CN114790485B - MNP (MNP) marking site of Acinetobacter genus, primer composition, kit and application of MNP marking site - Google Patents
MNP (MNP) marking site of Acinetobacter genus, primer composition, kit and application of MNP marking site Download PDFInfo
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Abstract
The invention discloses an MNP (MNP) marking site, a primer composition, a kit and application thereof, wherein the MNP marking site refers to a genome region which is screened on the genome of an Acinetobacter strain, is separated from other species and has a plurality of nucleotide polymorphisms in the species, and comprises marking sites of 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 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 the sequence analysis can be performed on all the marker loci of multiple samples at one time, the method has the advantages of high flux, multiple targets, high sensitivity and culture-free performance, can be applied to the identification and genetic variation detection of acinetobacter in large-scale samples, and has important significance for scientific research and epidemic prevention monitoring of the acinetobacter.
Description
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to an MNP (MNP) marking site 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 include 6 species, namely Acinetobacter calcoaceticus (A.calcoaceticus), acinetobacter Lu Fei (A.lwoffi), acinetobacter baumannii (A.baumannii), acinetobacter haemolyticus (A.haemolytius), acinetobacter agaricus (A.junii) and Acinetobacter johnsonii (A.johnsonii). Acinetobacter is easy to adhere to various medical materials and becomes a bacterial storage source. Contaminated medical equipment and staff hands are important propagation media in hospitals. The susceptible person is elderly patient, premature infant and neonate, operation wound, severe burn, tracheotomy or intubation, person using artificial respirator, intravenous catheter and peritoneal dialysis, broad-spectrum antibacterial or immunosuppressant applicator, etc. In addition, acinetobacter is also present in healthy human skin (25%), pharynx (7%), and conjunctiva, saliva, gastrointestinal tract, and vaginal secretions. The source of infection may be the patient himself (endogenous infection), or may be an acinetobacter infection or a carrier, especially medical personnel with both hands. The propagation paths are contact propagation and air propagation. In the case of the ventilator, the incidence of pneumonia is about 3% to 5%. Bacteremia is the most serious clinical type in acinetobacter infection, and the death rate is more than 30%.
Classical acinetobacter detection methods, including isolation and culture, serological analysis, PCR techniques, whole genome and metagenome sequencing, and the like, suffer from one or more limitations in terms of duration, operational complexity, detection throughput, accuracy and sensitivity of detection variation, cost, and the like. 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 a large amount of data waste and background noise caused by sequencing of a whole genome and a metagenome, and has the advantages of small sample requirement, accurate diagnosis result, data quantity saving and low-frequency variation detection.
The molecular markers detected by the existing targeted detection technology mainly comprise SNP and SSR markers. SSR markers are the most well-accepted markers for polymorphism, but are small in number in microorganisms; the number of SNP markers is huge, the distribution is dense, and the polymorphism of single SNP marker is insufficient to capture the potential allelic diversity in microorganism population.
Therefore, development of a novel molecular marker of a high polymorphism of a pathogenic microorganism Acinetobacter genus and a detection technology thereof becomes a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to provide an MNP (MNP) marking site of Acinetobacter, a primer composition, a kit and application thereof, which can carry out 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 achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the invention there is provided an MNP marker locus of the genus acinetobacter, said MNP marker locus being a species specific genomic region screened on the genome of the genus acinetobacter and having a plurality of nucleotide polymorphisms within the species, comprising the marker locus of MNP-1 to MNP-15 on the CP009257 genome.
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 CP009257 sequence.
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 nucleotide sequences of the 15 pairs of primers being shown as 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 present invention, there is provided a detection kit for detecting the MNP marker locus of acinetobacter, the kit comprising the primer composition.
Further, the kit further comprises a multiplex PCR premix.
In a fourth aspect of the invention, there is provided the use of said MNP marker locus of acinetobacter or said multiplex PCR primer composition or said detection kit in the qualitative detection of acinetobacter and in the preparation of a preparation for detection of acinetobacter.
In a fifth aspect of the invention, there is provided the use of said MNP marker locus of acinetobacter or said multiplex PCR primer composition or said detection kit for detecting genetic variations within and between acinetobacter strains.
In a sixth aspect of the invention, there is provided the use of said MNP marker locus of acinetobacter or said multiplex PCR primer composition or said detection kit for the construction of an acinetobacter database.
In a seventh aspect of the invention, there is provided the use of said MNP marker locus of acinetobacter or said multiplex PCR primer composition or said detection kit for the detection of microscopic species within acinetobacter.
In the above application, firstly, the total DNA of the bacteria of the sample to be tested is obtained; performing a first round of multiplex PCR amplification on the total DNA and the blank control by using the kit, wherein the number of cycles is not higher than 25; after the amplified product is purified, adding a label and a second generation sequencing joint of the sample through a second round of 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; the sequencing results are aligned to the reference sequence of the Acinetobacter genus, and the number of detection sequences and genotype data of the total DNA are obtained. And carrying out data quality control and data analysis on the sequencing data of the total DNA according to the number of the sequencing sequences of the Acinetobacter genus obtained in the total DNA and the blank control and the number of the detected MNP sites, and obtaining the number of the detected MNP sites, the number of the sequencing sequences covering each MNP site and the genotype data of the MNP sites.
When used for the identification of Acinetobacter, the quality control is performed according to the number of the sequencing sequences of Acinetobacter detected in the sample to be detected and the blank control and the number of MNP sites detected, and then whether the sample to be detected contains the nucleic acid of Acinetobacter or not is judged. The quality control scheme and the judging method are characterized in that DNA of the Acinetobacter strain with known copy number is taken as a detection sample, the sensitivity, accuracy and specificity of the kit for detecting the Acinetobacter strain are evaluated, and the quality control scheme and the judging method for the kit for detecting the Acinetobacter strain are formulated.
When used for the detection of genetic variation of Acinetobacter, 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 15 MNP sites of each strain to be compared by using the kit and the method. By genotype comparison, the strains to be compared are analyzed for differences in major genotypes at the 15 MNP sites. If the strain to be compared has a variation in the main genotype of at least one MNP site, it is determined that there is a genetic variation in both. Alternatively, 15 loci of the strain to be compared may be amplified by single PCR, and then Sanger sequencing is performed on the amplified products to obtain sequences, and then the genotypes of each MNP locus of the strain to be compared are aligned. If there are MNP sites of inconsistent main genotypes, there are variations between the strains to be compared. When detecting genetic variation inside the strain, determining whether the secondary genotype other than the primary genotype is detected at the MNP locus of the strain to be detected through a statistical model. If the strain to be tested has the subgenotype at least one MNP site, judging that the strain to be tested has genetic variation.
When used for constructing the DNA fingerprint database of the Acinetobacter, the genotype data of the MNP locus of the Acinetobacter identified from a sample is recorded into a database file to form the DNA fingerprint database of the Acinetobacter; each time a different sample is identified, it is identified whether the Acinetobacter genus in the sample has a difference in major genotype (with more than 50% of the genotypes supported by the sequencing fragments at one MNP site) from the strains in the database by comparison with the DNA fingerprint database of the Acinetobacter genus, and the Acinetobacter genus having a difference in major genotype at least 1 MNP site is a new variant type and is recorded in the DNA fingerprint database. Therefore, the DNA fingerprint database can be continuously enriched by utilizing the primer combination.
When the method is used for the typing of the Acinetobacter, the identification of the Acinetobacter in a sample to be tested is carried out, and the genotype of each MNP locus is obtained; collecting genome sequences of Acinetobacter disclosed on the net and constructing an Acinetobacter DNA fingerprint database to form an Acinetobacter reference sequence library; comparing the genotype of the Acinetobacter in the sample to be detected with a reference sequence library of the Acinetobacter, and screening strains which are identical or closest in genetic conduction to obtain the typing of the Acinetobacter strain in the sample to be detected. And identifying the existing type or new type of the Acinetobacter strain in the sample according to the comparison result with the reference sequence library, and realizing the fine-subdivision type of the Acinetobacter.
The invention is initiated in the field of Acinetobacter and is not reported in related documents; the MNP marker is mainly mined based on reported resequencing data of the acinetobacter representative species, and MNP marker sites which are special for the acinetobacter and have high distinction degree for each species of the acinetobacter are searched; the sequences on two sides of MNP marker are highly conserved among various small species of Acinetobacter, and the conserved region is used for designing multiplex PCR amplification primers; and then according to the test result of the standard substance, a set of primer combination with the largest polymorphism, high specificity MNP locus and the best compatibility and a detection kit are screened.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
the invention provides an MNP labeling site of Acinetobacter, a primer composition, a kit and application thereof. The provided 15 MNP loci of the Acinetobacter and the primer combination thereof can be used for multiplex PCR amplification, and the amplification products are sequenced by fusing a second generation sequencing platform, so that the requirements of high-throughput, high-efficiency, high-accuracy and high-sensitivity detection of the Acinetobacter are met, and the requirements of accurate detection of genetic variation among Acinetobacter strains are met; meets the requirement of identifying the degradation of the Acinetobacter genus population; meets the requirement of the standard and sharable fingerprint data construction of the Acinetobacter and provides technical support for scientific research, scientific monitoring and prevention and control of the Acinetobacter.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of MNP marker polymorphism;
FIG. 2 is a flow chart of screening and primer design of the MNP marker locus of Acinetobacter;
FIG. 3 is a flow chart of detection of MNP marker loci.
Detailed Description
The advantages and various effects of the embodiments of the present invention will be more clearly apparent from the following detailed description and examples. Those skilled in the art will appreciate that these specific implementations and examples are provided to illustrate, but not limit, examples of the present invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or may be prepared by existing methods.
The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:
MNP markers suitable for detection of the population organisms are screened as detection targets. MNP markers refer to polymorphic markers caused by multiple nucleotides in a region of the genome. MNP markers have the following advantages over SSR markers and SNP markers: (1) The alleles are abundant, and 2 are arranged on single MNP locus n Species alleles, higher than SSR and SNP, are suitable for detection of microorganisms, a typical population organism; (2) The species distinguishing capability is strong, the species identification can be realized by only a small amount of MNP marks, and the detection error rate is reduced. The MNP labeling method for detecting MNP labeling fuses the ultra-multiplex PCR and the second-generation high-throughput sequencing technology, and has the following advantages: (1) The output is a base sequence, and a standardized database can be constructed for sharing without parallel experiments; (2) The method has high efficiency, breaks through the limitation of the number of sequencing samples by using the sample DNA bar code, and can type tens of thousands of MNP sites of hundreds of samples at one time; (3) High sensitivity, multiple targets are detected at one time by using multiple PCR, and high false negative and low sensitivity caused by single target amplification failure are avoided; (4) High accuracy, and sequencing the amplified product hundreds of times by using a second-generation high-throughput sequencer.
In view of the advantages and the characteristics, the MNP marking and the detection technology thereof can realize classification and tracing of the multi-allele types of the group organisms, and have application potential in the aspects of identification of pathogenic microorganisms, construction of fingerprint databases, genetic variation detection and the like. At present, no report about MNP labeling exists in microorganisms, and corresponding technology is lacking. The development, screening and application of MNP labeling method has better application foundation in plants.
Thus, the present invention developed MNP marker loci of Acinetobacter, which are genomic regions screened on the Acinetobacter genome that are distinct from other species and have multiple nucleotide polymorphisms within the species, including the marker loci of MNP-1-MNP-15 on the CP009257 genome.
Next, the present invention developed a multiplex PCR primer composition for detecting the MNP labeling site of Acinetobacter, comprising 15 pairs of primers, the nucleotide sequences of the 15 pairs of primers being shown in SEQ ID NO.1 to SEQ ID NO. 30. The primers do not collide with each other, and efficient amplification can be performed through multiplex PCR;
the multiplex PCR primer composition can be used for a detection kit for detecting the MNP labeling site of the Acinetobacter genus.
The kit provided by the invention can accurately and sensitively detect the Acinetobacter with the concentration of 10 copies/reaction, and detect the Acinetobacter with the concentration of 1 copy/reaction, so that the risk of false positive exists.
In the reproducibility test of the invention, the difference logarithm of MNP marking main genotypes among different libraries and different library construction batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%.
The MNP markers and the kits of the invention have high specificity in detecting target microorganisms in complex templates.
The MNP marker locus of a genus acinetobacter, the primer composition, the kit and the 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 loci of Acinetobacter and design of multiplex PCR amplification primers
S1, screening of MNP marker loci of Acinetobacter
Based on complete or partial sequences of genomes of 901 different isolates of Acinetobacter genus disclosed on the net, 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 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 genus Acinetobacter 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 genus Acinetobacter;
on the reference genome, carrying out window translation by taking 100-300 bp as a window and taking 1bp as a step length, and screening to obtain a plurality of candidate MNP site areas, wherein the candidate MNP site areas contain more than or equal to 2 single nucleotide variation sites, and the single nucleotide polymorphism sites do not exist on sequences of 30bp at both ends;
screening a region with the discrimination DP more than or equal to 0.2 from the candidate polynucleotide polymorphism site region as an MNP marking site; wherein dp=d/t, t is the log of comparisons when all the minor species are compared pairwise in the candidate polynucleotide polymorphic site region, and d is the log of samples of differences in at least two single nucleic acid polymorphisms in the candidate polynucleotide polymorphic site region.
As an optional implementation mode, when screening is performed on the reference genome by taking 100-300 bp as a window, other step sizes can be selected, and the implementation mode adopts the step size of 1bp, so that the comprehensive screening is facilitated.
S2, design of multiplex PCR amplification primer
The multiplex PCR amplification primers of the MNP locus are designed through primer design software, the primer design follows that the primers are not interfered with each other, all the primers can be combined into a primer pool for multiplex PCR amplification, namely, all the designed primers can be amplified normally in one amplification reaction.
S3, evaluating detection efficiency of primer combination
Using a reference sample of Acinetobacter with known copy number, adding the reference sample into human genome DNA to prepare a 1000-copy/reaction simulated template, detecting by the MNP mark detection method, constructing 4 repeated sequencing libraries, screening a primer combination with uniform amplification and optimal compatibility according to the detection condition of MNP sites in the 4 libraries, and finally screening the primer combination of 15 MNP sites.
Example 2 detection of Acinetobacter by MNP site and primer
1. Detection of MNP markers
Acinetobacter species mock samples were prepared at 1 copy/reaction, 10 copy/reaction and 100 copy/reaction using a known copy number Acinetobacter species counting standard, added to human genomic DNA. An equal volume of sterile water was set at the same time as a blank. A total of 4 samples, each of which was constructed as 3 replicate libraries per day, were tested continuously for 4 days, i.e., 12 sets of sequencing data were obtained per sample, as shown in table 2. The detection flow of MNP markers is shown in fig. 3. And evaluating the reproducibility, accuracy and sensitivity of the detection method according to the number of sequencing fragments and the number of sites of the MNP locus of the Acinetobacter detected in the blank control and the nucleic acid standard of the Acinetobacter in 12 repeated experiments, and formulating a quality control system pollution and a threshold value for detecting a target pathogen.
TABLE 2 detection sensitivity and stability analysis of MNP labeling method of Acinetobacter
As shown in Table 2, the kit can stably detect 7 MNP sites in a 10-copy/reaction sample and at most 1 MNP site in a 0-copy/reaction sample, and 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.
2. Reproducibility and accuracy assessment of detection of Acinetobacter by MNP (MNP) marker detection kit
Based on whether the genotype of the co-detected site is reproducible or not in the two replicates, the reproducibility and accuracy of detection of Acinetobacter by the MNP-marker detection method are evaluated. Specifically, the data of 12 sets of 100 copies of the sample were compared in pairs, and the results are shown in Table 3.
TABLE 3 reproducibility and accuracy assessment of the method for detecting the MNP markers of Acinetobacter
As can be seen from Table 3, the number of MNP sites having a difference in the main genotypes was 0; according to the principle that the reproducible genotypes are considered to be accurate between 2 repeated experiments, the accuracy a=1- (1-r)/2=0.5+0.5r, and r represents the reproducibility, namely the ratio of the reproducible site number of the main genotype to the common site number. In the reproducibility test of the invention, the difference logarithm of MNP marking main genotypes among different libraries and different library construction batches of each sample is 0, the reproducibility rate r=100% and the accuracy rate a=100%.
3. Threshold value judgment for detection of Acinetobacter by MNP marker detection kit
Sequences aligned to Acinetobacter can be detected in 1 copy/reaction samples, covering at least 1 MNP site. The sequence of Acinetobacter was also detected in a partial blank. Because of the extreme sensitivity of MNP marker detection methods, contamination of the data in the detection is prone to false positives. Therefore, the quality control scheme is formulated in this example, and is specifically as follows:
1) The amount of sequencing data is greater than 4.5 megabases. The measurement and calculation basis is that the number of MNP loci 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 locus reaches 1000 times by one experiment, and ensure the accurate analysis of the base sequence of each MNP locus.
2) Determining whether the contamination is acceptable based on the signal index S of the genus acinetobacter in the test sample and the noise index P of the genus acinetobacter in the blank, wherein:
the blank noise index p=nc/Nc, where Nc and Nc represent the number of sequenced fragments and the total number of sequenced fragments, respectively, of the genus acinetobacter in the blank.
The signal index s=nt/Nt of the test sample, where Nt and Nt represent the number of sequenced fragments of acinetobacter and the total number of sequenced fragments, 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 Acinetobacter in test samples
As a result, as shown in Table 4, the average value of signal-to-noise ratios of the 10 copies of the sample and the blank was 44.6, and at least 7 MNP sites were stably detected in the 10 copies/reaction 12 sets of data, accounting for 46.7% of the total sites. Therefore, under the condition of ensuring accuracy, the standard prescribes that the signal-to-noise ratio judgment threshold of the Acinetobacter genus is 20, namely when the signal-to-noise ratio of the Acinetobacter genus in the sample is more than 20 and the site detection rate is more than or equal to 30%, the detection of the nucleic acid of the Acinetobacter genus in the sample is judged. Therefore, the kit provided by the invention can sensitively detect the acinetobacter with the concentration as low as 10 copy/reaction.
4. Specific evaluation of detection of Acinetobacter by MNP marker detection kit
Artificially mixing the Acinetobacter strain with DNA of mycobacterium tuberculosis, staphylococcus aureus, pertussis baud bacteria, huo Shibao terus, chlamydia pneumoniae, mycoplasma pneumoniae, EB virus, haemophilus influenzae, varicella zostera virus, cytomegalovirus, herpes simplex virus, klebsiella pneumoniae, legionella, moraxella catarrhalis, pseudomonas aeruginosa, rickettsia, staphylococcus aureus, streptococcus pneumoniae and streptococcus pyogenes according to the equimolar amount to prepare a mixed template, and detecting the Acinetobacter in the mixed template by adopting the method provided by the invention by using a blank template as a control. After 3 repeated experiments are carried out and analysis is carried out according to the quality control scheme and the judgment threshold, MNP loci of the Acinetobacter genus can be specifically detected in the 3 repeated experiments, 15 loci are all detected, and signal to noise ratios are 354.6, 323.7 and 367.3 in sequence, so that high specificity of MNP markers and the kit for detecting target microorganisms in complex templates is shown.
Example 3 detection of genetic variation between Acinetobacter strains
The collected 6 sub-generation strains of one Acinetobacter strain are detected by using the kit and the MNP marking site detection method, samples are sequentially named as S1-S6, the average coverage multiple of sequencing of each MNP site is 1103 times, and all 15 MNP marks can be detected by each strain (table 5). The fingerprints of 6 strains were aligned in pairs, and the results are shown in Table 5, in which 1 part (S-2) and 5 parts of Acinetobacter detected together in the same batch all had a major genotype difference at part of the sites (Table 5), and there was a variation between strains.
TABLE 5-6 detection analysis of Acinetobacter genus
As can be seen from Table 5, the application of the kit for identifying the genetic variation among strains by detecting MNP markers can be used for ensuring the genetic consistency of the same named Acinetobacter strains in different laboratories, so that the comparability of research results is ensured, and the kit has important significance for scientific research of the Acinetobacter. 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 in Acinetobacter
As a group organism, the individuals in the inner part of the Acinetobacter 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 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 e calculated based on binom. Inv function under the probability guarantee of α=99.9999% max (n=1) and e max (n.gtoreq.2) is 1.03% and 0.0994%, respectively, and the true hypogenotype is judged only when the number of sequences of the hypogenotype exceeds the critical value. When a plurality of candidate minor alleles exist, multiple correction is carried out on the P value of each candidate allele type, and FDR is carried out<0.5% of candidate alleles are judged to be true minor genotypes.
Parameter e related to Table 6 max (n=1) and e max (n.gtoreq.2) refers to the highest proportion of the total sequence of the locus of the sequence of the wrong allele carrying n SNPs. e, e max (n=1) and e max (n.gtoreq.2) 1.03% and 0.0994%, respectively, are obtained from the frequency of all minor genotypes detected at 930 homozygous MNP sites.
TABLE 6 critical values for determining the hypo-isogenotypes at partial sequencing depth
According to the above parameters, nucleic acids of two strains having a difference in genotypes shown in Table 5 were mixed in the following 8 ratios of 1/1000,3/1000,5/1000,7/1000,1/100,3/100,5/100,7/100, and artificial heterozygous samples were prepared, each sample was tested for 3 replicates, and 24 sequencing data were obtained in total. By accurately comparing the genotypes of MNP loci of the two strains, loci with heterozygous genotypes are detected in 24 artificial heterozygous samples, and the applicability of the developed MNP marker detection method of Acinetobacter in detecting genetic variation inside a strain population is demonstrated.
EXAMPLE 5 construction of Acinetobacter DNA fingerprint database
All strains used for constructing the Acinetobacter DNA fingerprint database or DNA of samples are extracted by using the conventional CTAB method, commercial kit and other methods, and agarose gel and ultraviolet spectrophotometry are adopted to detect the quality of the DNA. If the ratio of the absorbance values of the extracted DNA at 260nm and 230nm is more than 2.0, the ratio of the absorbance values of 260nm and 280nm is between 1.6 and 1.8, the DNA electrophoresis main band is obvious, no obvious degradation and RNA residues exist, the genome DNA reaches the relevant quality requirements, and the subsequent experiments can be carried out.
And (3) carrying out sequence comparison on the sequencing data of the 6 strains to obtain the main genotype of each site of each strain, forming MNP fingerprint of each strain, and recording a database file to form 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 of the strain obtained by each detection is compared with the constructed MNP fingerprint database, and the MNP fingerprint database constructed by the MNP fingerprint of the strain with the main genotype difference is realized, so that the co-construction sharing and the random updating of the database are realized.
EXAMPLE 6 use in the Fine typing of Acinetobacter
The above 6 Acinetobacter strains were typed using the primer combination and MNP marker locus detection method described in example 2. And comparing the DNA fingerprint of each strain with the constructed fingerprint database in pairs, defining the existing variant as the same as the existing fingerprint database, defining the new variant as the main genotype difference at least one MNP locus, and realizing the fine subdivision of the Acinetobacter genus.
The results of the detection on 6 parts of Acinetobacter are shown in Table 5, and are expected to be consistent, and 1 part of the detected 6 parts of Acinetobacter are different from the other 5 parts of the detected 6 parts of Acinetobacter at 2 MNP sites, and the detected 6 parts of Acinetobacter are possibly different variants. Therefore, the resolution of the method for the Acinetobacter reaches the level of single base, and the method can realize the fine subdivision of the Acinetobacter in the sample.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiment and all such alterations and modifications as fall within the scope of the embodiments of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, the embodiments of the present invention are intended to include such modifications and alterations insofar as they come within the scope of the embodiments of the invention as claimed and the equivalents thereof.
Sequence listing
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Claims (7)
1. A multiplex PCR primer composition for detecting Acinetobacter, which 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 detection kit for detecting acinetobacter, said kit 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 to 3 for qualitative detection of acinetobacter for non-diagnostic purposes.
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 variation within and among acinetobacter strains.
6. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 for constructing an acinetobacter database.
7. Use of a primer composition according to claim 1 or a detection kit according to any one of claims 2 to 3 in a finely divided form within the genus acinetobacter.
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| 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 |
| 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 |
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| 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 |
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| 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 |
| 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)
| Title |
|---|
| Global evolution of multidrug-resistant Acinetobacter baumannii clonal lineages;Raffaele Zarrilli等;Int J Antimicrob Agents .;第41卷(第1期);11-19 * |
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