pair combination, kit and application
<130> 20210925
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<212> DNA
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ttggtctgaa atagccatgg cttcttcttg gtcgtgggtt acataaacag ttgtaattcc 60
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<210> 4
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tagacatgct tacagaggat gacttt 146
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<212> DNA
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acttttgtca aggttctgtc gcgtgtcata ggtaaaaaac tagatcagca aggtctggaa 60
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<210> 6
<211> 141
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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cctgcttcat cgtgtccatg tacttgttga tgattttaac ccaagtgtct gtatcacttg 60
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acaggttcat attgtatttc t 141
<210> 7
<211> 150
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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aaacaagctc cgaatatctc tccccttggt agctgcaccg atagacagac gcagattttc 60
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<210> 8
<211> 149
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
ttcacctcgt taatcaatcc tttgatgtca atctttccat ttggagttag tggcaaactg 60
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ttgatggcct tggtaatatc gatatctcg 149
<210> 9
<211> 150
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
agaattccaa gaattttgca aggatacggt cctagttgcc cacaatgcta cctttgacgt 60
tggctttatg aatgctaatt atgagcgtca tgatcttcca aagattagtc agccagttat 120
tgatacgctg gagtttgcta gaaacctcta 150
<210> 10
<211> 150
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
caaagtggaa gccttctgga ttttcaacca tggttccagc taccaaagct cgagtatctg 60
catctgtgat ttcttacttc ttatcggcca gtgccttgaa cttagcaaag agtggtttga 120
tatcctcttc tgtaaaatct agggccaatt 150
<210> 11
<211> 150
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
cactagggct ccgatgacaa tacttgcgat aaatagaagg acagttccag ggtttggagc 60
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ggctttgggc gcaatccaca taagcaagat 150
<210> 12
<211> 146
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
aggttctgaa tatgcaaata ctgtcactca aggtatcgta tccagtctca atagaaatgt 60
atccttaaaa tcggaagatg gacaagctat ttctacaaaa gccatccaaa ctgatactgc 120
tattaaccca ggtaactctg gcggcc 146
<210> 13
<211> 150
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
tgattcctca tcagcagtag caaagtgggt aaagattcct tcaacacaaa caccgtgttg 60
ttggagcaaa tcttgagcct gctcaacctc acttgcctct ctaaaaccaa tccgtcccat 120
ccctgaatca atcttgaggt ggactgtcaa 150
<210> 14
<211> 143
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
tcgctaatac catattagta tcctttcttt tatctacaca aagaataaca cacttatgtt 60
aaccctatat gaactttaat aaaaaactaa tctgtctaca agctaatctt aaattccata 120
ccgtgttcat aagggagaaa act 143
<210> 15
<211> 150
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
agtaccagta attcctttgg tttgagatca atttcttcat ttttataatg tgctttgtaa 60
gaggtaaaat ccactgttac atcctgatat cgccaaatat cctctatgac gtagtaacgt 120
ctgataagcg ccttaatacg cttgacaagc 150
<210> 16
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
cgtttctaca aagactggaa cctat 25
<210> 17
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
tcgctttgtt gctggtagc 19
<210> 18
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
ttggtctgaa atagccatgg c 21
<210> 19
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
tgttagcaga tggtattttg caatca 26
<210> 20
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
acttttgtca aggttctgtc gc 22
<210> 21
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
cctgcttcat cgtgtccatg ta 22
<210> 22
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
aaacaagctc cgaatatctc tccc 24
<210> 23
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
ttcacctcgt taatcaatcc tttga 25
<210> 24
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
agaattccaa gaattttgca aggat 25
<210> 25
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
caaagtggaa gccttctgga ttttc 25
<210> 26
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
cactagggct ccgatgacaa tac 23
<210> 27
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
aggttctgaa tatgcaaata ctgtc 25
<210> 28
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
tgattcctca tcagcagtag caa 23
<210> 29
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
tcgctaatac catattagta tcctttct 28
<210> 30
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
agtaccagta attcctttgg tttga 25
<210> 31
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
ggcaacaatg accgtattac aa 22
<210> 32
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
cagttgttgg caatcaaaga gc 22
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
gttctcctca tggacgagcc 20
<210> 34
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
aaagtcatcc tctgtaagca tgtct 25
<210> 35
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
caggtaaaac tcctgtttgg cag 23
<210> 36
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
agaaatacaa tatgaacctg tcttaccc 28
<210> 37
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
ttttcaaaag caaccctaac caaat 25
<210> 38
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
cgagatatcg atattaccaa ggcca 25
<210> 39
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
tagaggtttc tagcaaactc cagc 24
<210> 40
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
aattggccct agattttaca gaaga 25
<210> 41
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
atcttgctta tgtggattgc gc 22
<210> 42
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
ggccgccaga gttacctg 18
<210> 43
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
ttgacagtcc acctcaagat tgatt 25
<210> 44
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
agttttctcc cttatgaaca cggta 25
<210> 45
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
gcttgtcaag cgtattaagg cg 22
Claims (9)
1. The core MNP marker combination of streptococcus pneumoniae is characterized by comprising 15 markers, and the specific nucleotide sequences of the core MNP marker combination are shown as SEQ ID NO.1-SEQ ID NO. 15.
2. A multiplex PCR primer pair combination for detecting the streptococcus pneumoniae core MNP marker combination according to claim 1, wherein the multiplex PCR primer pair combination comprises 15 pairs of primers, the specific primer nucleotide sequences are shown in SEQ ID No.16-SEQ ID No. 45.
3. A test kit for detecting the streptococcus pneumoniae core MNP marker combination according to claim 1, wherein the kit comprises the primer pair combination according to claim 2.
4. The test kit of claim 3, wherein the kit further comprises a multiplex PCR premix.
5. Use of a core MNP marker combination of streptococcus pneumoniae according to claim 1 or a primer pair combination according to claim 2 or a detection kit according to any one of claims 3-4 for qualitative detection of streptococcus pneumoniae of non-diagnostic interest.
6. Use of a core MNP marker combination of streptococcus pneumoniae according to claim 1 or a primer pair combination according to claim 2 or a detection kit according to any one of claims 3-4 in the preparation of a qualitative detection product of streptococcus pneumoniae.
7. Use of a core MNP marker combination of streptococcus pneumoniae according to claim 1 or a primer pair combination according to claim 2 or a detection kit according to any one of claims 3-4 for the non-diagnostic detection of streptococcus pneumoniae micro-types.
8. Use of a core MNP marker combination of streptococcus pneumoniae according to claim 1 or a primer pair combination according to claim 2 or a detection kit according to any one of claims 3-4 for constructing a streptococcus pneumoniae database.
9. Use of a core MNP marker combination of streptococcus pneumoniae according to claim 1 or a primer pair combination according to claim 2 or a detection kit according to any one of claims 3-4 for the detection of genetic variation within and between streptococcus pneumoniae strains for non-diagnostic purposes.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6800744B1 (en) * | 1997-07-02 | 2004-10-05 | Genome Therapeutics Corporation | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| WO2011048104A1 (en) * | 2009-10-19 | 2011-04-28 | Azienda Ospedaliero-Universitaria Meyer | Method for streptococcus pneumoniae diagnosis and serotyping |
| CN112481408A (en) * | 2020-12-16 | 2021-03-12 | 武汉市农业科学院 | MNP core primer combination for molecular identification of eggplant DNA varieties and application thereof |
| CN112501343A (en) * | 2020-12-19 | 2021-03-16 | 中国农业科学院油料作物研究所 | MNP (MNP) marker primer combination for identifying rape varieties and substantive derived varieties thereof and application of MNP marker primer combination |
-
2021
- 2021-11-06 CN CN202111309341.3A patent/CN114277164B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6800744B1 (en) * | 1997-07-02 | 2004-10-05 | Genome Therapeutics Corporation | Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics |
| WO2011048104A1 (en) * | 2009-10-19 | 2011-04-28 | Azienda Ospedaliero-Universitaria Meyer | Method for streptococcus pneumoniae diagnosis and serotyping |
| CN112481408A (en) * | 2020-12-16 | 2021-03-12 | 武汉市农业科学院 | MNP core primer combination for molecular identification of eggplant DNA varieties and application thereof |
| CN112501343A (en) * | 2020-12-19 | 2021-03-16 | 中国农业科学院油料作物研究所 | MNP (MNP) marker primer combination for identifying rape varieties and substantive derived varieties thereof and application of MNP marker primer combination |
Non-Patent Citations (3)
| Title |
|---|
| Comparative genomic analysis of multidrug-resistant Streptococcus pneumoniae isolates;Fen Pan等;Infection and Drug Resistance;第11卷;659-670 * |
| DNA身份精准鉴定技术MNP标记法在蕲艾鉴定中的应用初探;张英等;2019中国针灸学会年会暨40周年回顾;摘要 * |
| Multiple nucleotide polymorphism DNA markers for the accurate evaluation of genetic variations;Zhiwei Fang等;BioRxiv;1-29 * |
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