CN114703301B - Primer group and kit for identifying three types of Bowden bacteria and application of primer group and kit - Google Patents

Primer group and kit for identifying three types of Bowden bacteria and application of primer group and kit Download PDF

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CN114703301B
CN114703301B CN202210036282.5A CN202210036282A CN114703301B CN 114703301 B CN114703301 B CN 114703301B CN 202210036282 A CN202210036282 A CN 202210036282A CN 114703301 B CN114703301 B CN 114703301B
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CN114703301A (en
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彭海
张险峰
吕静
高利芬
李论
方斌
杨红梅
李国明
江永忠
雷亚克
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Hubei Provincial Center For Disease Control And Prevention (hubei Academy Of Preventive Medicine)
Wuhan Mingming Biotechnology Co ltd
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Wuhan Mingming Biotechnology Co ltd
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Abstract

The invention discloses a primer group and a kit for identifying three abalone bacteria and application thereof, and belongs to the technical field of biology. The primer set includes: at least one of a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, and a tenth primer pair, each of the primer pairs including a forward primer and a reverse primer. The primer group provided by the invention has higher specificity and stability, and the kit using the primer group has good sensitivity, reproducibility and accuracy. The kit detects 10 MNP marker loci through a primer group, and can be used for identification and differentiation of three types of Bowder bacteria for non-diagnosis purposes, identification of genetic variation among strains and inside strains, and construction of MNP fingerprint databases of the three types of Bowder bacteria.

Description

Primer group and kit for identifying three types of Bowden bacteria and application of primer group and kit
Technical Field
The invention relates to the technical field of biology, in particular to a primer group and a kit for identifying three types of Bowden bacteria and application thereof.
Background
Bao Te bacteria (boretella) mainly comprise: pertussis (b. Pertussis), pertussis parapertussis (b. Para pertussis), bordetella bronchiseptica (b. Branchiseptica), and the like. The three species of baud bacteria are relatively common and all are pathogenic bacteria associated with human respiratory tract infections. The existing detection method mainly detects the 16S sequence in a sample based on PCR technology, and the method usually detects the three bacteria at the same time, but pertussis baud bacteria, parapertussis Bao Te bacteria and bronchogenic Bao Te bacteria are highly similar in inheritance, so that the detection based on the 16S sequence is difficult to accurately distinguish the three bacteria.
Disclosure of Invention
In order to solve the problems in the prior art, the embodiment of the invention provides a primer group and a kit for identifying three types of Botrytis cinerea and application thereof. The technical scheme is as follows:
in one aspect, the invention provides a primer set for identifying three types of abalone bacteria, the primer set comprising: at least one of a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, and a tenth primer pair, each of the primer pairs including a forward primer and a reverse primer,
the forward primer of the first primer pair is shown as SEQ ID NO:1, wherein the reverse primer of the first primer pair is shown as SEQ ID NO:2, the forward primer of the second primer pair is shown as SEQ ID NO:3, the reverse primer of the second primer pair is shown as SEQ ID NO:4, wherein the forward primer of the third primer pair is shown as SEQ ID NO:5, the reverse primer of the third primer pair is shown as SEQ ID NO:6, the forward primer of the fourth primer pair is shown as SEQ ID NO:7, the reverse primer of the fourth primer pair is shown as SEQ ID NO:8, the forward primer of the fifth primer pair is shown as SEQ ID NO:9, the reverse primer of the fifth primer pair is shown as SEQ ID NO:10, the forward primer of the sixth primer pair is shown as SEQ ID NO:11, the reverse primer of the sixth primer pair is shown as SEQ ID NO:12, the forward primer of the seventh primer pair is shown as SEQ ID NO:13, the reverse primer of the seventh primer pair is shown as SEQ ID NO:14, the forward primer of the eighth primer pair is shown as SEQ ID NO:15, the reverse primer of the eighth primer pair is shown as SEQ ID NO:16, the forward primer of the ninth primer pair is shown as SEQ ID NO:17, the reverse primer of the ninth primer pair is shown as SEQ ID NO:18, the forward primer of the tenth primer pair is shown as SEQ ID NO:19, the reverse primer of the tenth primer pair is shown as SEQ ID NO: shown at 20.
On the other hand, the embodiment of the invention provides a kit for identifying three types of abalone bacteria, which comprises the primer group.
Specifically, the kit further comprises a multiplex PCR premix.
In yet another aspect, an embodiment of the present invention provides an application of the primer set described above, where the application includes: carrying out differentiation and identification of three types of baud bacteria, identification of genetic variation among strains and inside strains and construction of MNP fingerprint databases of the three types of baud bacteria for non-diagnosis purpose on at least one of 10 MNP marking loci on each baud bacteria genome by utilizing the primer group, wherein the sequences of the 10 MNP marking loci are shown as SEQ ID NO:21 to SEQ ID NO:30, wherein the three types of baud bacteria are pertussis baud bacteria, parapertussis Bao Te bacteria and baud bacteria.
The technical scheme provided by the embodiment of the invention has the beneficial effects that: the primer set provided by the embodiment of the invention has higher specificity and stability, and the kit using the primer set has good sensitivity, reproducibility and accuracy. The kit detects 10 MNP marking sites through a primer group, and can be used for identification and differentiation of three types of Bowder bacteria for non-diagnosis purposes, identification of genetic variation among strains and inside strains, and construction of MNP fingerprint databases of the three types of Bowder bacteria.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present disclosure, and other drawings may be obtained according to these drawings without inventive effort for a person of ordinary skill in the art.
Fig. 1 is a sequence similarity diagram of a primer set provided in an embodiment of the present disclosure for detecting mnp_5 marker loci on bordetella pertussis, bordetella parapertussis Bao Te and bordetella bronchiseptica Bao Te.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail below.
The three sources of the abalone fungus provided by the embodiment of the invention are all given to the disease prevention control center in Hubei province.
Example 1
The embodiment of the invention provides a primer group of three types of Bowden bacteria, wherein the three types of Bowden bacteria are pertussis Bowden bacteria, parapertussis Bao Te bacteria and bronchogenic Bao Te bacteria, and the primer group comprises: a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, and a tenth primer pair, each primer pair comprising a forward primer and a reverse primer.
The forward primer of the first primer pair is shown as SEQ ID NO:1, the reverse primer of the first primer pair is shown as SEQ ID NO:2, the forward primer of the second primer pair is shown as SEQ ID NO:3, the reverse primer of the second primer pair is shown as SEQ ID NO:4, the forward primer of the third primer pair is shown as SEQ ID NO:5, the reverse primer of the third primer pair is shown as SEQ ID NO:6, the forward primer of the fourth primer pair is shown as SEQ ID NO:7, the reverse primer of the fourth primer pair is shown as SEQ ID NO:8, the forward primer of the fifth primer pair is shown as SEQ ID NO:9, the reverse primer of the fifth primer pair is shown as SEQ ID NO:10, the forward primer of the sixth primer pair is shown as SEQ ID NO:11, the reverse primer of the sixth primer pair is shown as SEQ ID NO:12, the forward primer of the seventh primer pair is shown as SEQ ID NO:13, the reverse primer of the seventh primer pair is shown as SEQ ID NO:14, the forward primer of the eighth primer pair is shown as SEQ ID NO:15, the reverse primer of the eighth primer pair is shown as SEQ ID NO:16, the forward primer of the ninth primer pair is shown as SEQ ID NO:17, the reverse primer of the ninth primer pair is shown as SEQ ID NO:18, the forward primer of the tenth primer pair is shown as SEQ ID NO:19, the reverse primer of the tenth primer pair is shown as SEQ ID NO: shown at 20.
When implemented, the bases of the primer set may be modified.
The embodiment of the invention provides a kit for identifying three types of abalone bacteria, which comprises primer groups shown in a table 1.
In particular, the kit may further comprise a multiplex PCR premix.
The primers provided by the embodiment of the invention are specifically shown in table 1.
Table 1 shows the sequences of the primer sets
The performance of the primer set was evaluated as follows:
three baud nucleic acid standard substances with known copy numbers are respectively added into human genome DNA, the source of the human genome DNA is a human Huh-7 cell line stored in the laboratory, three mixed simulated samples of three baud bacteria with 1 copy/reaction, 10 copy/reaction and 100 copy/reaction of pathogenic bacteria are prepared, and meanwhile, the set equal volume of sterile water is used as a blank control (namely, a sample with 0 copy/reaction). Results each Bowder bacteria tested a total of 4 samples, 0, 1, 10 and 100 copies/reaction as shown in Table 2, each sample was constructed for 4 consecutive days using the kit provided by the examples of the present invention, i.e., 12 libraries were obtained per sample and 48 libraries were obtained for a total of 4 samples. The 48 libraries were mixed in equimolar amounts, and after mixing, second generation sequencing was performed, 12 sequencing fragment sets were obtained for each sample, and a total of 48 sequencing fragment sets were obtained for 4 samples.
When the library is constructed, the kit provided by the embodiment of the invention is adopted to amplify a sample to obtain an amplified product, the amplified product is connected with a commercial sample label purchased, the library suitable for high-throughput sequencing is obtained, the library is subjected to high-throughput sequencing, and the sequencing result is specifically shown in table 2.
Table 2 shows the sensitivity and stability analysis of the kit
In table 2: the samples of 0 copies/reaction are all blank control groups, and the other 3 gradient samples are all positive sample groups.
Assessment for stability and sensitivity
As can be seen from table 2, in the 12-set repeat experiments (3 replicates per day for a total of 4 days), the close numbers of the sequences as set forth in SEQ ID NOs: 21 to SEQ ID NO:30, the kit provided by the embodiment of the invention is stable in amplification of three types of Bowden bacteria and good in stability in detection of the three types of Bowden bacteria. The 10 copies/reaction sample can stably detect more than 7 MNP marking sites, genotypes can be compared with three types of Bowder bacteria, and the sensitivity of the kit for detecting the three types of Bowder bacteria provided by the embodiment of the invention can be reduced to 10 copies/reaction.
Evaluation for reproduction and accuracy
Based on 12 repeated experiments of each sample, in any two repeated experiments, whether the genotypes of the jointly detected MNP loci are reproducible or not is used for evaluating and detecting the reproducibility and the accuracy of three Botrytis cinerea. Specifically, the paired comparison was performed on 12 sets of data of 100 copies/reaction samples shown in Table 2, and at the same time, the number of MNP marker loci differing in genotype was 0; the evaluation is carried out according to the principle that the reproducible genotypes are considered to be accurate among 2 repeated experiments, wherein the accuracy a=1- (1-r)/2=0.5+0.5r, r represents the reproducibility, and the reproducibility is the ratio of the number of loci with reproducible genotypes to the number of common loci. The results are shown in Table 3 and are specifically as follows:
table 3 shows the evaluation of reproduction rate and accuracy
As can be seen from table 3, the log of the difference between MNP marker main genotypes between different libraries and between different library batches in each sample in the reproducibility experiment is 0, i.e., the reproducibility r=100% and the accuracy a=100%.
Quality control for pollution
Three sequences of Bowden bacteria can be detected in the 1 copy/reaction sample covering at least 1 MNP marker locus. Due to the extreme sensitivity of the detection method using MNP marker loci, a part of Bao Te bacteria sequence was also detected in a part of blank control, and the data pollution in the detection process is easy to generate false positive results. Therefore, the embodiment of the invention sets a blank control sample of 0 copy/reaction sample, 1 copy/reaction and 10 copy/reaction weak positive sample, and carries out repeated detection for 4 continuous days, thereby being capable of being used for preparing a threshold value and a judgment standard for quality control system pollution and target pathogen detection.
The quality control scheme is as follows:
1) The amount of sequencing data is greater than 20 megabases. According to the multiple tests of samples containing one or three of 0 copies/reaction, 1 copy/reaction, 10 copies/reaction and 100 copies/reaction of Bowden, when the sequencing data amount is not more than 20 megabases, the samples containing 10 copies/test and more of Bao Te bacteria can have less than 3 MNP marking sites detected, and the situation that the number of MNP marking sites detected is 3 cannot be satisfied by increasing the sequencing amount; when the sequencing data amount is more than 20 megabases, all samples containing 10 copies/test or more of the Botrytis cinerea have no less than 3 MNP labeling sites detected.
2) According to the signal index S of each Bowden in the test sample T Noise index S of each Bowden in the control c Determining whether the contamination is acceptable, wherein:
noise figure S of blank control c ＝n c /N c Wherein n is c And N c The number of sequenced fragments and the number of total sequenced fragments for each of the baud bacteria in the blank control are represented, respectively.
Signal index S of test sample t ＝n t /N t Wherein n is t And N t Representing the number of sequenced fragments and the number of total sequenced fragments, respectively, for each of the baud bacteria in the test sample.
Setting a test sample containing each Bowden bacteria in a gradient manner according to the expected lower detection limit, and according to the signal-to-noise ratio R, namely S, of the test sample and the blank control t And S is c Is set, a threshold of whether contamination is acceptable.
In order to ensure accuracy and give consideration to sensitivity, the R value set in the embodiment of the present invention is at least 10, i.e. the signal index in the sample is at least 10 times the noise index in the blank.
3) The number of MNP marker loci detected in the test sample is calculated.
In this example, pertussis Bao Te is taken as an example, wherein the R value of the target pathogen is shown in table 4, and in table 4 of this example, the signal index and the noise index in the signal-to-noise ratio calculation are both average values of 12 experimental data.
Table 4 shows the signal to noise ratio of pertussis-baud in the sample
As can be seen from Table 4, the maximum value of the signal-to-noise ratio (maximum signal index 5/average noise index 0.6) of the pertussis Bao Te strain at 1 copy of the sample and the blank is 8.3 and the average value is 4.7, and therefore, the present invention provides that the contamination in the detection system can be judged to be acceptable when the signal-to-noise ratio is not less than 10. In order to avoid high noise ratio caused by excessive amplification of few polluted sites, the invention provides that the number of detection marker sites is added simultaneously as a judging condition for detecting a certain pallium nucleic acid of a sample to be detected. At most 2 MNP marker loci can be detected in 1 copy/reaction samples. The invention therefore provides that:
1. when the signal to noise ratio of a certain Bowder in the sample is not less than 10 times and the detection mark sites are not less than 3, determining that the nucleic acid of the Bowder is detected in the sample.
2. When the signal-to-noise ratio is not more than 1 and the number of detection marks is 0, judging that the nucleic acid of the Botrytis cinerea is not detected in the sample;
3. the other cases are judged as the suspected detection of the nucleic acid of the Bowden in the sample.
By adopting the method for detecting and judging the pathogenic bacteria, the pertussis Bao Te bacteria and the bordetella bronchiseptica contained in the sample can be accurately judged. Therefore, the primer group can sensitively detect three types of Bowden bacteria with the copy number as low as 10 copies/reaction, and has extremely high detection sensitivity and stability.
Assessment of specificity
The DNA of pertussis, pertussis Bao Te, bordetella bronchiseptica Bao Te, mycobacterium tuberculosis, acinetobacter strain, huo Shibao termitis, chlamydia pneumoniae, mycoplasma pneumoniae, EB virus, haemophilus influenzae, varicella zoster virus, cytomegalovirus, herpes simplex virus, human bocavirus, klebsiella pneumoniae, legionella, moraxella catarrhalis, pseudomonas aeruginosa, rickettsia, staphylococcus aureus, streptococcus pneumoniae and streptococcus pyogenes are mixed together according to equal copy numbers to prepare a mixed template, a blank sample is used as a control, three abalone bacteria in the mixed template are detected by adopting the primer set provided by the embodiment of the invention, and 3 repeated experiments are carried out. After analysis is performed according to the quality control scheme and the judgment threshold, the sequencing sequences detected in 3 repeated experiments can only be compared with 10 MNP marking sites provided by the embodiment of the invention; through sequence similarity analysis, three types of Bowden pathogenic bacteria can be detected in 3 repeated experiments, and the minimum signal-to-noise ratio value reaches 402 in 9 signal-to-noise ratio values obtained through 3 repeated experiments respectively carried out by 3 types of Bowden, which shows that the detection rate of the three types of Bowden in the 3 repeated experiments reaches 100%, and therefore, the specificity of identifying and distinguishing the three types of Bowden in the complex template is higher.
Example two
The embodiment of the invention provides an application of a primer group, which comprises the following steps: detecting genetic variation among strains of three types of baud bacteria with non-diagnostic purposes by using primer groups to 10 MNP (MNP) marker loci on the genome of the three types of baud bacteria, wherein the sequences of the 10 MNP marker loci are shown as SEQ ID NO:21 to SEQ ID NO: shown at 30.
The following briefly describes the preparation of MNP (polynucleotide polymorphism) marker loci:
based on complete or partial sequences of genome of 898 different isolates of pertussis baud bacteria, pertussis Bao Te bacteria and bronchogenic Bao Te bacteria disclosed on the net, 10 MNP (MNP) marker loci are obtained through sequence comparison. 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 representative isolates are generally used as reference. Selecting a genome sequence of one representative strain of three types of abalone bacteria as a reference genome, and comparing the genome sequence with the reference genome to obtain single nucleic acid polymorphic loci of each strain of the three types of abalone bacteria;
screening a region with the discrimination DP more than or equal to 0.2 from the candidate polynucleotide polymorphic site regions as MNP marking sites; where dp=d/t, t is the log of comparison when all the small species are compared pairwise in the candidate polynucleotide polymorphic site region, and d is the log of samples of at least two single nucleic acid polymorphism differences in the candidate polynucleotide polymorphic site region.
As an alternative 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 step size is 1bp in the embodiment, so that comprehensive screening is facilitated. Obtaining the sequence shown as SEQ ID NO:21 to SEQ ID NO:30 (mnp_1 to mnp_10), and the starting positions of mnp_1 to mnp_10 on the reference sequence are shown in table 5.
Table 5 shows the initial position of MNP marker loci on the reference sequence
The primer group provided by the embodiment of the invention is used for detecting 6 sub-generation strains of the same pertussis Bao Te bacteria in different periods, 6 samples are sequentially named as K-1-K-6, the average coverage of sequencing of each sample is 1103 times, and all 10 MNP (MNP) marker loci can be detected in each sample. Fingerprint spectra of 6 samples were aligned pairwise, and the results are shown in table 6.
Table 6 shows the detection analysis of 6 samples
As is clear from Table 6, 1 part of (K-2) and 5 parts of pertussis baud bacteria detected together in the same batch all have a major genotype difference of partial sites, and it is seen that there is a variation between strains at different times of the same pertussis Bao Te bacteria. The use of the primer group to identify the genetic variation among strains can be used for ensuring the genetic consistency of the pertussis baud strains named in different laboratories, so that the comparability of research results is ensured, and the method has important significance for scientific research of the pertussis Bao Te bacteria. In clinical terms, one can take into account the diagnostic regimen as to whether the site of the difference affects resistance.
Example III
The embodiment of the invention provides an application of a primer group, which comprises the following steps: carrying out non-diagnostic genetic variation identification on 10 MNP (MNP) marker loci on three Bowden genomes by using a primer group, wherein the sequences of the 10 MNP marker loci are shown as SEQ ID NO in a sequence table: 21 to SEQ ID NO: shown at 30.
The bacteria are used as colony organisms, and during the preservation and propagation process, part of individuals can be mutated to form heterozygous colonies, so that the stability and consistency of the phenotype of the strain for test are affected. Such variants, when detected by molecular marker detection on the population, exhibit an allelic form other than the major genotype of the MNP marker locus. Variant individuals exhibit a low frequency of allelic forms when they make up a significant portion of the population. The low frequency of allele and technical errors, which result in the difficulty of distinguishing between current techniques, requires a determination of the authenticity of the detected minor allele.
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 provided in table 7. Table 7 lists the threshold values that the number of sequenced sequences of the sub-isogenotypes at each site should meet at partial sequencing depth.
Table 7 is the threshold for determining the hypo-isogenotypes at partial sequencing depth
The parameters emax (n=1) and emax (n.gtoreq.2) referred to in Table 7 refer to the maximum value of the frequency of minor alleles of n SNPs obtained and present in the major genotype among the 930 homozygous MNP marker loci investigated in this example. The frequency of the hypogenotype refers to the ratio of the number of sequences of the hypogenotype to the total number of sequences of the MNP marker locus.
Specifically, as can be seen from table 7, when emax (n=1) and emax (n+.2) are 1.03% and 0.0994%, respectively, under the guarantee of α=99.9999% probability calculated based on binom.inv function, the critical value of the number of sequences for sequencing the hypo-genotype in each site is determined as the true hypo-genotype only when the number of sequences for sequencing the hypo-genotype exceeds the critical value. When there are a plurality of candidate minor alleles, multiple corrections are made to the P value of each candidate allele, and FDR (False Discovery Rate ) <0.5% of the candidate alleles are judged to be true minor genotypes.
According to the above parameters, nucleic acids of two strains having a difference in major genotypes provided in table 6 were respectively in the following 8 concentration ratios: 1/1000, 3/1000, 5/1000, 7/1000, 1/100, 3/100, 5/100 and 7/100 to obtain artificial heterozygous samples, and repeatedly detecting each artificial heterozygous sample for 3 times to obtain 24 sequencing data in total. The nucleotide sequence of the two strains shown in SEQ ID NO:21 to SEQ ID NO:30, and detecting the loci with heterozygous genotypes in 24 manual heterozygous samples, which indicates that the loci with heterozygous genotypes exist in the genotypes of MNP marker loci shown in 30, wherein the loci are shown as SEQ ID NO:21 to SEQ ID NO:30 is suitable for the identification of genetic variation inside three types of Botrytis cinerea. In this embodiment, the 10 MNP marker loci are used to identify genetic variation in three types of baud bacteria together, and in other embodiments, any one or a combination of several of the 10 MNP marker loci may be used to identify genetic variation in three types of baud bacteria.
Example IV
The embodiment of the invention provides an application of a primer group, which comprises the following steps: detecting 10 MNP marking sites on each Bowder genome by using the primer group, constructing MNP fingerprint databases of three Bowder bacteria according to the sequences of the obtained MNP marking sites, wherein the sequences of the 10 MNP marking sites are shown as SEQ ID NO in a sequence table: 21 to SEQ ID NO: shown at 30.
And (3) extracting DNA of all strains used for constructing the pertussis baud bacteria MNP fingerprint database by using a conventional CTAB method from 6 copies of the strains K-1 to K-6 provided in the second embodiment, collecting the DNA by agarose gel electrophoresis to obtain a product, and detecting the quality of the DNA by using an ultraviolet spectrophotometer. If the ratio of absorbance values of the extracted DNA at 230nm and 260nm is greater than 2.0, the ratio of absorbance values of 260nm and 280nm is between 1.6 and 1.8, and the DNA electrophoresis main band is obvious, no obvious degradation and RNA residues exist, the genome DNA is proved to reach the relevant quality requirements, and the subsequent sequence comparison can be carried out.
Sequence comparison is carried out on the sequencing data of 6 copies of strains K-1 to K-6 provided in the second embodiment, and SEQ ID NO in a sequence table of each strain is obtained: 21 to SEQ ID NO:30, thereby forming MNP fingerprint of each strain, recording a database file, and constructing an MNP fingerprint database of pertussis Bao Te bacteria.
The constructed MNP fingerprint database of each bacterium is compatible with all high-throughput sequencing data based on the detected gene sequences of each bacterium. 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 real-time updating of the database are realized. In this embodiment, the MNP fingerprint database is constructed by using the 10 MNP marker loci together, and in other embodiments, the MNP fingerprint database may be constructed by using any one or a combination of any several of the 10 MNP marker loci.
Example five
The embodiment of the invention provides an application of a primer group, which comprises the following steps: detecting 10 MNP (MNP) marker loci on each Bowden genome by using the primer group, and performing the application of distinguishing and identifying three Bowden according to the sequence of the obtained MNP marker loci, wherein the sequences of the 10 MNP marker loci are shown as SEQ ID NO in a sequence table: 21 to SEQ ID NO: shown at 30.
As shown in Table 2 of the example and experiments aiming at specificity evaluation, the kit provided by the example of the invention is obtained to obtain sequencing data of nucleic acid samples containing three types of Bowden and genotypes of MNP marker loci, sequencing sequences supporting each genotype are compared with a genome sequence library of three types of Bowden, and the genotypes obtained by the 10 MNP marker loci can be matched with one of the three types of Bowden through sequence similarity analysis. The 48 sets of sequencing data in Table 2 all matched three types of Bowden simultaneously. According to the result of sequence comparison, the signal-to-noise ratio of each Bowden is calculated by using the quality control scheme provided by the first embodiment of the invention, and whether each Bowden is detected is determined.
As shown in the specificity evaluation experiment in the first embodiment, three types of Bowder bacteria can be detected in the mixed template containing three types of Bowder bacteria, and 3 times of repeated experiments are performed, meanwhile, in the embodiment, the sequence similarity of the three types of Bowder bacteria on the genome is verified by taking the MNP_5 marker locus as an example, FIG. 1 shows the sequence similarity, and the pertussis Bowder bacteria, the pertussis Bao Te bacteria and the bronchogenic Bowder bacteria in sequence from left to right, and as can be seen from FIG. 1, the pertussis Bowder bacteria, the pertussis Bao Te bacteria and the bronchogenic Bao Te bacteria can be distinguished aiming at the MNP_5 marker locus, and in the embodiment, the three types of Bowder bacteria can be detected by one MNP marker locus of the MNP_5 marker locus. In addition, three types of abalone bacteria may be identified and distinguished by using any one or a combination of any two of the 10 MNP marker loci, or three types of abalone bacteria may be identified and distinguished by using the 10 MNP marker loci together. The primer group can be used for identifying and distinguishing three abalone bacteria.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
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Sequence listing
<110> wuhan-institute of biotechnology limited, hubei province center for disease prevention and control (national academy of preventive medicine, hubei province)
<120> a primer set and kit for identifying three abalone bacteria and application thereof
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atcggccggc ccttgataaa a 21
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tacgcctggt aagccaaacg 20
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gcaaagtagt gcccccac 18
<210> 9
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<213> Artificial sequence (Artificial Sequence)
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acagttcctc cagccattgc 20
<210> 10
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
atcgagcaga tcacgcatac c 21
<210> 11
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
tacatcccgc tactgcaatc caa 23
<210> 12
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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cgtcttcccc tctgcgttt 19
<210> 13
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<213> Artificial sequence (Artificial Sequence)
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agaaaccata gccaccgcc 19
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gtcagccagc agactcgc 18
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<212> DNA
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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cgtagatctc ggcgcggg 18
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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gaagccgatg agcaccttgg 20
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ctgcgccatg gcctctac 18
<210> 21
<211> 137
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ttggaccgct gcgccaatcc ggctttttca agctccctca gggagctttt ttttttgccg 60
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<210> 22
<211> 143
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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cgccacgccc ttcgacatca agctgaagga atgcccccag gcgctgggcg cgctcaagct 60
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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<210> 24
<211> 150
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
tacgcctggt aagccaaacg cgtcgtgcca ccgcgcgccg gaacacccgg cgcgcggttt 60
tttttgccgc cgggccgccc caaggcaaaa aagccccctc ggggggcagc aagcccgcat 120
agcgggcgca gcgtgggggc actactttgc 150
<210> 25
<211> 139
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
acagttcctc cagccattgc cgattagcgc gcgggtacgc caccttcacc ggcgcttgct 60
catggtgttg catcaccgac tgatgctcca ccgtcgggat gttcttgcgc ttggcttcgg 120
tatgcgtgat ctgctcgat 139
<210> 26
<211> 149
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
tacatcccgc tactgcaatc caacacggca tgaacgctcc ttcggcgcaa agtcgcgcga 60
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<210> 27
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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tgcccagatc gttgatgagc gtgtccc 147
<210> 28
<211> 150
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
gtcagccagc agactcgcgc caatcccaac ccctacacat cgcgaaggtc cgtagcgtcg 60
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<210> 29
<211> 149
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
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cgaagaggtc gcccgcgccg agatctacg 149
<210> 30
<211> 150
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
gaagccgatg agcaccttgg cgccctttag gtcgtgcttg aggcgcacgc ggtcgatcag 60
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ttcctcggcc ttgtagaggc catggcgcag 150

Claims (4)

1. A primer set for identifying three types of baud bacteria, which are pertussis baud bacteria, parapertussis Bao Te bacteria and baud bacteria, characterized in that the primer set comprises: a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, a fifth primer pair, a sixth primer pair, a seventh primer pair, an eighth primer pair, a ninth primer pair, and a tenth primer pair, each of the primer pairs comprising a forward primer and a reverse primer,
the forward primer of the first primer pair is shown as SEQ ID NO:1, wherein the reverse primer of the first primer pair is shown as SEQ ID NO:2, the forward primer of the second primer pair is shown as SEQ ID NO:3, the reverse primer of the second primer pair is shown as SEQ ID NO:4, wherein the forward primer of the third primer pair is shown as SEQ ID NO:5, the reverse primer of the third primer pair is shown as SEQ ID NO:6, the forward primer of the fourth primer pair is shown as SEQ ID NO:7, the reverse primer of the fourth primer pair is shown as SEQ ID NO:8, the forward primer of the fifth primer pair is shown as SEQ ID NO:9, the reverse primer of the fifth primer pair is shown as SEQ ID NO:10, the forward primer of the sixth primer pair is shown as SEQ ID NO:11, the reverse primer of the sixth primer pair is shown as SEQ ID NO:12, the forward primer of the seventh primer pair is shown as SEQ ID NO:13, the reverse primer of the seventh primer pair is shown as SEQ ID NO:14, the forward primer of the eighth primer pair is shown as SEQ ID NO:15, the reverse primer of the eighth primer pair is shown as SEQ ID NO:16, the forward primer of the ninth primer pair is shown as SEQ ID NO:17, the reverse primer of the ninth primer pair is shown as SEQ ID NO:18, the forward primer of the tenth primer pair is shown as SEQ ID NO:19, the reverse primer of the tenth primer pair is shown as SEQ ID NO: shown at 20.
2. A kit for identifying three types of baud bacteria, comprising the primer set of claim 1.
3. The kit of claim 2, further comprising a multiplex PCR premix.
4. The use of the primer set of claim 1, wherein the use comprises: detecting 10 MNP (MNP) marker loci on three types of baud bacteria genome by using the primer group, and carrying out identification, differentiation, inter-strain and intra-strain genetic variation identification of three types of baud bacteria for non-diagnosis purposes according to the sequences of the obtained MNP marker loci and constructing an MNP fingerprint database of the three types of baud bacteria, wherein the sequences of the 10 MNP marker loci are shown as SEQ ID NO:21 to SEQ ID NO:30, wherein the three types of baud bacteria are pertussis baud bacteria, parapertussis Bao Te bacteria and baud bacteria.
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CN113862384A (en) * 2021-08-20 2021-12-31 江汉大学 MNP (protein marker) marker locus of Francisella tularensis, primer composition, kit and application
CN113718045A (en) * 2021-09-18 2021-11-30 上海伯杰医疗科技有限公司北京分公司 DNA fragment, primer, probe and kit for detecting 4 kinds of Bordetella pertussis and specifically detecting Bordetella pertussis and application

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