CN115029454B - MNP (MNP) marking site of Moraxella catarrhalis, primer composition, kit and application of MNP marking site - Google Patents
MNP (MNP) marking site of Moraxella catarrhalis, primer composition, kit and application of MNP marking site Download PDFInfo
- Publication number
- CN115029454B CN115029454B CN202111355986.0A CN202111355986A CN115029454B CN 115029454 B CN115029454 B CN 115029454B CN 202111355986 A CN202111355986 A CN 202111355986A CN 115029454 B CN115029454 B CN 115029454B
- Authority
- CN
- China
- Prior art keywords
- mnp
- moraxella catarrhalis
- detection
- kit
- artificial sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B25/20—Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B30/10—Sequence alignment; Homology search
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics
- G16B50/30—Data warehousing; Computing architectures
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses MNP (MNP) marking sites of Moraxella catarrhalis, a primer composition, a kit and application thereof, wherein the MNP marking sites refer to genome regions which are screened on the genome of the Moraxella catarrhalis and are separated from other species and have a plurality of nucleotide polymorphisms in the species, and the marking sites comprise MNP-1-MNP-15; the primer is shown as SEQ ID NO. 1-SEQ ID NO. 30. The MNP marker locus can specifically identify the Moraxella catarrhalis 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 marking sites of multiple samples at one time; the kit has the advantages of high throughput, multiple targets, high sensitivity and culture-free detection, can be applied to identification and genetic variation detection of Moraxella catarrhalis in large-scale samples, and has important significance for scientific research and epidemic prevention monitoring of Moraxella catarrhalis.
Description
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to MNP (MNP) marking sites of Moraxella catarrhalis, a primer composition, a kit and application thereof.
Background
Moraxella catarrhalis (Moraxella catarrhalis, abbreviated as MC) was first discovered in 1896 and has been considered in the past to be an upper respiratory normal colonising bacteria that is non-pathogenic to humans. However, researches for more than 20 years have found that the bacterium can cause upper respiratory tract infection of children and old people, and also an important pathogenic bacterium causing lower respiratory tract infection of adults, is the 3 rd most common pathogenic bacterium of children's maxillary sinusitis, tympanitis, pneumonia and chronic lower respiratory tract infection of adults, is next to haemophilus influenzae and streptococcus pneumoniae, and the incidence rate of the bacterium is increasing year by year.
Moraxella catarrhalis is also becoming a common pathogenic microorganism in the laboratory. As a population organism, individuals in the population can undergo variation during unavoidable culture and passage. For laboratory studies, such undetectable variations can result in strains of the same name in different laboratories or different times in the same laboratory being virtually different, resulting in irreproducible and incomparable experimental results. Heterogeneity between human hela cell laboratories has resulted in a significant amount of incomparable experimental results and wasted data. Therefore, the development of the accurate, sensitive and mutation-monitoring detection and analysis method for the Moraxella catarrhalis has important significance for scientific research and monitoring of the Moraxella catarrhalis.
Laboratory diagnostic methods for Moraxella catarrhalis mainly include bacterial culture and identification, nucleic acid detection and serological tests, among which the most common are nucleic acid detection based on specific polymerase chain reaction (polymerase chain reaction, PCR). With the development of sequencing technology, whole genome and metagenome sequencing are also used in different sample assays. These existing techniques have one or more limitations in terms of isolation culture, length of operation, complexity of operation, throughput of detection, accuracy and sensitivity of detecting variations, cost, etc., which depend on the strain. 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 limitation of massive data waste and background noise caused by the separation culture of the whole genome depending on pathogenic bacteria and metagenome sequencing, and has the advantages of small sample requirement, accurate diagnosis result, data quantity conservation, low-frequency variation detection and culture-free. 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, developing a novel molecular marker of high polymorphism of Moraxella catarrhalis and a high-efficiency, accurate, sensitive and culture-free detection technology thereof becomes a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a specific MNP (MNP) marking site of Moraxella catarrhalis, a primer composition, a kit and application thereof, which can carry out qualitative identification and mutation detection on the Moraxella catarrhalis and have the effects of multiple targets, high flux, high sensitivity and fine typing.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect of the invention there is provided a MNP marker locus specific for Moraxella catarrhalis, the MNP marker locus being a genomic region screened on the Moraxella catarrhalis genome that is distinct from other species and has multiple nucleotide polymorphisms within the species, comprising the marker loci of MNP-1 to MNP-15 of the reference genome with CP 002005.
In the above technical scheme, the marking sites of MNP-1 to MNP-15 are specifically shown in the specification table 1, and the starting and ending positions of the MNP marks marked in the table 1 are determined based on the reference sequences corresponding to the same row of MNPs in the table 1.
In a second aspect of the present invention, there is provided a multiplex PCR primer composition for detecting the MNP marker loci, the multiplex PCR primer composition comprising 15 pairs of primers, the specific primer sequences being shown in SEQ ID NO.1 to SEQ ID NO. 30.
In the above technical solution, the primers of each MNP marker locus include an upper primer and a lower primer, and are specifically shown in table 1 of the specification.
In a third aspect of the invention, there is provided a detection kit for detecting the MNP marker locus of moraxella catarrhalis, 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-tagged site of Moraxella catarrhalis or said multiplex PCR primer composition or said detection kit for qualitative detection of Moraxella catarrhalis for non-diagnostic purposes and for preparing a qualitative detection product of Moraxella catarrhalis.
In a fifth aspect of the invention, there is provided the use of said MNP marker locus of moraxella catarrhalis or said multiplex PCR primer composition or said detection kit for detecting genetic variations within and between moraxella catarrhalis strains.
In a sixth aspect of the invention, there is provided the use of said MNP marker locus of moraxella catarrhalis or said multiplex PCR primer composition or said detection kit in the construction of a moraxella catarrhalis database.
In a seventh aspect of the invention, there is provided the use of said MNP marker locus of moraxella catarrhalis or said multiplex PCR primer composition or said detection kit in the accurate and finely divided detection of moraxella catarrhalis.
In the application, the specific operation steps are as follows: firstly, obtaining total bacterial DNA of a sample to be detected; performing a first round of multiplex PCR amplification on the total DNA and the blank control by using the kit, wherein the number of cycles is not higher than 25; purifying the amplified product, and then adding a sample tag and a second generation sequencing joint based on the second-round PCR amplification; quantifying after purifying the second round of amplification products; detecting a plurality of strains by mixing the amplification products of the second round in equal amounts and then performing high throughput sequencing; and comparing the sequencing result with the reference sequence of the Moraxella catarrhalis 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 sequences of Moraxella catarrhalis obtained in the total DNA and the blank control and the number of the detected MNP markers, and obtaining the number of the detected MNP markers, the number of the sequencing sequences covering each MNP marker and the MNP marker genotype data.
When the method is used for identifying the Moraxella catarrhalis, whether the sample to be detected contains nucleic acid of the Moraxella catarrhalis or not is judged after quality control according to the number of sequencing sequences of the Moraxella catarrhalis detected in the sample to be detected and the blank control and the number of MNP marks detected. The quality control scheme and the judging method are characterized in that DNA of the Moraxella catarrhalis with known copy number is taken as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting the Moraxella catarrhalis are evaluated, and the quality control scheme and the judging method when the kit detects the Moraxella catarrhalis are formulated.
When used in the detection of genetic variation of Moraxella catarrhalis, it includes the detection of genetic variation between strains and within strains. The detection of genetic variation among strains comprises the steps of obtaining genotype data of each strain to be compared in the MNP mark by using the kit and the method. And analyzing whether the main genotypes of the strains to be compared on the MNP markers are different or not through genotype comparison. If the strain to be compared has a variation in the major genotype of at least one MNP marker (i.e. one MNP marker has more than 50% of the genotypes supported by the sequencing fragments), then it is determined that there is a genetic variation in both. Alternatively, 15 sites of the strain to be compared may be amplified by single PCR, respectively, and then Sanger sequencing may be performed on the amplified products to obtain sequences, and then the genotypes of each MNP marker of the strain to be compared may be aligned. If MNP markers of non-identical major genotypes are present, variations are present between the strains to be compared. 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.
When the method is used for constructing the MNP fingerprint database of Moraxella catarrhalis, genotype data of the MNP marks of Moraxella catarrhalis identified from a sample are recorded into a database file to form the MNP fingerprint database of Moraxella catarrhalis; and when different samples are identified each time, comparing the samples with an MNP fingerprint database of the Moraxella catarrhalis, identifying whether the Moraxella catarrhalis in the samples has a main genotype difference with the strains in the database in the MNP marks, and recording the samples in the MNP fingerprint database, wherein the Moraxella catarrhalis with the main genotype difference in at least 1 MNP mark is a new variant.
When the method is used for parting the Moraxella catarrhalis, the Moraxella catarrhalis in the sample to be tested is identified, and the genotype of each MNP mark is obtained. And comparing the genome sequence of the Moraxella catarrhalis with a reference sequence library formed by the published genome sequence of the Moraxella catarrhalis and an MNP fingerprint database of the Moraxella catarrhalis, identifying whether the Moraxella catarrhalis in the sample is an existing strain or a new variant strain, and recording the MNP fingerprint of the new variant strain into the MNP fingerprint database. Therefore, the MNP fingerprint database can be continuously enriched by utilizing the primer combination.
The invention belongs to the first creation in the field of Moraxella catarrhalis, and is not reported in related literature; MNP markers are mainly developed based on reference sequences, and MNP markers which are large-scale and are distinguished from other species, polymorphic in the Moraxella catarrhalis species and conserved in sequence at two sides can be mined according to reported resequencing data of the Moraxella catarrhalis representative species; MNP marker detection primers suitable for multiplex PCR amplification can be designed through conserved sequences at two sides of MNP markers; and then a set of MNP mark with maximum polymorphism and high specificity and a set of primer combination with best compatibility can be screened out according to the test result of the standard substance.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
the invention provides MNP (MNP) marking sites of Moraxella catarrhalis, a primer composition, a kit and application thereof. The provided 15 MNP markers of the Moraxella catarrhalis and the primer combination thereof can be used for multiplex PCR amplification, and the amplification products are sequenced by fusing with a second generation sequencing platform, so that the requirements of high-throughput, high-efficiency, high-accuracy and high-sensitivity detection of the mycobacterium to be combined are met, and the requirements of the Moraxella catarrhalis standard and the sharable fingerprint data construction are met; the need to accurately detect genetic variation between strains of Moraxella catarrhalis; the identification of the homozygous and heterozygous requirements of Moraxella catarrhalis provides technical support for scientific research, monitoring and control of Moraxella catarrhalis.
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 MNP marker loci of Moraxella catarrhalis;
FIG. 3 is a flow chart of detection of MNP marker loci.
Detailed Description
The advantages and various effects of the embodiments of the present invention will be more clearly apparent from the following detailed description and examples. Those skilled in the art will appreciate that these specific implementations and examples are provided to illustrate, but not limit, examples of the present invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or may be prepared by existing methods.
The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:
the invention develops a novel molecular marker-MNP marker which is suitable for detecting group organisms and is specific to species. MNP markers refer to polymorphic markers caused by multiple nucleotides in a region of the genome. MNP markers have the following advantages over SSR markers and SNP markers: (1) Allele-rich, single MNP markers with 2 n Species alleles higher than SSR and SNP; (2) The species distinguishing capability is strong, the species identification can be realized by only a small amount of MNP marks, and the detection error rate is reduced. The MNP labeling method for detecting MNP labels based on the combination of super multiplex PCR and a second generation high throughput sequencing technology has the following advantages: (1) The output is a base sequence, a standardized database can be constructed for sharing without parallel experiments; (2) The method has high efficiency, breaks through the limitation of the number of sequencing samples by using the sample DNA bar code, and can type tens of thousands of MNP labels of hundreds of samples at one time; (3) High sensitivity, multiple targets are detected at one time by using multiple PCR, and high false negative and low sensitivity caused by single target amplification failure are avoided; (4) High accuracy, and sequencing the amplified product hundreds of times by using a second-generation high-throughput sequencer.
In view of the advantages and the characteristics, the MNP marking and the detection technology thereof can realize classification and tracing of the multi-allele types of the group organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, genetic variation detection and the like. At present, no report about MNP labeling exists in microorganisms, and corresponding technology is lacking. Thus, the present invention developed MNP marker loci for Moraxella catarrhalis that are genomic regions screened on the Moraxella catarrhalis genome that are distinct from other species and have multiple nucleotide polymorphisms within the species, including the marker loci for MNP-1-MNP-15 of the reference genome with CP 002005.
Next, the present invention has developed a multiplex PCR primer composition for detecting MNP marker loci of Moraxella catarrhalis, characterized in that the multiplex PCR primer composition comprises 15 pairs of primers, the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID NO.1 to SEQ ID NO. 30. The primers do not collide with each other, and efficient amplification can be performed by multiplex PCR.
The multiplex PCR primer composition can be used as a detection kit for detecting the MNP labeling site of Moraxella catarrhalis.
The kit of the invention can accurately and sensitively detect the catarrhal Moraxella with the concentration as low as 10 copies/reaction.
The MNP marker and the kit have high specificity in detecting the Moraxella catarrhalis in a complex template.
The MNP marker loci, primer compositions, kits and uses thereof of one of the Moraxella catarrhalis of the present application will be described in detail below in conjunction with examples, comparative examples and experimental data.
Example 1 screening of MNP marker loci of Moraxella catarrhalis and design of multiplex PCR amplification primers
S1, screening MNP (MNP) marker locus of Moraxella catarrhalis
Based on complete or partial sequences of genomes of 898 different isolates of Moraxella catarrhalis disclosed on the network, 15 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 15 MNP marker loci screened are shown in table 1:
TABLE 1 MNP marker loci and detection primers starting position on the reference sequence
The step S1 specifically includes:
selecting a genome sequence of a representative strain of the Moraxella catarrhalis as a reference genome, and comparing the genome sequence with the reference genome to obtain single nucleic acid polymorphism sites of each strain of the Moraxella catarrhalis;
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 (MNP) marking areas, wherein the candidate MNP marking 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 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 sites are 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 detected sites.
DNA of a commercially available Moraxella catarrhalis standard strain ATCC 25240 (product number: CL 1231) was used, and after quantitative analysis by digital PCR, the DNA was added to human genomic DNA (2 ng/reaction) to prepare 1000 copies/reaction templates, and by using the above-mentioned primer combinations, a detection primer combination having high species discrimination, uniform amplification and optimal compatibility of the species-specific MNP markers was screened according to the detection conditions of the MNP markers in the 4 libraries, and finally, 15 MNP markers and detection marker primer combinations as described in Table 1 of the present invention were screened.
Threshold settings and Performance assessment for MNP markers and primers identification of Moraxella catarrhalis described in example 2
In this example, 1 copy/reaction, 10 copy/reaction and 100 copy/reaction of Moraxella catarrhalis simulated samples were prepared using DNA of the Moraxella catarrhalis standard strain ATCC 25240 (product number: CL 1231) having a known copy number, quantified by digital PCR, and added to human genomic DNA. An equal volume of sterile water was set at the same time as a blank. A total of 4 samples, each of which was constructed as 3 replicate libraries per day, were tested continuously for 4 days, i.e., 12 sets of sequencing data were obtained per sample, as shown in table 2. 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 Moraxella catarrhalis MNP markers detected in the blank control and Moraxella catarrhalis nucleic acid standard in 12 repeated experiments, and formulating thresholds for quality control system pollution and target pathogen detection. The detection flow of MNP markers is shown in fig. 3.
1. Sensitivity and stability analysis for MNP (MNP) markers and kit detection of Moraxella catarrhalis
As shown in Table 2, the kit can stably detect more than 7 MNP markers in a 10-copy/reaction sample, and can detect at most 1 MNP marker in a 0-copy/reaction sample, and the kit 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 Moraxella catarrhalis
2. Reproducibility and accuracy assessment of MNP markers and kits for detection of Moraxella catarrhalis
Based on whether the genotype of the co-detected site is reproducible in the two repetitions, the reproducibility and accuracy of detection of Moraxella catarrhalis by the MNP marker detection method are evaluated. Specifically, the paired comparison was performed on 12 sets of data of 100 copies/reaction sample, respectively, and the results are shown in table 3.
TABLE 3 reproducibility and accuracy assessment of Moraxella catarrhalis MNP marker detection method
As can be seen from Table 3, the number of MNP markers having a difference in the main genotypes was 0; according to the principle that the reproducible genotypes are considered to be accurate between 2 repeated experiments, the accuracy a=1- (1-r)/2=0.5+0.5r, and r represents the reproducibility, namely the ratio of the reproducible site number of the main genotype to the common site number. In the project reproducibility test, the difference logarithm of MNP marking main genotypes among different libraries and different library construction batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%. Thus, the kit of the invention is capable of accurately detecting as low as 10 copies/reaction of Moraxella catarrhalis.
3. Threshold determination of MNP markers and kit detection of Moraxella catarrhalis
As shown in Table 2, the sequences aligned to Moraxella catarrhalis could be detected in 1 copy/reaction samples, covering at least 1 MNP tag. The sequences of Moraxella catarrhalis were also detected in the partial blank. Because of the extreme sensitivity of MNP marker detection methods, contamination of the data in the detection is prone to false positives. Therefore, the quality control scheme is formulated in this example, and is specifically as follows:
1) The amount of sequencing data is greater than 4.5 megabases. The measurement and calculation basis is that the number of MNP markers 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 site reaches 1000 times by one experiment, and the accurate analysis of the base sequence of each MNP marker is ensured.
2) Determining whether the contamination is acceptable based on the signal index S of moraxella catarrhalis in the test sample and the noise index P of moraxella catarrhalis 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 number of sequenced fragments of moraxella catarrhalis, 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 total number of sequenced fragments of moraxella catarrhalis, 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 Moraxella catarrhalis in samples to be tested
As a result, as shown in Table 4, the average value of the noise index of Moraxella catarrhalis in the control was 0.04%, the average value of the signal index in the 1-copy/reaction sample was 0.22%, and the average value of the signal-to-noise ratio of the 1-copy/reaction sample and the control was 6.00, and therefore, the present invention provides that when the signal-to-noise ratio is more than 10 times, it can be judged that the contamination in the detection system is acceptable.
The average signal to noise ratio at 10 copies/reaction and the blank was 72.52 with a minimum of 52.50; at least 7 MNP markers were stably detected in the 10 copies/reaction 15 sets of data, accounting for 43.8% of the total sites. Therefore, under the condition of ensuring accuracy, the standard prescribes that the positive judgment standard of the Moraxella catarrhalis is as follows: when the signal-to-noise ratio of the Moraxella catarrhalis in the sample is greater than 52.5 and the site detection rate is greater than or equal to 40%, determining that the nucleic acid of the Moraxella catarrhalis is detected in the sample.
Therefore, the kit provided by the invention can accurately and sensitively detect the catarrhal Moraxella with the concentration as low as 10 copies/reaction.
4. Specific evaluation for detecting Moraxella catarrhalis by MNP (MNP) mark detection method
The DNA of Moraxella catarrhalis, mycobacterium tuberculosis, acinetobacter strain, pertussis baud bacteria, huo Shibao termates, chlamydia pneumoniae, mycoplasma pneumoniae, EB virus, haemophilus influenzae, varicella zoster virus, cytomegalovirus, herpes simplex virus, human bocavirus, klebsiella pneumoniae, legionella, pseudomonas aeruginosa, rickettsia, staphylococcus aureus, streptococcus pneumoniae and Streptococcus pyogenes are mixed together according to the equimolar amount to prepare a mixed template, and the equal volume of sterile water is used as a blank control, and the method provided by the invention is adopted to detect Moraxella catarrhalis in the mixed template and the blank control, so that 3 repeated experiments are carried out. Through sequence comparison, 15 MNP markers of the Moraxella catarrhalis can be specifically detected in 3 repeated experiments; after analysis according to the quality control scheme and the judgment threshold, the nucleic acid of the Moraxella catarrhalis is judged to be positive in 3 repeated experiments, which indicates that the MNP mark and the kit detect the high specificity of the Moraxella catarrhalis in complex templates.
Example 3 detection of genetic variation between Moraxella catarrhalis strains
The detection of 1 part of Moraxella catarrhalis strains provided by the Hubei province disease prevention control center and 6 parts of offspring strains preserved in different periods is carried out by using the kit and the MNP marking site detection method, samples are sequentially named as S1-S6, the average coverage of sequencing of each sample is 1416 times, and all 15 MNP marks can be detected by each strain (Table 5). The fingerprint of 6 strains was aligned pairwise, and the results are shown in Table 5, where 1 part (S-2) and 5 parts of Moraxella catarrhalis detected together in the same lot had major genotype differences at 2 MNP markers (Table 5), indicating the presence of inter-strain variation.
TABLE 5 detection analysis of 6 Moraxella catarrhalis
As can be seen from Table 5, the application of the kit of the invention in identifying genetic variation among strains by detecting MNP markers can be used for ensuring the genetic consistency of the same named Moraxella catarrhalis strains in different laboratories, thereby ensuring the comparability of research results, and having great significance to scientific research of Moraxella catarrhalis. In clinical terms, one can take into account the diagnostic regimen as to whether the site of the difference affects resistance.
Example 4 detection of genetic variation inside Moraxella catarrhalis strains
As a group organism, the individuals in the Moraxella catarrhalis group are mutated so that the group is no longer homozygous to form a heterogeneous heterozygous group, and the stability and consistency of the phenotype of the microorganism for 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.
Genotypes without strand preference were judged for authenticity based on the number and proportion of sequenced sequences in table 6. Table 6 lists the calculated probabilities at a=99.9999% based on the binom. Inv functionE under the guarantee of the rate 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 markers.
TABLE 6-threshold for determining the hypo-isogenotypes at partial sequencing depth
According to the above parameters, nucleic acids of two strains S1 and S2 having a difference in genotype were mixed in the following 8 ratios of 1/1000,3/1000,5/1000,7/1000,1/100,3/100,5/100,7/100, and artificial heterozygous samples were prepared, each sample was tested 3 times for repetition, and 24 sequencing data were obtained in total. Through the accurate comparison with the MNP marked genotypes of the two strains, the loci with heterozygous genotypes are detected in 24 artificial heterozygous samples, and the applicability of the developed MNP marked detection method of the Moraxella catarrhalis in detecting genetic variation inside a strain population is demonstrated.
Example 5 construction of Moraxella catarrhalis DNA fingerprint database
All strains or DNA of samples used for constructing a Moraxella catarrhalis MNP 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.
And (3) comparing the sequencing data of the 6 strains with the reference genotype, and obtaining the main genotype of each site of each strain to form the MNP fingerprint of each strain. And recording the obtained MNP fingerprint of each strain into a database file to form a Moraxella catarrhalis DNA fingerprint database.
The constructed MNP fingerprint database is based on the gene sequence of the detected strain, is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed and shared and being updated at any time. And comparing the MNP fingerprint of the strain obtained by each detection with an MNP fingerprint database constructed based on the existing genome data, and inputting the MNP fingerprint of the strain with the main genotype difference into the constructed MNP fingerprint database to achieve real-time updating and co-construction sharing of the database.
Example 6 use in the fine subdivision of Moraxella catarrhalis
Firstly, constructing a reference sequence library of Moraxella catarrhalis, which consists of a published genome sequence of Moraxella catarrhalis and a constructed fingerprint database of Moraxella catarrhalis; obtaining MNP fingerprints of Moraxella catarrhalis in each sample to be detected by using the primer combination and MNP marker locus detection method described in example 2; comparing the DNA fingerprint of each strain with a constructed reference sequence library, wherein the DNA fingerprint is 100% identical to the genotype of the existing strain, and is a new variant strain with main genotype difference in at least one MNP mark, so that the precise typing of Moraxella catarrhalis is realized. By comparison, 6 strains tested in Table 5 can be classified into 2 types, wherein 5 strains with the same genotype are consistent with ATCC 25240 strains, and S-2 strains and ATCC 25240 strains have the main genotype difference at 2 MNP markers, and are judged as new variant strains. Therefore, the kit and MNP mark provided by the invention can be used for subtly typing Moraxella catarrhalis to a single base level.
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
<110> Jiang Handa science
<120> MNP labeling site of Moraxella catarrhalis, primer composition, kit and application thereof
<160> 30
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tgatactgtc attagtgatg atgtga 26
<210> 2
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
accgcaccac caatcagg 18
<210> 3
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
cccaaacaac ctgtaacaat cactt 25
<210> 4
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
tcaagttgcc aataattatg atggtg 26
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
tcctcaccca atcgccaaaa tag 23
<210> 6
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
cgattggcta aaacccatgc c 21
<210> 7
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
ctggcacaat cgcacgatt 19
<210> 8
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
agccaagtca agccagtatt ga 22
<210> 9
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
aaaagccaac cttgccaaat ccatg 25
<210> 10
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
acagtccaag cagcactttt aga 23
<210> 11
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
gctgaagcta cctgccaaaa tta 23
<210> 12
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
ctggcgtcat cagccattca ta 22
<210> 13
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
acaaacatgg gagctataaa tgaca 25
<210> 14
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
gataagacgg ctttgcatga caag 24
<210> 15
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
atatccatgc caagcaccat actg 24
<210> 16
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
tgagcttaag tgtttcggca tttag 25
<210> 17
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
gatggatttg ccttggttgc acaa 24
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
cctctggtac aaccgctcct 20
<210> 19
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
atggttgctt taaatacctc tgagt 25
<210> 20
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
gtttcgggca aaagaccatg tg 22
<210> 21
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
aaacaatcgt tataaataac tgtgtcg 27
<210> 22
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
aataggtcaa taaaatgatg atcataatag ac 32
<210> 23
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
cccaaaaagc aggcattgaa gataa 25
<210> 24
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
acctgggata ttcaccagtt tttct 25
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
ccttggtgtc tttggcgttg 20
<210> 26
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
ccaatccaga tatcgccatg gtag 24
<210> 27
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
tatgccaaga ggttgcccaa aaat 24
<210> 28
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
gagcttatgg gctatagtac tgaca 25
<210> 29
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
cacggtgcat ggcagaga 18
<210> 30
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
taatgcaggc tgtcataatg ttgac 25
Claims (7)
1. The multiplex PCR primer composition for detecting Moraxella catarrhalis is characterized by comprising 15 pairs of primers, wherein the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID NO. 1-SEQ ID NO. 30.
2. A test kit for detecting moraxella catarrhalis, 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 a primer composition according to claim 1 or a detection kit according to any one of claims 2-3 for qualitative detection of a non-diagnostic purpose of a moraxella catarrhalis and for the preparation of a qualitative detection product of a moraxella catarrhalis.
5. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for detecting genetic variations within and between strains of moraxella catarrhalis.
6. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2-3 for the construction of a moraxella catarrhalis database.
7. Use of the primer composition of claim 1 or the detection kit of any one of claims 2 to 3 in the finely divided detection of moraxella catarrhalis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111355986.0A CN115029454B (en) | 2021-11-16 | 2021-11-16 | MNP (MNP) marking site of Moraxella catarrhalis, primer composition, kit and application of MNP marking site |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111355986.0A CN115029454B (en) | 2021-11-16 | 2021-11-16 | MNP (MNP) marking site of Moraxella catarrhalis, primer composition, kit and application of MNP marking site |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115029454A CN115029454A (en) | 2022-09-09 |
| CN115029454B true CN115029454B (en) | 2023-06-20 |
Family
ID=83117707
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111355986.0A Active CN115029454B (en) | 2021-11-16 | 2021-11-16 | MNP (MNP) marking site of Moraxella catarrhalis, primer composition, kit and application of MNP marking site |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115029454B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004104170A2 (en) * | 2003-05-13 | 2004-12-02 | The Research Foundation Of State University Of New York | Tools for detecting moraxella catarrhalis. |
| CN109439780A (en) * | 2018-12-25 | 2019-03-08 | 江苏和创生物科技有限公司 | Moraxelle catarrhalis fluorescence PCR detection reagent kit |
| CN112481401A (en) * | 2020-12-25 | 2021-03-12 | 郑州安图生物工程股份有限公司 | Kit for simultaneously detecting streptococcus pneumoniae, legionella pneumophila and moraxella catarrhalis |
-
2021
- 2021-11-16 CN CN202111355986.0A patent/CN115029454B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004104170A2 (en) * | 2003-05-13 | 2004-12-02 | The Research Foundation Of State University Of New York | Tools for detecting moraxella catarrhalis. |
| CN109439780A (en) * | 2018-12-25 | 2019-03-08 | 江苏和创生物科技有限公司 | Moraxelle catarrhalis fluorescence PCR detection reagent kit |
| CN112481401A (en) * | 2020-12-25 | 2021-03-12 | 郑州安图生物工程股份有限公司 | Kit for simultaneously detecting streptococcus pneumoniae, legionella pneumophila and moraxella catarrhalis |
Non-Patent Citations (3)
| Title |
|---|
| 儿童呼吸道卡他莫拉菌分离株耐药性与BRO基因分型研究;王频佳;谢成彬;吴雨露;;临床儿科杂志(第08期);719-722 * |
| 卡他莫拉菌的检测及药物敏感性;胡付品,朱德妹,李胜利,吴湜,张婴元;中国抗感染化疗杂志(第04期);218-221 * |
| 卡他莫拉菌耐药性与β内酰胺酶分型;王艳,张美和,沈叙庄,袁林,杨永弘;中华检验医学杂志(第06期);645-647 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115029454A (en) | 2022-09-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113718057B (en) | MNP (MNP) marking site of EB (Epstein-Barr) virus, primer composition, kit and application | |
| CN113862384B (en) | MNP (MNP) marking site of Francisella tularensis, primer composition, kit and application | |
| CN115029454B (en) | MNP (MNP) marking site of Moraxella catarrhalis, primer composition, kit and application of MNP marking site | |
| CN114214435B (en) | MNP (MNP) labeling combination of mycoplasma pneumoniae, primer pair combination, kit and application of MNP labeling combination | |
| CN114790489B (en) | MNP (MNP) marking site of haemophilus influenzae, primer composition, kit and application of MNP marking site | |
| CN115029452B (en) | MNP (MNP) marking site of Legionella, primer composition, kit and application of MNP marking site | |
| CN114790488B (en) | MNP (MNP) marking site of staphylococcus aureus, primer composition, kit and application of MNP marking site | |
| CN115029453B (en) | MNP (MNP) marking site of streptococcus pyogenes, primer composition, kit and application of MNP marking site | |
| CN114107525B (en) | MNP (MNP) marking site of pseudomonas aeruginosa, primer composition, kit and application of MNP marking site | |
| CN114836574B (en) | MNP (MNP) marking site of mumps virus, primer composition, kit and application of MNP marking site | |
| CN114836572B (en) | MNP (MNP) marker locus of paraenterovirus, primer composition, kit and application of MNP marker locus | |
| CN114836550B (en) | MNP (MNP) marking site of klebsiella pneumoniae, primer composition, kit and application of MNP marking site | |
| CN114107562B (en) | MNP (human parainfluenza virus) marker locus, primer composition, kit and application of MNP marker locus | |
| CN114790487B (en) | MNP (MNP) marking site of Huo Shibao terylen, primer composition, kit and application of MNP marking site | |
| CN115029477B (en) | MNP (MNP-associated protein) marker locus of human rhinovirus, primer composition, kit and application of MNP marker locus | |
| CN113862383B (en) | MNP (MNP) marking site of bacillus subtilis, primer composition and application of MNP marking site | |
| CN114836573B (en) | MNP (MNP) marking site of measles virus, primer composition, kit and application of MNP marking site | |
| CN115029479B (en) | MNP (MNP) marking site of Zika virus, primer composition, kit and application of MNP marking site | |
| CN114790494B (en) | MNP (varicella-zoster virus) marking site, primer composition, kit and application thereof | |
| CN114277163B (en) | MNP (MNP) labeling combination of chlamydia pneumoniae, primer pair combination, kit and application of kit | |
| CN114790485B (en) | MNP (MNP) marking site of Acinetobacter genus, primer composition, kit and application of MNP marking site | |
| CN114107563B (en) | MNP (human metapneumovirus) marker locus, primer composition, kit and application of MNP marker locus | |
| CN114015793B (en) | MNP (MNP) marking site of rickettsia, primer composition, kit and application of MNP marking site | |
| CN114790486B (en) | MNP (MNP) marking site of bacillus anthracis, primer composition, kit and application of MNP marking site | |
| CN114277162B (en) | MNP (MNP) labeling combination of mycobacterium tuberculosis, primer pair combination, kit and application of kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |