CN113025761A - Multi-amplification matched high-throughput sequencing method and kit for pathogenic microorganism identification - Google Patents

Multi-amplification matched high-throughput sequencing method and kit for pathogenic microorganism identification Download PDF

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CN113025761A
CN113025761A CN202110582010.0A CN202110582010A CN113025761A CN 113025761 A CN113025761 A CN 113025761A CN 202110582010 A CN202110582010 A CN 202110582010A CN 113025761 A CN113025761 A CN 113025761A
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林东旭
朱方何
袁光孝
李新慧
张陈陈
陈杰
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Guangzhou Sagene Biotech Corp
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Abstract

The invention discloses a multiple amplification matching high-throughput sequencing method and a kit for pathogenic microorganism identification, and the method comprises two steps of amplification, wherein in the first step of amplification, an upstream long primer pool is used for single-ended amplification; and adding a downstream long primer pool, a primer P1 and a primer P2 into a reaction system in the second amplification step, wherein the total three primers form a primer for the second amplification step, and then performing quality control, purification, high-throughput sequencing and data analysis. The invention uses the two-step multiple amplification method for the first time to match with the second-generation sequencing method to detect the pathogenic microorganisms which can not be identified in the traditional clinical method in the human respiratory tract alveolar lavage fluid sample and the blood or plasma sample, has high sensitivity, has 50 pathogenic genome copies per ml at the detection limit, is quick and simple, has less sequencing data amount and reduces the detection cost.

Description

Multi-amplification matched high-throughput sequencing method and kit for pathogenic microorganism identification
Technical Field
The invention relates to the technical field of infectious disease clinical detection, in particular to a multiplex amplification and high-throughput sequencing method and a kit for pathogenic microorganism identification.
Background
The pathogenic microorganisms capable of infecting human are various, and most of them are viruses, bacteria, fungi, parasites, mycoplasma, chlamydia and the like, and they are widely present in human body. Infection through the respiratory tract is the most common form and has a high mortality rate. According to 2016 statistics published by the World Health Organization (WHO) 2018, the lower respiratory tract infection is higher in the fourth of the top ten death causes worldwide and the first rank in infectious diseases, resulting in about 300 million deaths.
The screening of infectious pathogens mainly depends on a separation culture method and an automatic microorganism identification system, although the method has the advantages of simplicity and convenience in operation, low cost and the like, the method also has the defects of low flux, long time consumption, easiness in subjective factor influence on the separation process and the like, and the whole process usually comprises pathogen culture, identification and antimicrobial drug sensitivity test, and needs 2-4 days or even longer. The loop-mediated isothermal amplification is an isothermal rapid nucleic acid amplification method based on DNA polymerase with strand displacement activity, is applied to pathogen detection, and has the advantages of high sensitivity, strong specificity, high reaction speed, no need of special instruments and the like. The micro-fluidic technology has the advantages of miniaturization, integration and automation, and has the advantages of less reagent dosage, low energy consumption and less pollution, but the method has high technical cost and is not beneficial to clinical large-scale pathogenic microorganism screening and popularization in small and medium hospitals. Besides, the methods also comprise common clinical pathogen detection means such as fluorescence qPCR, immunofluorescence and the like, and although the methods are widely accepted in clinic, the methods have the defects of low flux, narrow application disease spectrum and the like. The traditional clinical diagnosis of pathogenic microorganisms considers that the gold standard is still a culture method, but the culture positive rate is very different, for example, the positive rate of blood culture is only 10%.
In recent years, with the maturation and clinical application of the second-generation sequencing technology, the metagenome second-generation sequencing method (mNGS) based on the Illumina Nextseq550 sequencing platform is also gradually accepted clinically and used for detecting a plurality of systemic infectious diseases, including upper and lower respiratory tract infection, blood infection or sepsis, infectious encephalitis or meningitis, infections of the thoracic cavity, the abdominal cavity, the bone joint, the oral cavity and the like. The mNGS method is a technology for carrying out second-generation sequencing detection on sample nucleic acid, belongs to undifferentiated detection, has wide coverage on pathogen detection types, can be used for detecting fungi, bacteria, viruses, parasites and the like, has strong detection capability on new or rare infections, and detects new crown viruses which are outbreaked globally in 2019 years firstly through the mNGS technology. While the mNGS technique has numerous advantages, there are several drawbacks and bruises: 1. the sensitivity is low, the sensitivity of the mNGS detection is always subjected to the following defects due to the limitation of a sequencing method, and although the detection types are many, the detection omission condition occurs for the pathogens with low copy number, such as intracellular bacteria, RNA virus and the like. 2. Host influence, mNGS is the detection of all nucleic acids in clinical samples, so that a large amount of human host nucleic acids have large influence on method interference, a host removing technology is needed to remove the human host nucleic acids, but the host removing technology often influences pathogenic nucleic acids, and finally influences the detection accuracy. 3. The operation time is long and tedious, the sample needs to be subjected to wall breaking, extraction, host removal, interruption, terminal repair, joint connection, multiple purification and other operation links before sequencing of the mNGS metagenome, so that the sequencing can be carried out on the machine, the TAT period is increased, the operation is tedious, and part of pathogenic nucleic acid is lost when an experiment link is added, and the detection rate is finally directly influenced. 4. The cost is high, the detection result accuracy can be ensured only by the mNGS needing the sequencing data volume of basically 20M reads, so the sequencing cost accounts for a large proportion of the total cost, and the cost of the above-mentioned multiple experimental links is added. The high cost also directly leads to high detection service cost on the market, increases the medical expenditure of patients, and also influences the large-scale popularization and application of the technology. Therefore, a pathogenic microorganism identification method which can detect pathogenic species or flux and ensure high detection sensitivity, is rapid and simple and has relatively low cost is required to be developed at present.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a multiplex amplification and high-throughput sequencing method and a kit for pathogenic microorganism identification. The invention relates to a two-step amplification method, wherein in the first step, single-ended amplification is carried out by using an upstream long primer pool; and in the second-step amplification, a downstream long primer pool and joint sequences P1 and P2 are added into a reaction system, and a total of three primers form a primer for the second-step amplification, and then quality control, purification, high-throughput sequencing and data analysis are carried out.
The invention provides a multiplex amplification and high-throughput sequencing method for pathogenic microorganism identification, which comprises the following steps:
s1: manufacturing a primer pool;
mixing the upstream long primer and the downstream long primer which are required to be identified and have the specificity of various pathogens according to an equimolar ratio to obtain an upstream long primer pool and a downstream long primer pool; the upstream long primer sequentially has the following structures: the sequence comprises a joint sequence P1, an index sequence, a sequencing primer 1 and a targeted pathogen sequence upstream amplification primer; the structure of the downstream long primer is as follows in sequence: a joint sequence P2, a sequencing primer 2, a molecular tag UMI sequence, a linker sequence and a downstream amplification primer of a targeted pathogen sequence; the nucleotide sequence of the linker sequence P1 is shown as SEQ ID NO.81, and the nucleotide sequence of the linker sequence P2 is shown as SEQ ID NO. 82; the index sequence is used for distinguishing different samples, one sample corresponds to one index sequence, and multiple index sequences are needed if multiple samples are made at one time. The function of the UMI sequence is to remove amplification-induced interference. The upstream amplification primer of the targeted pathogen sequence and the downstream amplification primer of the targeted pathogen sequence are a section of specific sequence designed by using a bioinformatics method aiming at a target amplification sequence, and meet the requirements of a conventional primer sequence.
S2: amplifying by a two-step method;
(1) the first step of amplification uses the upstream long primer pool to carry out single-ended amplification;
(2) the second step of amplification is to add the downstream long primer pool, a primer P1 and a primer P2 into the reaction system of the step (1), wherein the nucleotide sequences of the primer P1 and the primer P2 are respectively identical to the nucleotide sequence P1 and the nucleotide sequence P2 of the adaptor, and the primers exist in the form of independent primers; three kinds of primers constitute the primer for the second step of amplification;
s3: quality control;
carrying out electrophoresis detection on the product amplified by the two-step method;
s4: purifying;
purifying the products after the two amplifications by using magnetic beads to obtain a purified sequencing library; carrying out accurate quantification and fragment size analysis on the purified sequencing library;
s5: sequencing;
diluting the library according to the requirement of a sequencing platform, mixing samples according to the requirement of data volume by taking the quantitative result of the step S4 as a standard, and sequencing by adopting Illumina Miniseq SE 150;
s6: analyzing data;
and filtering and combining sequencing original data, then performing database comparison, and determining species differentiation and quantitative analysis of pathogens.
Further, the step S1 is preceded by the steps of collecting clinical samples, pre-treating samples, and extracting nucleic acids.
Furthermore, the total length of the upstream long primer is 80-90 bp, and the length of the index sequence is 8-10 bp.
Further, the total length of the downstream long primer is 90-100 bp, the molecular label UMI sequence is composed of 8-15 random bases, the length of the linker sequence is 2-5 bp, and the base composition is variable;
further, the length of the product obtained after the second amplification step is 100-150 bp.
The invention also provides a primer pool, which comprises an upstream long primer and a downstream long primer;
the upstream long primer sequentially has the following structures: the kit comprises a joint sequence P1, an index sequence, a sequencing primer 1 and a targeted pathogen sequence upstream amplification primer, wherein the total length of the upstream primer is 80-90 bp, and the length of the index sequence is 8-10 bp; the nucleotide sequence of the linker sequence P1 is shown as SEQ ID NO. 81;
the structure of the downstream long primer is as follows in sequence: the primer sequence comprises a joint sequence P2, a sequencing primer 2, a molecular tag UMI sequence, a linker sequence and a targeted pathogen sequence downstream amplification primer, wherein the total length of the downstream primer is 90-100 bp, the molecular tag UMI sequence consists of 8-15 random bases, and the length of the linker sequence is 2-5 bp; the nucleotide sequence of the joint sequence P2 is shown as SEQ ID NO. 82.
Further, the structure of the upstream long primer can also be as follows in sequence: the kit comprises a joint sequence P1, an index sequence, a sequencing primer 1, an index sequence and a targeted pathogen sequence upstream amplification primer, wherein the total length of the upstream amplification primer is 80-90 bp, and the length of the index sequence is 8-10 bp; the nucleotide sequence of the linker sequence P1 is shown in SEQ ID NO. 81.
Further, the structures of the downstream long primer can also be as follows in sequence: the primer sequence comprises a joint sequence P2, a molecular tag UMI sequence, a linker sequence, a sequencing primer 2, a molecular tag UMI sequence, a linker sequence and a targeted pathogen sequence downstream amplification primer, wherein the total length of the downstream long primer is 90-100 bp, the molecular tag UMI sequence consists of 8-15 random bases, and the length of the linker sequence is 2-5 bp; the nucleotide sequence of the joint sequence P2 is shown as SEQ ID NO. 82.
The invention also provides a pathogenic microorganism detection kit, which comprises the following components:
DNA polymerase, dNTP, upstream long primer mixture, downstream long primer mixture, primer P1, primer P2 and water; the upstream long primer sequentially has the following structures: the sequence comprises a joint sequence P1, an index sequence, a sequencing primer 1 and a targeted pathogen sequence upstream amplification primer; the structure of the downstream long primer is as follows in sequence: a joint sequence P2, a sequencing primer 2, a molecular tag UMI sequence, a linker sequence and a downstream amplification primer of a targeted pathogen sequence; the nucleotide sequences of the primer P1 and the primer P2 are identical to the linker sequence P1 and the linker sequence P2, respectively, and exist as separate primers; the upstream long primer mixture comprises 40 primers of SEQ ID NO. 1-40, the downstream long primer mixture comprises 40 primers of SEQ ID NO. 41-80, the nucleotide sequence of the primer P1 is shown as SEQ ID NO.81, and the nucleotide sequence of the primer P2 is shown as SEQ ID NO. 82.
In summary, compared with the prior art, the invention achieves the following technical effects:
1. the unique two-step amplification method of the invention has the advantages that the upstream long primer is subjected to one-way amplification in the first step, so that each amplified single-stranded product is ensured to take the input nucleic acid as an amplification template, and amplification taking the amplified product as the template is not generated; the UMI sequence is added in the second amplification step, so that PCR preference can be removed by combining sequences with the same label after subsequent P1 and P2 exponential amplification, and the abundance and the proportion of the pathogen to be detected can be obtained more accurately through analysis and quantification.
2. The unique primer structure design of the invention can complete the library construction link through simple amplification, avoids the traditional steps of nucleic acid breaking, tail end repairing, A adding, joint connecting and the like, greatly improves the library construction efficiency and simplifies the operation flow.
3. The method realizes the matched development with an illumina Miniseq sequencing platform, has low Miniseq flux and short sequencing time, can complete the sequencing within 5 hours, and shortens the period of pathogen infection detection.
4. The invention is a methodology of target amplification matched with second-generation sequencing, so the sequencing data amount is small, and the detection cost is lower than that of the similar methodology.
5. The invention is a nucleic acid segment for highly specific target amplification of the genome of the pathogenic microorganism, so that the clinical sample does not need to be subjected to human source removal operation, and is not influenced by human DNA or RNA, thereby reducing the operation and avoiding the influence of the human source removal host technology on the pathogen ratio.
6. Although only 20 pathogen detections are developed, the number of pathogen species can be increased to 100 or more actually based on the multiplex amplification technology, and the increased pathogen species flux can provide more information for clinical judgment.
7. The invention can specifically amplify weak signals by adopting a PCR amplification technology, and has obvious detection effect on pathogens such as fungi, intracellular bacteria or RNA viruses with low copy number or difficult detection compared with a metagenome sequencing method.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic diagram of the structure and specific sequence example of the long upstream primer and the long downstream primer of the present invention.
FIG. 2 is a schematic flow diagram of the two-step amplification method of the present invention.
FIG. 3 is a diagram of agarose gel electrophoresis of the purified amplified magnetic beads of example 2 of the present invention.
FIG. 4 is a schematic flow chart of sequencing data analysis according to the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, shall fall within the scope of protection of the present invention.
The experimental procedures used in the following examples are conventional unless otherwise specified. The materials, reagents and the like used are commercially available unless otherwise specified.
The microorganism identification method of the present invention specifically comprises the steps of:
1. sample treatment:
(1) collecting and transporting: clinical samples of the patient were collected.
(2) Pretreatment: performing extraction pretreatment according to the type requirements of the sample, performing wall breaking treatment if the sample is an alveolar lavage fluid sample, and performing plasma separation treatment if the sample is a blood sample.
(3) Nucleic acid extraction: extracting DNA/RNA. And detecting the concentration and purity of the extracted nucleic acid.
2. Before amplification:
(1) manufacturing a primer pool: mixing the upstream long primer and the downstream long primer which are required to be identified and are specific to various pathogens according to an equimolar ratio to obtain an upstream long primer pool and a downstream long primer pool.
(2) RNA reverse transcription: random primer reverse transcription was performed according to kit instructions to generate the first strand of cDNA.
3. Amplification:
according to the invention, library construction is completed by two-step amplification, as shown in FIG. 2, magnetic bead purification is not needed after the first-step amplification, amplification products are directly subjected to secondary amplification, and gel cutting recovery is not needed, so that the possibility of pollution in the operation process is avoided.
(1) The first step of amplification is single-ended amplification using an upstream long primer pool. The upstream long primer structure: the sequence comprises a joint sequence P1, an index sequence (used for distinguishing different samples), a sequencing primer 1 and a pathogen-targeted sequence upstream amplification primer.
(2) And in the second step of amplification, a downstream long primer is added for amplification in the same reaction, and primers P1 and P2 at two ends are also added, wherein three primers in total form a primer for secondary amplification (the primer sequences are shown in a sequence table, and the nucleotide sequences of P1 and P2 are respectively SEQ ID NO.81 and SEQ ID NO. 82). The structure of the downstream long primer is as follows: the kit comprises a linker sequence P2, a sequencing primer 2, a molecular tag UMI sequence (used for removing interference introduced by amplification) consisting of 8-15 random bases, a linker sequence (2-5 bp), and a targeted pathogen sequence downstream amplification primer. The structures of the sequences of the upstream long primer and the downstream long primer are shown in FIG. 1.
The long primer structure design also comprises a single-ended or double-ended added molecular label (UMI), a single-ended or double-ended added index sequence and a linker sequence.
The upstream long primer can also have the following structure: the kit comprises a joint sequence P1, an index sequence, a sequencing primer 1, an index sequence and a targeted pathogen sequence upstream amplification primer, wherein the total length of a long primer is 80-90 bp, and the length of the index sequence is 8-10 bp; the nucleotide sequence of the linker sequence P1 is shown in SEQ ID NO. 81.
The downstream long primer may also have the following structure: the primer sequence comprises a joint sequence P2, a molecular tag UMI sequence, a linker sequence, a sequencing primer 2, a molecular tag UMI sequence, a linker sequence and a targeted pathogen sequence downstream amplification primer, wherein the total length of the downstream long primer is 90-100 bp, the molecular tag UMI sequence consists of 8-15 random bases, the length of the linker sequence is 2-5 bp, and the base composition is variable; the nucleotide sequence of the joint sequence P2 is shown as SEQ ID NO. 82.
4. Quality control QC
And (4) carrying out electrophoresis detection on the product amplified by the two-step method.
5. Purification of
(1) And purifying the products obtained after the two times of amplification by using magnetic beads to obtain a purified library.
(2) The purified sequencing library was subjected to precise quantitation and fragment size analysis.
Pooling sequencing
Diluting the library according to the requirements of a sequencing platform, mixing samples according to the data quantity requirements by taking the quantitative result in the step 5 as a standard, and sequencing by adopting Illumina Miniseq SE 150.
7. Data analysis
The sequencing raw data was filtered and all UMI-repeated and sequence-repeated reads were pooled. And then performing database comparison, and determining the differentiation and quantitative analysis of the detected reads species.
Example 1 procedure for identification of pathogenic microorganisms of the present invention
The method specifically comprises the following steps:
1. sample treatment:
(1) collecting and transporting: clinical samples of patients are collected, and different collection and transportation modes are adopted according to different sample types.
(2) Pretreatment: performing extraction pretreatment according to the type requirements of the sample, performing wall breaking treatment if the sample is an alveolar lavage fluid sample, and performing plasma separation treatment if the sample is a blood sample.
(3) Nucleic acid extraction: DNA/RNA was extracted using a DNA and RNA co-extraction kit developed by Secho (Yue ear instrument 20200771) according to the procedures of the instructions. The concentration and purity of the extracted nucleic acid were measured using nanodrop2000/qubit 3.0.
2. Before amplification:
(1) manufacturing a primer pool: and mixing the 20 kinds of pathogen specific upstream long primers and downstream long primers to be identified according to an equimolar ratio to prepare an upstream long primer pool and a downstream long primer pool.
(2) RNA reverse transcription: random primer reverse transcription was performed according to kit instructions to generate the first strand of cDNA.
3. Amplification:
the invention completes library construction by two-step amplification, the flow is shown in figure 2, magnetic bead purification is not needed after the first-step amplification, amplification products are directly amplified for the second time, and gel cutting recovery is not needed, so that the possibility of pollution in the operation process is avoided.
(1) The amplification of the first step is single-ended amplification by using 40 upstream long primer pools (the nucleotide sequences of the primers are shown in SEQ ID No. 1-40). The upstream long primer structure: the sequence comprises a joint sequence P1, an index sequence (used for distinguishing different samples), a sequencing primer 1 and a pathogen-targeted sequence upstream amplification primer. The total length of the long upstream primer is 80-90 bp, wherein the index sequence is 8-10 bp, and 8 bp is adopted in the embodiment.
First step amplification PCR reaction (22. mu.l):
Figure 16751DEST_PATH_IMAGE001
the first amplification step sets the following reaction program:
Figure 154471DEST_PATH_IMAGE002
(2) in the second step of amplification, 40 downstream long primers (the nucleotide sequence of the primers is shown in SEQ ID NO. 41-80) are added to perform amplification in the same reaction, and primers P1 and P2 at two ends are added, wherein a total of the three primers form a primer for secondary amplification. The structure of the downstream long primer is as follows: the primer sequence comprises a linker sequence P2, a sequencing primer 2, a molecular tag UMI sequence (for removing interference introduced by amplification, 10 bp is adopted in the embodiment), a linker sequence (2-5 bp is adopted in the embodiment, 3 bp is adopted in the embodiment) consisting of 8-15 random bases, and a targeted pathogen sequence downstream amplification primer. The total length of the downstream long primer is 90-100 bp, and the length of the amplification product of the long primer and the short primers at two ends is 100-150 bp.
Second step amplification PCR reaction (50. mu.l):
Figure 351097DEST_PATH_IMAGE003
the second amplification step sets the following reaction program:
Figure 826465DEST_PATH_IMAGE004
4. quality control QC
And 5-10 mul of products amplified by the two-step method is taken for agarose gel electrophoresis detection. The electrophorogram showed that the sample amplified bands were of a size consistent with that expected, and the bands were bright and free of non-specific amplified bands.
5. Purification of
(1) And purifying the product after the two times of amplification by using AMPureXP magnetic beads of Beckman & Coulter company, adding 75 mu l of magnetic bead suspension into a reaction system, fully and uniformly blowing, uniformly mixing, and standing at room temperature for 5 min. And (3) sucking the supernatant, discarding, washing twice by using 80% ethanol, standing at room temperature for 5min, airing the magnetic beads, and adding 20 mu l of lowTE to elute the magnetic beads to obtain a purified library.
(2) The purified sequencing library was subjected to exact quantitation and fragment size analysis using qubit3.0 and Agilent 2100.
Pooling sequencing
Diluting the library according to the requirement of a sequencing platform, mixing samples according to the data quantity requirement by taking the quantitative result of the Qubit as a standard, and sequencing by adopting Illumina Miniseq SE 150.
7. Data analysis
The sequencing raw data was filtered and all UMI-repeated and sequence-repeated reads were pooled. And then performing database comparison, and determining the differentiation and quantitative analysis of the detected reads species.
Example 2 multiplex amplification reference Strain Studies
1. The pathogen composition is as follows: according to the data accumulated before and some published literatures or research reports, 20 pathogenic microorganisms common to respiratory tract and blood infections are selected as research objects, and the types are shown in the following table:
Figure 829057DEST_PATH_IMAGE005
2. preparation of reference strain nucleic acid: all the standard reference strains of 20 pathogenic microorganisms are purchased from Guangdong province strain preservation center, corresponding strains are respectively inoculated on corresponding culture dishes in a super clean bench, and the strains are cultured for 12 to 24 hours at respective proper temperature. Individual colonies were picked, inoculated into 5 ml of liquid medium and cultured overnight. Nucleic acid was extracted from 1ml of the bacterial suspension using QIAamp DNA Microbiome Kit according to the instructions, and the concentration and purity of the extracted nucleic acid were measured using Qubit3.0 and Agilent2100, and the nucleic acid was stored at-20 ℃ for further use.
3. Primer design and verification: downloading whole genome reference sequences of 20 pathogens from a public database, screening and obtaining a plurality of homologous conserved regions dispersed on a genome as primer design targets through bioinformatics analysis of sequence ratio pairs, performing multiple primer design by using autonomous development software, obtaining 2 target regions specifically representing the pathogens through analysis of each pathogen, designing 2 pairs of primers for each target region, and obtaining 40 pairs of specific primers in total, wherein the following table shows that:
Figure 24546DEST_PATH_IMAGE006
the amplicon length was kept around 100 bp. 40 pairs of primers are designed, and two primers specific to each pathogen are used as templates for carrying out single amplification.
The reaction system is as follows:
Figure 719969DEST_PATH_IMAGE007
the PCR reaction procedure was as follows:
Figure 430436DEST_PATH_IMAGE008
results all strains amplified the desired size, bright single band product, no non-specific amplification, and the amplification products were confirmed by Sanger sequencing. In order to further verify the specificity of the primers, 20 pathogenic nucleic acids are mixed to serve as an amplification template, and a single-pair primer amplification result shows no non-specific amplification band.
4. Examination of detection limits
(1) A long primer pool: the long primer is formed by adding the tested primer sequence to the adaptor sequence, and then synthesized. And (3) mixing the synthesized 40 upstream long primers (the nucleotide sequences of the 40 upstream long primers are shown in SEQ ID No. 1-SEQ ID No. 40) and 40 downstream long primers (the nucleotide sequences of the 40 downstream long primers are shown in SEQ ID No. 41-SEQ ID No. 80) according to an equimolar ratio to obtain upstream and downstream long primer pools, wherein the upstream and downstream long primer pools are fully mixed on a shaking instrument.
(2) Quantification and dilution: performing absolute quantification on nucleic acids of 4 target strains of staphylococcus aureus, pseudomonas aeruginosa, escherichia coli and candida albicans, and sequentially diluting to 10 degrees in a gradient manner6、105、104、103、102、101copy/ml, blank control module was sterile water.
(3) Amplification: the amplification was carried out using a two-step amplification method, the amplification conditions and steps being as follows:
first step amplification PCR reaction (22. mu.l):
Figure 158090DEST_PATH_IMAGE009
the first amplification step sets the following reaction program:
Figure 67140DEST_PATH_IMAGE010
second step amplification PCR reaction (50. mu.l):
Figure 871148DEST_PATH_IMAGE011
the second amplification step sets the following reaction program:
Figure 68911DEST_PATH_IMAGE012
dilution gradient of 106The electrophoresis after the purification of the amplified magnetic beads is shown in figure 3, and the four pathogen amplified bands are single and bright on the electrophoresis picture without nonspecific amplification and dimer pollution. Will 104、103、102、101And (5) carrying out on-machine sequencing on a Miniseq sequencer after the amplification product is purified.
(4) Sequencing data analysis: the flow of analysis of the off-line data is shown in fig. 4:
and filtering and controlling the sequencing off-line data by using Fastp software, comparing filtered reads with a pathogen database, and outputting a comparison result by using samtools software. Comparing with pathogen database of bacteria and fungi respectively by using BWA, and outputting all comparison results. And then combining reads with the same sequence of the same UMI by using Fastp software, finally annotating the combined comparison result, and counting the number of the reads of each pathogenic bacterium. The statistical results are as follows:
Figure 350988DEST_PATH_IMAGE013
as can be seen from the above table, the detection limit of the methodology of the present invention is 50 copies of the pathogen genome per ml, and the detection limit is very low, so that the method can achieve higher sensitivity.
Example 3 multiplex amplification clinical sample Studies
To demonstrate the higher sensitivity of the method of the invention compared to conventional clinical methods, the following comparative experiments were performed.
1. And (3) collecting clinical samples:
alveolar Lavage Fluid (BALF) of 10 severe patients and blood samples of 10 severe patients were collected from the ICU of the cooperative hospital, the patients were between 35 and 80 years of age, and 10 men and women, respectively, had a SOFA score of 1 to 13.
2. Sample treatment:
all 20 samples were identified using conventional clinical methodology: BALF and blood cultures, antibody detection and qPCR detection. Another specimen from the same patient was subjected to the methodological pretreatment of the invention:
1) alveolar lavage fluid: taking 0.5 ml of sample, firstly carrying out treatment for 30 min by using a shaker and glass bead wall breaking, and then extracting by using a taimen nucleic acid extraction kit (Yuexian mechanical equipment 20200771) according to an operation instruction.
2) Blood: fresh 8 ml blood samples were first centrifuged at high speed in two steps to separate plasma, and 3 ml of plasma was extracted for cfDNA according to the QiagencfDNAextraction kit instructions.
3. Amplification sequencing analysis: the same as in example 1.
4. Pathogen identification comparison:
Figure 567074DEST_PATH_IMAGE014
(B: alveolar lavage fluid; P: plasma)
As can be seen from the comparison results, the pathogens detected by the common clinical method are all detected by the method (see B2, B4, B7, B10, P2, P3, P7 and P9), wherein the mixed infection of the two pathogens can be detected by using the method of the invention on the B2 sample and the B10 sample, but only one pathogen is successfully detected by the common clinical method; pathogenic bacteria which are not detected by the common clinical method can also be detected by the method of the invention (see B1, B3, B6, B8, P4 and P5). In addition, pathogenic microorganisms which are failed to culture or have limited detection types and are missed can be detected through multiple amplification sequencing. The method of the present invention is therefore an effective complement to clinical testing.
EXAMPLE 4 detection kit for pathogenic microorganisms prepared by the method of the present invention
The kit comprises the following components: DNA polymerase, dNTP, upstream long primer mixture, downstream long primer mixture, primer P1, primer P2 and water; the upstream long primer mixture comprises 40 primers of SEQ ID NO. 1-40, the downstream long primer mixture comprises 40 primers of SEQ ID NO. 41-80, the nucleotide sequence of the primer P1 is shown as SEQ ID NO.81, and the nucleotide sequence of the primer P2 is shown as SEQ ID NO. 82.
Wherein the DNA polymerization and dNTPs can also be present in the kit as a mixture (Mix) in order to save sample addition steps. Firstly, extracting a clinical sample, carrying out corresponding pretreatment, then extracting nucleic acid, and measuring the concentration and purity of the nucleic acid, wherein the measured nucleic acid is used as a template. Two-step amplification was then performed. The first step of amplification is single-ended amplification using the upstream long primer mixture; in the second-step amplification, the downstream long primer mixture is added into the reaction system of the first-step amplification, the primers P1 and P2 are also added, and a total of three primers form a primer of the second-step amplification; quality control, purification, high throughput sequencing and data analysis were then performed, and the steps after the two-step amplification are detailed in example 1. The kit disclosed by the invention is used for detecting 20 pathogenic microorganisms with numbers of 1-20 mentioned in the table of the embodiment 2, but according to the method disclosed by the invention, specific upstream long primers and downstream long primers can be designed for other pathogenic microorganisms (such as RNA viruses) so as to widen the detection range.
In conclusion, in the two-step amplification method, the upstream long primer is subjected to unidirectional amplification in the first step, so that each amplified single-stranded product is ensured to take the input nucleic acid as an amplification template, and amplification taking the amplified product as the template is not generated; the addition of UMI sequences for the second amplification step ensures that PCR bias can be removed by pooling the same tagged sequences after subsequent P1 and P2 exponential amplifications. The invention uses the two-step multiple amplification method for the first time to match with the second-generation sequencing method to detect the pathogenic microorganisms which can not be identified in the traditional clinical method in the human respiratory tract alveolar lavage fluid sample and the blood or plasma sample, the sensitivity is high, the detection limit is 50 pathogenic genome copies per ml, compared with the prior art, the method is quick and simple, and the result can be obtained only in 5 hours. The invention is a methodology of targeted amplification coupled with second-generation sequencing, so that the amount of sequencing data is small, and the detection cost is lower than that of the similar methodology.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
SEQ ID NO.1
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCATCAAAGCGGATACCCAGT
SEQ ID NO.2
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCTTGAGATGACCTGAGGACGA
SEQ ID NO.3
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGAGTCGCTTTTTCGGGACATC
SEQ ID NO.4
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCGAAATGGCGGTGGCGGTA
SEQ ID NO.5
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAATCGGGATATCCTTCGGTAA
SEQ ID NO.6
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTGCACTTCCACTCGAACTTG
SEQ ID NO.7
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTATGAGCACGTTGAGAGTTT
SEQ ID NO.8
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTCTATCTCGAATGCGAACC
SEQ ID NO.9
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTAGCAGCACCTTAGCATCCT
SEQ ID NO.10
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTCTACCGGTCTAGTTGT
SEQ ID NO.11
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAAGACACCGAGACAGGG
SEQ ID NO.12
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCGACACTCGTTTCGCAGG
SEQ ID NO.13
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGAGTACCATGAGGCGCT
SEQ ID NO.14
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTATCTGTTGATCTTACGGAT
SEQ ID NO.15
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTCCCATACCACGCTCCACG
SEQ ID NO.16
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGATCCGGGATTGGGGAT
SEQ ID NO.17
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTACGTTCTCGTTGTAGGC
SEQ ID NO.18
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCAAACGACGCAACGTT
SEQ ID NO.19
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGACTTCCTCATAATCGT
SEQ ID NO.20
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAATTCGCACCTTCGAACG
SEQ ID NO.21
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGCTGTACGCGCGCTATTA
SEQ ID NO.22
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACAAGGCCTGAGCATGGC
SEQ ID NO.23
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCATGCGCAGGTTCTCGT
SEQ ID NO.24
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTTCAGCTGGATCGGCAG
SEQ ID NO.25
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGTCTGACCTGATCTTGCGTA
SEQ ID NO.26
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATTCATGCGACTACATGCACC
SEQ ID NO.27
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCTGCCGGAGATGGTGAG
SEQ ID NO.28
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCAACACCATCTTATTGCTCCTTG
SEQ ID NO.29
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCTGTCGACACAAGCTCTTAATCA
SEQ ID NO.30
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACATTAAGTTATTGATGTCGGTTTCT
SEQ ID NO.31
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGACGCTTCTGGTTGAACTCCAT
SEQ ID NO.32
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAACAAGGGGGTTTTGGCTG
SEQ ID NO.33
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCCTAGACTGCTTCTCGAACC
SEQ ID NO.34
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAAGGGCCGACGAACTCG
SEQ ID NO.35
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCCGACTACGTCACCCCG
SEQ ID NO.36
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAGCATTTCAGCCAAATTTGCC
SEQ ID NO.37
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTGGAAATGCACGCAGACT
SEQ ID NO.38
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAAAGCTGGCTACGTAAGGGA
SEQ ID NO.39
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGTAGCTGTTTTTATCGCGCT
SEQ ID NO.40
CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGCACGGCGTATCCTATCG
SEQ ID NO.41
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGGGGATAACACCTTCAGGC
SEQ ID NO.42
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGCCTCCATCACCTCTAACCC
SEQ ID NO.43
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAACCATGGATGCTCAGCACGGA
SEQ ID NO.44
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGTGACGGCGAGATGTTCCT
SEQ ID NO.45
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGCCAATCACAGGCGGTGATG
SEQ ID NO.46
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACAAGCACTGGGTCAGAAGGT
SEQ ID NO.47
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAACTTTCCATTGCCTCGCGGTT
SEQ ID NO.48
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGGCGAAACCTTCGAGTTTAACG
SEQ ID NO.49
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGTGTCTCGTTTCCAACAGCTTT
SEQ ID NO.50
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGGACCACCAACTATGGCCG
SEQ ID NO.51
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACAGCAAGGACCTTAACAATG
SEQ ID NO.52
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGATGTGGCTCATCTCCAAGAGG
SEQ ID NO.53
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACTCATCTGCACCAGAATGAC
SEQ ID NO.54
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAAATACCACGGTACCGAGAT
SEQ ID NO.55
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGTTGCACGTGGCCTTTGC
SEQ ID NO.56
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAACTACGTATCGAGCCTTTTGGGTT
SEQ ID NO.57
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGGCTGCTCCTTCGTGCATC
SEQ ID NO.58
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGCGGTACTTGATTATGTCTACATCA
SEQ ID NO.59
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACGAGTGAATTGTAAGCTGTGCC
SEQ ID NO.60
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACTTAAGTCGTGGCCTACCAT
SEQ ID NO.61
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACATGACCAGCTGGATCAGGT
SEQ ID NO.62
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAACCTTGGTCAGCTGGTCCA
SEQ ID NO.63
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACATCGAAGGCCGCGACAT
SEQ ID NO.64
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGGCGAAAACTCGTAGTGCG
SEQ ID NO.65
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACATGACCAGCTGGATCAGGT
SEQ ID NO.66
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGGAAAATCTTCATTGGCTTTGGC
SEQ ID NO.67
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACCAACAGTGTAATAAGTAACTCGGC
SEQ ID NO.68
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGCGGTACTTGATTATGTCTATTCATC
SEQ ID NO.69
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACGGGGCAGAAGAGATAGAATCA
SEQ ID NO.70
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGATGCTGATAGGAGATAGGTTGGGTA
SEQ ID NO.71
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACCGACGGTCAGAAACTGGTC
SEQ ID NO.72
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACCGCACGCCATAAATCCCTTCACC
SEQ ID NO.73
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAATCAGTCGACAAATTAGACCCAAAA
SEQ ID NO.74
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAAGACGATGTCACCGGCGAAG
SEQ ID NO.75
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAGTGGTCAATATCGAATGCGTTTAAA
SEQ ID NO.76
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACACGCCATAAATCCCTTCACC
SEQ ID NO.77
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAACCGCTACAGCAAAGAAAGTTT
SEQ ID NO.78
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACGATATGATCGCGGTCGGAT
SEQ ID NO.79
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGAACAACTTATCGCACGTCTCCT
SEQ ID NO.80
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNCGACAAAATCGTCATAAAAATCCAACCG
SEQ ID NO.81
CAAGCAGAAGACGGCATACGAGAT
SEQ ID NO.82
AATGATACGGCGACCACCGAGATC
SEQUENCE LISTING
<110> Guangzhou Setaimen Biotechnology GmbH
<120> pathogenic microorganism identification multiple amplification and high-throughput sequencing method and kit
<130> 2021.05.20
<160> 82
<170> PatentIn version 3.5
<210> 1
<211> 86
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 1
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctcatc aaagcggata cccagt 86
<210> 2
<211> 88
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 2
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctcctt gagatgacct gaggacga 88
<210> 3
<211> 88
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 3
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctagag tcgctttttc gggacatc 88
<210> 4
<211> 86
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 4
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatcttcga aatggcggtg gcggta 86
<210> 5
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 5
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctaatc gggatatcct tcggtaa 87
<210> 6
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 6
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctttgc acttccactc gaacttg 87
<210> 7
<211> 86
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 7
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatcttatg agcacgttga gagttt 86
<210> 8
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 8
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctactc tatctcgaat gcgaacc 87
<210> 9
<211> 86
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 9
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatcttagc agcaccttag catcct 86
<210> 10
<211> 86
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 10
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctgtct ctaccggtct agttgt 86
<210> 11
<211> 84
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 11
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctgaag acaccgagac aggg 84
<210> 12
<211> 86
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 12
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctctcg acactcgttt cgcagg 86
<210> 13
<211> 84
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 13
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatcttgag taccatgagg cgct 84
<210> 14
<211> 86
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 14
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatcttatc tgttgatctt acggat 86
<210> 15
<211> 86
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 15
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctttcc cataccacgc tccacg 86
<210> 16
<211> 84
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 16
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctggat ccgggattgg ggat 84
<210> 17
<211> 85
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 17
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctctac gttctcgttg taggc 85
<210> 18
<211> 84
<212> DNA
<213> Artificial sequence
<220>
<223> CAAGCAGAAGACGGCATACGAGATCTAAGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC
TCTCAAACGACGCAACGTT
<400> 18
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctctca aacgacgcaa cgtt 84
<210> 19
<211> 84
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 19
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctcgac ttcctcataa tcgt 84
<210> 20
<211> 84
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 20
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctaatt cgcaccttcg aacg 84
<210> 21
<211> 85
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 21
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatcttgct gtacgcgcgc tatta 85
<210> 22
<211> 84
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 22
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctacaa ggcctgagca tggc 84
<210> 23
<211> 84
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 23
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctccat gcgcaggttc tcgt 84
<210> 24
<211> 85
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 24
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatcttgtt cagctggatc ggcag 85
<210> 25
<211> 88
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 25
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctaagt ctgacctgat cttgcgta 88
<210> 26
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 26
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctattc atgcgactac atgcacc 87
<210> 27
<211> 84
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 27
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctgctg ccggagatgg tgag 84
<210> 28
<211> 90
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 28
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatcttcaa caccatctta ttgctccttg 90
<210> 29
<211> 90
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 29
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatcttctg tcgacacaag ctcttaatca 90
<210> 30
<211> 92
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 30
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctacat taagttattg atgtcggttt ct 92
<210> 31
<211> 88
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 31
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctgacg cttctggttg aactccat 88
<210> 32
<211> 86
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 32
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctaaac aagggggttt tggctg 86
<210> 33
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 33
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctccct agactgcttc tcgaacc 87
<210> 34
<211> 84
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 34
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctgaag ggccgacgaa ctcg 84
<210> 35
<211> 84
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 35
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatcttccg actacgtcac cccg 84
<210> 36
<211> 88
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 36
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctcagc atttcagcca aatttgcc 88
<210> 37
<211> 86
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 37
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctcttg gaaatgcacg cagact 86
<210> 38
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 38
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctcaaa gctggctacg taaggga 87
<210> 39
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 39
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctcgta gctgttttta tcgcgct 87
<210> 40
<211> 85
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 40
caagcagaag acggcatacg agatctaagt cggtgactgg agttcagacg tgtgctcttc 60
cgatctggca cggcgtatcc tatcg 85
<210> 41
<211> 90
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 41
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aggggataac accttcaggc 90
<210> 42
<211> 91
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 42
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg agcctccatc acctctaacc c 91
<210> 43
<211> 92
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 43
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aaccatggat gctcagcacg ga 92
<210> 44
<211> 90
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 44
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg agtgacggcg agatgttcct 90
<210> 45
<211> 91
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 45
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg agccaatcac aggcggtgat g 91
<210> 46
<211> 91
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 46
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg acaagcactg ggtcagaagg t 91
<210> 47
<211> 92
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 47
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aactttccat tgcctcgcgg tt 92
<210> 48
<211> 93
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 48
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aggcgaaacc ttcgagttta acg 93
<210> 49
<211> 93
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 49
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg agtgtctcgt ttccaacagc ttt 93
<210> 50
<211> 90
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 50
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aggaccacca actatggccg 90
<210> 51
<211> 91
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 51
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg acagcaagga ccttaacaat g 91
<210> 52
<211> 91
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 52
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg atgtggctca tctccaagag g 91
<210> 53
<211> 91
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 53
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg actcatctgc accagaatga c 91
<210> 54
<211> 90
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 54
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aaataccacg gtaccgagat 90
<210> 55
<211> 89
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 55
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg agttgcacgt ggcctttgc 89
<210> 56
<211> 95
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 56
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aactacgtat cgagcctttt gggtt 95
<210> 57
<211> 90
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 57
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aggctgctcc ttcgtgcatc 90
<210> 58
<211> 96
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 58
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg agcggtactt gattatgtct acatca 96
<210> 59
<211> 93
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 59
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg acgagtgaat tgtaagctgt gcc 93
<210> 60
<211> 91
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 60
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg acttaagtcg tggcctacca t 91
<210> 61
<211> 91
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 61
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg acatgaccag ctggatcagg t 91
<210> 62
<211> 90
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 62
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aaccttggtc agctggtcca 90
<210> 63
<211> 89
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 63
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg acatcgaagg ccgcgacat 89
<210> 64
<211> 90
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 64
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aggcgaaaac tcgtagtgcg 90
<210> 65
<211> 91
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 65
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg acatgaccag ctggatcagg t 91
<210> 66
<211> 94
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 66
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aggaaaatct tcattggctt tggc 94
<210> 67
<211> 96
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 67
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg accaacagtg taataagtaa ctcggc 96
<210> 68
<211> 97
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 68
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg agcggtactt gattatgtct attcatc 97
<210> 69
<211> 93
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 69
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg acggggcaga agagatagaa tca 93
<210> 70
<211> 95
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 70
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg atgctgatag gagataggtt gggta 95
<210> 71
<211> 91
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 71
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg accgacggtc agaaactggt c 91
<210> 72
<211> 95
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 72
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg accgcacgcc ataaatccct tcacc 95
<210> 73
<211> 96
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 73
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aatcagtcga caaattagac ccaaaa 96
<210> 74
<211> 91
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 74
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aagacgatgt caccggcgaa g 91
<210> 75
<211> 96
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 75
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg agtggtcaat atcgaatgcg tttaaa 96
<210> 76
<211> 92
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 76
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg acacgccata aatcccttca cc 92
<210> 77
<211> 93
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 77
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aaccgctaca gcaaagaaag ttt 93
<210> 78
<211> 91
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 78
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg acgatatgat cgcggtcgga t 91
<210> 79
<211> 92
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 79
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg aacaacttat cgcacgtctc ct 92
<210> 80
<211> 96
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> misc_feature
<222> (59)..(68)
<223> n is a, c, g, or t
<400> 80
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatctnn 60
nnnnnnnncg acaaaatcgt cataaaaatc caaccg 96
<210> 81
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 81
caagcagaag acggcatacg agat 24
<210> 82
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Artificial sequence
<400> 82
aatgatacgg cgaccaccga gatc 24

Claims (9)

1. A multiplex amplification and high-throughput sequencing method for pathogenic microorganism identification is characterized by comprising the following steps:
s1: manufacturing a primer pool;
mixing the upstream long primer and the downstream long primer which are required to be identified and have the specificity of various pathogens according to an equimolar ratio to obtain an upstream long primer pool and a downstream long primer pool; the upstream long primer sequentially has the following structures: the sequence comprises a joint sequence P1, an index sequence, a sequencing primer 1 and a targeted pathogen sequence upstream amplification primer; the structure of the downstream long primer is as follows in sequence: a joint sequence P2, a sequencing primer 2, a molecular tag UMI sequence, a linker sequence and a downstream amplification primer of a targeted pathogen sequence; the nucleotide sequence of the linker sequence P1 is shown as SEQ ID NO.81, and the nucleotide sequence of the linker sequence P2 is shown as SEQ ID NO. 82;
s2: amplifying by a two-step method;
(1) the first step of amplification uses the upstream long primer pool to carry out single-ended amplification;
(2) the second step of amplification is to add the downstream long primer pool, a primer P1 and a primer P2 into the reaction system of the step (1), wherein the nucleotide sequences of the primer P1 and the primer P2 are respectively identical to the nucleotide sequence P1 and the nucleotide sequence P2 of the adaptor, and the primers exist in the form of independent primers; three kinds of primers constitute the primer for the second step of amplification;
s3: quality control;
carrying out electrophoresis detection on the product amplified by the two-step method;
s4: purifying;
purifying the products after the two amplifications by using magnetic beads to obtain a purified sequencing library; carrying out accurate quantification and fragment size analysis on the purified sequencing library;
s5: sequencing;
diluting the library according to the requirement of a sequencing platform, mixing samples according to the requirement of data volume by taking the quantitative result of the step S4 as a standard, and sequencing by adopting Illumina Miniseq SE 150;
s6: analyzing data;
and filtering and combining sequencing original data, then performing database comparison, and determining species differentiation and quantitative analysis of pathogens.
2. The method of claim 1, wherein the step of S1 is preceded by the steps of collecting clinical specimens, pre-treating specimens, and extracting nucleic acids.
3. The method according to claim 1, wherein the upstream primer has a total length of 80-90 bp, and the index sequence has a length of 8-10 bp.
4. The method according to claim 1, wherein the total length of the downstream long primer is 90-100 bp, the molecular tag UMI sequence is composed of 8-15 random bases, and the length of the linker sequence is 2-5 bp.
5. The method of claim 1, wherein the length of the product after the second amplification step is 100-150 bp.
6. A primer pool, wherein the primer pool comprises an upstream long primer and a downstream long primer;
the upstream long primer sequentially has the following structures: the kit comprises a joint sequence P1, an index sequence, a sequencing primer 1 and a targeted pathogen sequence upstream amplification primer, wherein the total length of the upstream primer is 80-90 bp, and the length of the index sequence is 8-10 bp; the nucleotide sequence of the linker sequence P1 is shown as SEQ ID NO. 81;
the structure of the downstream long primer is as follows in sequence: the primer sequence comprises a joint sequence P2, a sequencing primer 2, a molecular tag UMI sequence, a linker sequence and a targeted pathogen sequence downstream amplification primer, wherein the total length of the downstream primer is 90-100 bp, the molecular tag UMI sequence consists of 8-15 random bases, and the length of the linker sequence is 2-5 bp; the nucleotide sequence of the joint sequence P2 is shown as SEQ ID NO. 82.
7. The primer pool of claim 6, wherein the structure of the upstream long primer is further: the kit comprises a joint sequence P1, an index sequence, a sequencing primer 1, an index sequence and a targeted pathogen sequence upstream amplification primer, wherein the total length of the upstream amplification primer is 80-90 bp, and the length of the index sequence is 8-10 bp; the nucleotide sequence of the linker sequence P1 is shown in SEQ ID NO. 81.
8. The primer pool of claim 6, wherein the structure of the downstream long primer is further: the primer sequence comprises a joint sequence P2, a molecular tag UMI sequence, a linker sequence, a sequencing primer 2, a molecular tag UMI sequence, a linker sequence and a targeted pathogen sequence downstream amplification primer, wherein the total length of the downstream long primer is 90-100 bp, the molecular tag UMI sequence consists of 8-15 random bases, and the length of the linker sequence is 2-5 bp; the nucleotide sequence of the joint sequence P2 is shown as SEQ ID NO. 82.
9. A pathogenic microorganism detection kit is characterized by comprising the following components:
DNA polymerase, dNTP, upstream long primer mixture, downstream long primer mixture, primer P1, primer P2 and water; the upstream long primer sequentially has the following structures: the sequence comprises a joint sequence P1, an index sequence, a sequencing primer 1 and a targeted pathogen sequence upstream amplification primer; the structure of the downstream long primer is as follows in sequence: a joint sequence P2, a sequencing primer 2, a molecular tag UMI sequence, a linker sequence and a downstream amplification primer of a targeted pathogen sequence; the nucleotide sequences of the primer P1 and the primer P2 are identical to the linker sequence P1 and the linker sequence P2, respectively, and exist as separate primers; the upstream long primer mixture comprises 40 primers of SEQ ID NO. 1-40, the downstream long primer mixture comprises 40 primers of SEQ ID NO. 41-80, the nucleotide sequence of the primer P1 is shown as SEQ ID NO.81, and the nucleotide sequence of the primer P2 is shown as SEQ ID NO. 82.
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