CN114790485A - MNP (protein marker P) marker locus of acinetobacter, primer composition, kit and application of MNP marker locus - Google Patents

MNP (protein marker P) marker locus of acinetobacter, primer composition, kit and application of MNP marker locus Download PDF

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CN114790485A
CN114790485A CN202111300927.3A CN202111300927A CN114790485A CN 114790485 A CN114790485 A CN 114790485A CN 202111300927 A CN202111300927 A CN 202111300927A CN 114790485 A CN114790485 A CN 114790485A
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方治伟
高利芬
陈利红
肖华锋
周俊飞
彭海
李论
李甜甜
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Abstract

The invention discloses an MNP marker locus of acinetobacter, a primer composition, a kit and application thereof, wherein the MNP marker locus refers to a genome region which is screened on the genome of an acinetobacter strain, is distinguished from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marker loci of MNP-1-MNP-15; the primer is shown as SEQ ID NO. 1-SEQ ID NO. 30. The MNP marker site can specifically identify the Acinetobacter genus and finely distinguish different species in the genus; the primers are not interfered with each other, and the multiplex amplification and sequencing technology is integrated, so that sequence analysis can be performed on all marked sites of a plurality of samples at one time, the primers have the advantages of high throughput, multiple targets, high sensitivity and no culture, can be applied to identification and genetic variation detection of acinetobacter in large-scale samples, and have important significance on scientific research and epidemic prevention monitoring of acinetobacter.

Description

MNP (protein marker P) marker locus of acinetobacter, primer composition, kit and application of MNP marker locus
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to an MNP (MNP) marker locus of acinetobacter, a primer composition, a kit and application thereof.
Background
Acinetobacter (Acinetobacter) is widely distributed in the external environment and has extremely strong adhesion, and known Acinetobacter includes 6 species, i.e., Acinetobacter calcoaceticus (a. calco aceticus), Acinetobacter xylinum (a. lwoffi), Acinetobacter baumannii (a. baumanii), Acinetobacter haemolyticus (a. haemolytius), Acinetobacter junii (a. junii), and Acinetobacter johnsonii (a. johnsonii). The acinetobacter is easy to adhere to various medical materials and becomes a bacteria storage source. Contaminated medical instruments and staff hands are important transmission media in hospitals. The susceptible patients are elderly patients, premature infants and newborn infants, patients with operative trauma, severe burn, tracheotomy or intubation, patients with artificial respirator, venous catheter and peritoneal dialysis, patients with broad-spectrum antibacterial drugs or immunosuppressant, etc. In addition, acinetobacter is also present in healthy human skin (25%), pharynx (7%), and also in conjunctival, saliva, gastrointestinal and vaginal secretions. The source of infection may be the patient himself (endogenous infection) or an acinetobacter infected or bacteria-carrying person, in particular a medical person carrying bacteria from both hands. The propagation paths include contact propagation and air propagation. The incidence of pneumonia is about 3% to 5% among those using ventilators. Bacteremia is the most serious clinical type of acinetobacter infection, and the fatality rate reaches more than 30%.
Classical detection methods for acinetobacter include isolation culture, serological analysis, PCR techniques, whole and metagenome sequencing, etc., and have one or more limitations in terms of duration, complexity of operation, detection throughput, accuracy and sensitivity of detection variation, cost, etc. The targeted molecular marker detection technology combining the ultra-multiplex PCR amplification and the high-throughput sequencing can be used for enriching target microorganisms in a sample with low microorganism content in a targeted manner, avoids a large amount of data waste and background noise caused by whole genome and metagenome sequencing, and has the advantages of less sample requirement, accurate diagnosis result, data quantity saving and low-frequency variation detection.
The molecular markers detected by the existing targeted detection technology mainly comprise SNP (single nucleotide polymorphism) markers and SSR (simple sequence repeat) markers. SSR markers are generally accepted as the most polymorphic markers, but are few in microorganisms; SNP markers are large in number, densely distributed, and polymorphic, single SNP markers are insufficient to capture the potential allelic diversity in a microbial population.
Therefore, development of novel molecular markers with high polymorphism of pathogenic microorganism acinetobacter and detection techniques thereof have become an urgent technical problem to be solved.
Disclosure of Invention
The invention aims to provide an MNP (MNP) marker locus of acinetobacter, a primer composition, a kit and application thereof, which can perform qualitative identification and mutation detection on the acinetobacter and have the effects of multiple targets, high flux, high sensitivity and fine typing.
In order to realize the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided an MNP marker site of Acinetobacter, said MNP marker site being a genomic region specific to a species selected on the genome of Acinetobacter and having a plurality of nucleotide polymorphisms within the species, including marker sites of MNP-1-MNP-15 on the CP009257 genome.
In the technical scheme, the marking sites of MNP-1-MNP-15 are specifically shown in the specification of Table 1, and the starting and ending positions of the MNP marks marked in the Table 1 are determined based on a CP009257 sequence.
In a second aspect of the present invention, a multiplex PCR primer composition for detecting said MNP marker locus is provided, said multiplex PCR primer composition comprising 15 pairs of primers, the nucleotide sequences of said 15 pairs of primers are shown in SEQ ID No.1 to SEQ ID No. 30.
In the above technical scheme, the primer of each MNP marker site includes an upper primer and a lower primer, which are specifically shown in table 1 of the specification.
In a third aspect of the invention, a detection kit for detecting the MNP marker site of the Acinetobacter is provided, and the kit comprises the primer composition.
Further, the kit further comprises a multiplex PCR premix.
In the fourth aspect of the invention, the application of said MNP marker site of acinetobacter or said multiplex PCR primer composition or said detection kit in qualitative detection of acinetobacter and preparation of detection products of acinetobacter is provided.
In the fifth aspect of the invention, the application of said MNP marker site of Acinetobacter or said multiplex PCR primer composition or said detection kit in the detection of genetic variation within and among Acinetobacter strains is provided.
In the sixth aspect of the invention, the application of the MNP marker site of the acinetobacter, the multiplex PCR primer composition or the detection kit in constructing the acinetobacter database is provided.
In the seventh aspect of the invention, the MNP marker site of the acinetobacter genus or the multiplex PCR primer composition or the detection kit is provided for use in the fine typing detection of the acinetobacter genus.
In the application, firstly, the total bacterial DNA of a sample to be detected is obtained; carrying out first round of multiplex PCR amplification on the total DNA and a blank control by using the kit disclosed by the invention, wherein the cycle number is not higher than 25; after purifying the amplification product, adding a label and a second-generation sequencing joint of the sample through a second round of PCR amplification; purifying and quantifying the second round amplification product; when a plurality of strains are detected, performing high-throughput sequencing by equivalently mixing the second round amplification products; and aligning the sequencing result to the reference sequence of the acinetobacter to obtain the number and genotype data of the detected sequences in the total DNA. And performing data quality control and data analysis on the sequencing data of the total DNA according to the quantity of the obtained sequencing sequences of the Acinetobacter and the number of detected MNP sites of the blank control, so as to obtain the number of the detected MNP sites, the number of the sequencing sequences covering each MNP site and the genotype data of the MNP sites.
When the method is used for identifying the acinetobacter, whether the nucleic acid of the acinetobacter is contained in the sample to be detected or not is judged after quality control is carried out according to the quantity of sequencing sequences of the acinetobacter detected in the sample to be detected and a blank control and the quantity of MNP sites detected. The quality control scheme and the determination method are characterized in that DNA of the acinetobacter strain with known copy number is used as a detection sample, the sensitivity, the accuracy and the specificity of the kit for detecting the acinetobacter are evaluated, and the quality control scheme and the determination method are formulated when the kit is used for detecting the acinetobacter.
When used for detection of genetic variation in Acinetobacter, the detection of genetic variation between strains and within strains is included. The detection of genetic variation among strains comprises the steps of obtaining genotype data of each strain to be compared at 15 MNP sites by using the kit and the method. And analyzing whether the main genotypes of the strains to be compared on the 15 MNP sites are different or not by genotype comparison. If the strains to be compared have variation in the major genotype of at least one MNP site, the strains are judged to have genetic variation. As an alternative, 15 sites of the strains to be compared can be amplified respectively through single PCR, and then the amplification products are subjected to Sanger sequencing, and after the sequences are obtained, the genotypes of each MNP site of the strains to be compared are compared. If there are MNP sites that are inconsistent with the major genotype, there is variation between the strains to be compared. When the genetic variation in the strain is detected, whether a minor genotype other than the major genotype is detected at the MNP site of the strain to be detected is determined by a statistical model. If the to-be-detected strain has a minor genotype at least one MNP site, determining that genetic variation exists in the to-be-detected strain.
When the method is used for constructing the DNA fingerprint database of the acinetobacter, the genotype data of the MNP locus of the acinetobacter identified from the sample is recorded into a database file to form the DNA fingerprint database of the acinetobacter; and (2) when different samples are identified, comparing the different samples with the DNA fingerprint database of the acinetobacter to identify whether the acinetobacter in the samples has a main genotype difference (more than 50 percent of genotypes supported by sequencing fragments at one MNP site) with the strains in the database at the MNP site, wherein the acinetobacter with the main genotype difference at least 1 MNP site is a new variant type, and collecting the new variant type into the DNA fingerprint database. Therefore, the DNA fingerprint database can be continuously enriched by utilizing the primer combination.
When the method is used for typing the acinetobacter, identifying the acinetobacter in a sample to be tested to obtain the genotype of each MNP site; collecting genome sequences of acinetobacter disclosed on the network and a constructed acinetobacter DNA fingerprint database to form an acinetobacter reference sequence database; comparing the genotype of the acinetobacter in the sample to be detected with the reference sequence library of the acinetobacter, screening genetically identical or closest strains, and obtaining the typing of the acinetobacter strains in the sample to be detected. And identifying the existing type or the new variant of the acinetobacter strain in the sample according to the comparison result with the reference sequence library, thereby realizing the fine typing of the acinetobacter.
The invention belongs to the initiative in the field of acinetobacter and is not reported in related documents; the MNP marker is mainly mined based on reported retesting data of the acinetobacter representative microspecies, and MNP marker sites which are specific to the genus and have high discrimination on various microspecies of the acinetobacter are searched; the sequences at two sides of the MNP marker are highly conserved among various small species of the acinetobacter, and the conserved region is used for designing a multiple PCR amplification primer; and then according to the test result of the standard product, a set of MNP sites with the largest polymorphism and high specificity, a primer combination with the best compatibility and a detection kit are screened.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the invention provides an MNP marker locus of acinetobacter, a primer composition, a kit and application thereof. The provided 15 MNP sites of acinetobacter and the primer combination thereof can carry out multiplex PCR amplification, and are fused with a second-generation sequencing platform to carry out sequencing on an amplification product, thereby meeting the requirements of carrying out high-throughput, high-efficiency, high-accuracy and high-sensitivity detection on acinetobacter and the requirement of accurately detecting genetic variation among acinetobacter strains; the requirement of identifying the degeneration of the acinetobacter population is met; the method meets the requirement of shareable fingerprint data construction of the standard of the acinetobacter, and provides technical support for scientific research, scientific monitoring and prevention and treatment of the acinetobacter.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of MNP marker polymorphism;
FIG. 2 is a flow chart of the screening and primer design of the MNP marker site of Acinetobacter;
FIG. 3 is a flow chart of detection of MNP marker sites.
Detailed Description
The embodiments of the present invention will be specifically explained below with reference to specific embodiments and examples, and the advantages and various effects of the embodiments of the present invention will be more clearly presented thereby. It will be understood by those skilled in the art that the present embodiments and examples are illustrative of the present invention and are not to be construed as limiting the present invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or can be prepared by an existing method.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
and screening MNP markers suitable for detecting the population organisms as detection targets. MNP markers refer to polymorphic markers arising from multiple nucleotides within a region of the genome. Compared with SSR markers and SNP markers, MNP markers have the following advantages: (1) abundant alleles, 2 at a single MNP locus n Species alleles, higher than SSR and SNP, are suitable for detection of microorganisms, a typical population of organisms; (2) the species distinguishing capability is strong, species identification can be realized only by a small amount of MNP marks, and the detection error rate is reduced. The MNP labeling method for detecting the MNP label is fused with the ultra-multiplex PCR and the second-generation high-throughput sequencing technology, and has the following advantages: (1) the output is a base sequence, and a standardized database can be constructed for sharing without parallel experiments; (2) the method has high efficiency, breaks through the limitation of the quantity of sequencing samples by using the DNA barcodes of the samples, and can type tens of thousands of MNP sites of hundreds of samples at one time; (3) the sensitivity is high, multiple targets are detected at one time by utilizing multiple PCR, and high false negative and low sensitivity caused by amplification failure of a single target are avoided; (4) high accuracy, using the second generation high throughput sequencer to sequence the amplification product hundreds of times.
In view of the advantages and characteristics, the MNP marker and the detection technology thereof, namely the MNP marking method, can realize the classification and tracing of the multi-allelic genotypes of the population organisms, and have application potential in the aspects of identification of pathogenic microorganisms, fingerprint database construction, genetic variation detection and the like. At present, no report on MNP labeling exists in microorganisms, and corresponding technologies are lacked. The development, screening and application of the MNP marking method have better application foundation in plants.
Therefore, the invention develops MNP marker sites of acinetobacter, the MNP marker sites are genome regions which are screened on the genome of acinetobacter and are distinguished from other species and have a plurality of nucleotide polymorphisms in the species, including the marker sites of MNP-1-MNP-15 on the CP009257 genome.
Then, the invention develops a multiplex PCR primer composition for detecting the MNP marker locus of the acinetobacter, wherein the multiplex PCR primer composition comprises 15 pairs of primers, and the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID NO. 1-SEQ ID NO. 30. The primers are not conflicted with each other, and can be efficiently amplified through multiple PCR;
the multiplex PCR primer composition can be used for a detection kit for detecting the MNP marker locus of the acinetobacter.
The kit provided by the invention can accurately and sensitively detect 10 copies/reaction of acinetobacter and detect the risk of false positive in 1 copy/reaction of acinetobacter.
In the reproducibility test of the invention, the logarithm of difference of the MNP labeling main gene type among different libraries and different library establishing batches of each sample is 0, the reproducibility r is 100%, and the accuracy a is 100%.
The MNP marker and the kit of the present invention have high specificity in detecting a target microorganism in a complex template.
The MNP marker site of Acinetobacter, primer composition, kit and use thereof of the present application will be described in detail below with reference to examples, comparative examples and experimental data.
Example 1 screening of MNP marker sites of Acinetobacter and design of multiplex PCR amplification primers
S1 screening of MNP marker loci of Acinetobacter
Based on the complete or partial genome sequences of 901 different isolates of Acinetobacter published on the web, 15 MNP marker loci are obtained by sequence alignment. For species without genomic data on the net, the genomic sequence information of the representative microspecies of the microbial species to be detected can also be obtained by high-throughput sequencing, wherein the high-throughput sequencing can be whole genome or simplified genome sequencing. In order to ensure polymorphism of the selected marker, the genomic sequence of at least 10 isolates is generally used as a reference. The 15 MNP marker sites screened are shown in table 1:
TABLE 1-starting position of the MNP marker site and detection primer on the reference sequence
Figure BDA0003338339010000061
The step S1 specifically includes:
selecting a genome sequence of a representative strain of the Acinetobacter as a reference genome, and performing sequence comparison on the genome sequence and the reference genome to obtain single nucleic acid polymorphic sites of each strain of the Acinetobacter;
on the reference genome, performing window translation by taking 100-300 bp as a window and 1bp as a step length, and screening to obtain a plurality of candidate MNP site regions, wherein the candidate MNP site regions contain more than or equal to 2 single nucleotide variation sites, and the single nucleotide polymorphism sites do not exist on sequences of 30bp at two ends;
screening a region with the division DP of more than or equal to 0.2 in the candidate polynucleotide polymorphic site region as an MNP marker site; wherein DP is d/t, t is the log of comparisons for all the minor species in the candidate polynucleotide polymorphic site region when compared two by two, d is the log of samples for which at least two single nucleic acid polymorphisms in the candidate polynucleotide polymorphic site region are different.
As an optional implementation mode, when the reference genome is screened by taking 100-300 bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, which is beneficial to comprehensive screening.
S2 design of multiplex PCR amplification primers
The multiplex PCR amplification primers of the MNP sites are designed through primer design software, the design of the primers follows that the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely all the designed primers can be amplified normally in one amplification reaction.
S3 evaluation of detection efficiency of primer combination
Adding the count standard substance of Acinetobacter with known copy number into human genome DNA to prepare a 1000-copy/reaction simulation template, detecting by the MNP mark detection method, constructing 4 repeated sequencing libraries, screening primer combinations with uniform amplification and optimal compatibility according to the detection condition of MNP sites in the 4 libraries, and finally screening the primer compositions of 15 MNP sites.
Example 2 detection of Acinetobacter by MNP sites and primers
1. Detection of MNP markers
Acinetobacter genus mock samples of 1 copy/reaction, 10 copies/reaction, and 100 copies/reaction were prepared by adding to human genomic DNA using acinetobacter genus count standards of known copy numbers. An equal volume of sterile water was also set as a blank. A total of 4 samples were obtained, each sample was constructed into 3 duplicate libraries each day, and the assay was continued for 4 days, i.e. 12 sets of sequencing data were obtained for each sample, as shown in table 2. The flow of detection of MNP markers is shown in figure 3. According to the sequencing fragment number and the site number of MNP sites of the acinetobacter detected in blank control and the acinetobacter nucleic acid standard in 12 repeated experiments, the repeatability, the accuracy and the sensitivity of the detection method are evaluated, and the threshold value for detecting the pollution of a quality control system and the target pathogen is set.
TABLE 2 detection sensitivity and stability analysis of MNP labeling method of Acinetobacter
Figure BDA0003338339010000071
As shown in Table 2, the kit can stably detect 7 MNP sites in a sample of 10 copies/reaction and detect 1 MNP site at most in a sample of 0 copies/reaction, can obviously distinguish the samples of 10 copies/reaction and 0 copies/reaction, and has technical stability and detection sensitivity as low as 10 copies/reaction.
2. Evaluation of reproducibility and accuracy of MNP (MNP) marker detection kit for detecting Acinetobacter
The reproducibility and accuracy of the MNP marker detection method for detecting Acinetobacter sp was evaluated based on whether the genotype of the co-detected locus could be reproduced in two replicates. Specifically, two-by-two comparisons were made for each of 12 sets of data for 100 copies of the sample, and the results are shown in table 3.
TABLE 3 reproducibility and accuracy assessment of the detection method of the MNP marker of Acinetobacter
Figure BDA0003338339010000081
As can be seen from Table 3, the number of MNP sites with differences in major genotypes is 0; the accuracy rate a is 1- (1-r)/2 is 0.5+0.5r, and r represents the reproducibility rate, i.e., the ratio of the number of sites where the major genotype is reproducible to the number of common sites, which is considered to be the principle of accuracy among 2 repeated experiments. In the reproducibility test of the invention, the logarithm of difference of the MNP labeling main gene type among different libraries and different library establishing batches of each sample is 0, the reproducibility r is 100%, and the accuracy a is 100%.
3. Threshold judgment for detecting Acinetobacter by MNP (MNP) marker detection kit
In 1 copy/reaction sample, the sequence of the acinetobacter can be detected and aligned, and at least 1 MNP site is covered. While the sequence of Acinetobacter was also detected in the partial blank control. Due to the extreme sensitivity of MNP marker detection methods, contamination of the data during detection is likely to result in the generation of false positives. Therefore, in this example, a quality control scheme is established, specifically as follows:
1) the amount of sequencing data was greater than 4.5 megabases. The measurement and calculation are based on the fact that the number of MNP sites detected by each sample is 15, and the length of a sequencing fragment is 300 bases, so that when the data volume is more than 4.5 million bases, most samples can ensure that the number of the sequencing fragments covering each site reaches 1000 times through one-time experiment, and the accurate analysis of the base sequence of each MNP site is ensured.
2) Determining whether the contamination is acceptable based on a signal index S of Acinetobacter in the test sample and a noise index P of Acinetobacter in the blank, wherein:
the blank control noise index P ═ Nc/Nc, where Nc and Nc represent the number of sequencing fragments and the total number of sequencing fragments of acinetobacter genus, respectively, in the blank control.
The signal index S of the test sample is Nt/Nt, where Nt and Nt represent the number of sequencing fragments of acinetobacter and the total number of sequencing fragments in the test sample, respectively.
3) And calculating the detection rate of the MNP marker locus in the test sample, which is the ratio of the number of the detected locus to the number of the total design locus.
TABLE 4 SNR of Acinetobacter in the samples to be tested
Figure BDA0003338339010000091
The results are shown in table 4, where the average of the signal-to-noise ratios of the 10 copies of the sample and the blank is 44.6, and at least 7 MNP sites were stably detected in 12 sets of data of 10 copies/reaction, accounting for 46.7% of the total sites. Therefore, the present standard stipulates that the threshold value for determination of the signal-to-noise ratio of Acinetobacter is 20 with accuracy being ensured, i.e., that nucleic acid of Acinetobacter is detected in a sample when the signal-to-noise ratio of Acinetobacter in the sample is greater than 20 and the detection rate of sites is 30% or more. Therefore, the kit provided by the invention can sensitively detect the acinetobacter with the copy/response as low as 10.
4. Specific evaluation of MNP (MNP) marker detection kit for detecting acinetobacter
The DNA of the acinetobacter strain, the Mycobacterium tuberculosis, the staphylococcus aureus, the bordetella pertussis, the bordetella hollisae, the chlamydia pneumoniae, the mycoplasma pneumoniae, the EB virus, the haemophilus influenzae, the varicella zoster virus, the cytomegalovirus, the herpes simplex virus, the klebsiella pneumoniae, the legionella, the moraxella catarrhalis, the pseudomonas aeruginosa, the rickettsia, the staphylococcus aureus, the streptococcus pneumoniae and the streptococcus pyogenes are mixed together according to equal molar weight to prepare a mixed template, and the blank template is used as a reference to detect the acinetobacter in the mixed template by adopting the method provided by the invention. After 3 repeated experiments are carried out and analyzed according to the quality control scheme and the judgment threshold, MNP sites of acinetobacter can be specifically detected in all the 3 repeated experiments, 15 sites are all detected, and the signal-to-noise ratio is 354.6, 323.7 and 367.3 in sequence, so that the MNP marker and the kit can detect the high specificity of the target microorganism in the complex template.
Example 3 detection of genetic variation between Acinetobacter strains
The 6 progeny strains of one collected acinetobacter strain are detected by using the kit and the MNP marker locus detection method, samples are named as S1-S6 in sequence, the sequencing average coverage multiple of each MNP locus is 1103 times, and all 15 MNP markers can be detected from each strain (Table 5). The fingerprints of 6 strains are compared pairwise, and the results are shown in table 5, wherein 1 part (S-2) and 5 parts of acinetobacter detected together in the same batch have main genotype difference of partial sites (table 5), and variation among strains exists.
TABLE 5-6 detection assays for Acinetobacter genus
Figure BDA0003338339010000101
As can be seen from Table 5, the application of the kit for identifying genetic variation among strains by detecting MNP markers can be used for ensuring the genetic consistency of the same named acinetobacter strains in different laboratories, thereby ensuring the comparability of research results, which has important significance for scientific research of acinetobacter. Clinically, diagnostic protocols can be weighed against whether differential sites affect drug resistance.
Example 4 detection of genetic variation in Acinetobacter
As a group organism, partial individuals in the group of Acinetobacter have variation, so that the group is not homozygous any more, and a heterogeneous heterozygous group is formed, which influences the stability and consistency of the phenotype of the microorganism for testing in particular. The variant shows an allelic form outside the main genotype of the locus when the population is subjected to molecular marker detection. When the variant individuals have not accumulated, they account for a very small fraction of the population and are characterized by a low frequency of alleles. The low frequency of alleles is often confused with technical errors, making it difficult for the prior art to distinguish. The present invention detects highly polymorphic MNP markers. The technical error rate of MNP markers is significantly lower than that of SNP markers, based on the fact that the probability of multiple errors occurring simultaneously is lower than that of one error.
Authenticity assessment of the minor allelic genotype of this example was performed as follows: alleles with strand bias (ratio of the number of sequencing sequences overlaid on the DNA double strand) were first excluded according to the following rule: the strand preference is greater than 10-fold, or the difference from the strand preference of the dominant allele is greater than 5-fold.
Genotypes without strand preference were judged for authenticity based on the number and proportion of sequences sequenced in table 6. Inv function calculation under a 99.9999% probability guarantee, e max (n-1) and e max (n.gtoreq.2) 1.03% and 0.0994%, respectively, the number of sequenced sequences of the suballelic genotype at each locus is a critical value, and only when the number of sequenced sequences of the suballelic genotype exceeds the critical value, the true suballelic genotype is determined. When multiple candidate sub-alleles are present, the P-value for each candidate allele is multiply corrected, FDR<0.5% of the candidate alleles were judged to be true sub-allelic genotypes.
Table 6 relates to the parameter e max (n-1) and e max (n.gtoreq.2) meansThe highest proportion of the number of sequencing sequences carrying the wrong alleles of n SNPs to the total number of sequencing sequences at that site. e.g. of the type max (n-1) and e max (n.gtoreq.2) 1.03% and 0.0994%, respectively, were obtained from the frequency of all the minor alleles detected at 930 homozygous MNP sites.
TABLE 6 Critical values for determination of sub-allelic genotypes at partial sequencing depths
Figure BDA0003338339010000111
Figure BDA0003338339010000121
Nucleic acids of two strains differing in genotype as shown in Table 5 were mixed in the following 8 ratios 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/100, 5/100, 7/100 according to the above parameters to prepare artificial heterozygous samples, and each sample was examined for 3 replicates to obtain a total of 24 sequencing data. The loci with heterozygous genotypes are detected in 24 artificial heterozygous samples by accurately comparing the genotypes of the MNP loci of the two strains, which indicates the applicability of the developed MNP marker detection method of the acinetobacter in detecting genetic variation in the strain population.
Example 5 construction of Acinetobacter DNA fingerprint database
Extracting DNA of all strains or samples for constructing DNA fingerprint database of Acinetobacter by using methods such as a conventional CTAB method, a commercial kit and the like, and detecting the quality of the DNA by using agarose gel and an ultraviolet spectrophotometer. If the ratio of the absorbance values of the extracted DNA at 260nm to 230nm is more than 2.0, the ratio of the absorbance values of 260nm to 280nm is between 1.6 and 1.8, the main band of the DNA electrophoresis is obvious, and no obvious degradation or RNA residue exists, the genome DNA reaches the relevant quality requirement, and subsequent experiments can be carried out.
And (3) carrying out sequence comparison on the sequencing data of the 6 strains to obtain a main genotype of each site of each strain, forming an MNP fingerprint of each strain, and recording a database file to form an Acinetobacter DNA fingerprint database. The constructed MNP fingerprint database is based on the gene sequences of the detected strains and is therefore compatible with all high throughput sequencing data. The MNP fingerprint spectrum of the strain obtained by each detection is compared with the established MNP fingerprint database, and the MNP fingerprint database established by the MNP fingerprint spectrum of the strain with the difference of the main genotypes realizes the co-establishment sharing and the random updating of the database.
Example 6 application in Fine typing of Acinetobacter
The 6 Acinetobacter strains were typed using the primer combination and MNP marker locus detection method described in example 2. And comparing the DNA fingerprints of each strain pairwise and the constructed fingerprint database, defining the DNA fingerprints as existing variants which are the same as those of the existing fingerprint database, defining the variants as new variants with main genotype difference at least one MNP site, and realizing the fine typing of the acinetobacter.
The results of the detection of 6 acinetobacter species are shown in table 5, and in agreement with the expectation, 1 and 5 of the 6 acinetobacter species detected were different, possibly in different variants, in the major genotypes at the 2 MNP sites. Therefore, the resolution of the method for the acinetobacter reaches the level of single base, and the fine typing of the acinetobacter in the sample can be realized.
Finally, it should be further 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 of those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including the preferred embodiment and all changes and modifications that fall within the true scope of the embodiments of the present invention.
It will be apparent to those skilled in the art that various modifications and variations can be made in the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, if such modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to encompass such modifications and variations.
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Claims (8)

1. The MNP marker site of the acinetobacter is a genome region which is screened on the genome of the acinetobacter and is distinguished from other species and has various multiple nucleotide polymorphisms in the genus, and comprises the marker sites of MNP-1-MNP-15 on the CP009257 genome.
2. A multiplex PCR primer composition for detecting the MNP marker locus of the acinetobacter as claimed in claim 1, characterized in that the multiplex PCR primer composition comprises 15 pairs of primers, and the nucleotide sequences of the 15 pairs of primers are shown as SEQ ID No. 1-SEQ ID No. 30.
3. A test kit for detecting the MNP marker site of Acinetobacter according to claim 1, wherein the kit comprises the primer composition according to claim 2.
4. The test kit of claim 3, wherein the kit further comprises a multiplex PCR premix.
5. Use of the MNP marker site of acinetobacter of claim 1, or the primer composition of claim 2, or the detection kit of any one of claims 3-4 for the qualitative detection of acinetobacter.
6. Use of the MNP marker site of acinetobacter according to claim 1, or the primer composition according to claim 2, or the detection kit according to any of claims 3-4 for detecting genetic variations within and between acinetobacter strains.
7. Use of the MNP marker site of acinetobacter of claim 1, or the primer composition of claim 2, or the detection kit of any one of claims 3-4 for constructing an acinetobacter database.
8. Use of the MNP marker site of the acinetobacter of claim 1 or the primer composition of claim 2 or the detection kit of any one of claims 3-4 for fine typing within the acinetobacter.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002036827A1 (en) * 2000-11-01 2002-05-10 The General Hospital Corporation Bacterial strain typing
WO2006097636A1 (en) * 2005-03-17 2006-09-21 Universite De La Mediterranee (Aix-Marseille Ii) Nucleic acid fragments and specific detection method by molecular identification of different bacteria species of the genus acinetobacter
US20090253128A1 (en) * 2008-04-03 2009-10-08 Centers For Disease Control Department Of Healthy Nucleotide sequence for identifying of acinetobacter bacteria, and method and kit of identification of acinetobacter bacteria
US20110065111A1 (en) * 2009-08-31 2011-03-17 Ibis Biosciences, Inc. Compositions For Use In Genotyping Of Klebsiella Pneumoniae
WO2014157176A1 (en) * 2013-03-25 2014-10-02 国立大学法人名古屋大学 Genotype classification method for bacterium of genus acinetobacter and primer set to be used therein

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002036827A1 (en) * 2000-11-01 2002-05-10 The General Hospital Corporation Bacterial strain typing
WO2006097636A1 (en) * 2005-03-17 2006-09-21 Universite De La Mediterranee (Aix-Marseille Ii) Nucleic acid fragments and specific detection method by molecular identification of different bacteria species of the genus acinetobacter
US20090253128A1 (en) * 2008-04-03 2009-10-08 Centers For Disease Control Department Of Healthy Nucleotide sequence for identifying of acinetobacter bacteria, and method and kit of identification of acinetobacter bacteria
US20110065111A1 (en) * 2009-08-31 2011-03-17 Ibis Biosciences, Inc. Compositions For Use In Genotyping Of Klebsiella Pneumoniae
WO2014157176A1 (en) * 2013-03-25 2014-10-02 国立大学法人名古屋大学 Genotype classification method for bacterium of genus acinetobacter and primer set to be used therein

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RAFFAELE ZARRILLI等: "Global evolution of multidrug-resistant Acinetobacter baumannii clonal lineages", INT J ANTIMICROB AGENTS ., vol. 41, no. 1, pages 11 - 19 *

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