CN116287357A - Respiratory tract pathogenic bacteria detection kit based on targeted amplicon sequencing - Google Patents
Respiratory tract pathogenic bacteria detection kit based on targeted amplicon sequencing Download PDFInfo
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Abstract
The invention provides a respiratory tract pathogenic bacteria detection kit based on targeted amplicon sequencing, and relates to the technical field of microorganism detection. The method for detecting respiratory tract pathogenic bacteria adopts a targeting amplicon sequencing technology and combines a multiplex PCR technology aiming at clinical suspected respiratory tract infection samples, and compared with other targeting detection technologies, the method can realize semi-quantitative detection of different detection targets by introducing a single-molecule identification tag sequence, and has higher accuracy. The method disclosed by the invention is economical, rapid, high in specificity and accuracy, and meanwhile, the result is easy to interpret, so that the method has a great application value in the aspect of respiratory pathogen detection.
Description
Technical Field
The invention belongs to the technical field of microorganism detection, and particularly relates to a respiratory tract pathogenic bacteria detection kit based on targeted amplicon sequencing.
Background
Respiratory tract infection (Respiratory Tract Infections) is an infectious disease caused by invasion and multiplication of pathogens from nasal cavities, throats, trachea, bronchi and other parts of a human body, and is a common infectious disease. Pathogens of respiratory tract infections can be classified into bacterial, viral, fungal, mycoplasma, chlamydia, rickettsia, and the like. The traditional separation culture and serological detection method is time-consuming and tedious, and results in the defects of untimely detection, low sensitivity, low positive rate and the like.
Metagenomic second generation sequencing (mNGS) can be applied to respiratory disease pathogen detection, and mNGS has higher sensitivity and higher detection speed, but in practical application, there are some problems which are difficult to solve: the method has low detection sensitivity to microorganisms with low extraction efficiency of nucleic acid such as intracellular bacteria and fungi, low detection sensitivity to low-concentration intracellular infectious bacteria such as mycobacterium tuberculosis and Legionella, high host background, difficult unification of sequencing result standards, high cost and the like, and limits the wide application of the method.
Chinese patent CN202110024096.5 discloses a primer composition, a sequencing kit and a detection method, wherein the composition comprises a first primer composition, a second primer composition and a 16S primer pair, the first primer composition comprises a plurality of primer pairs for detecting viral pathogens causing respiratory tract infection, the second primer composition comprises a plurality of primer pairs for detecting bacterial pathogens causing respiratory tract infection, and the 16S primer pair is used for detecting pathogens causing respiratory tract infection simultaneously with the first primer composition or the second primer composition. The primer composition provided by the invention can detect more than 30 respiratory pathogens simultaneously, and partial pathogens can be subjected to subtype distinction according to a sequencing sequence.
Chinese patent CN202111356118.4 discloses a composition, kit, method and use for detecting pathogens causing respiratory tract infection and identifying pathogen species, said composition comprising an upstream and a downstream primer and probe of influenza a virus, an upstream and a downstream primer and probe of influenza b virus, an upstream and a downstream primer and probe of rhinovirus, an upstream and a downstream primer and probe of respiratory tract adenovirus, said composition of the invention in combination with fluorescent probe method can be carried out simultaneously in one test using two tubes, its cost is low and flux is high. The method has the advantages that a single test tube can give out information of 4 targets, the operation is simple, the result reading process can be judged through CT values, and false positive and environmental pollution caused by cross infection among samples are avoided.
The Chinese patent CN202011098442.6 discloses a respiratory tract infection pathogen nucleic acid joint detection kit, which comprises a primer probe combination, wherein the primer probe combination comprises an upstream primer and a downstream primer for detecting novel coronaviruses, influenza A viruses, influenza B viruses, respiratory syncytial viruses, human parainfluenza viruses, adenoviruses, mycoplasma pneumoniae and chlamydia pneumoniae and a probe, and the kit has the advantages of good detection accuracy, strong specificity, high sensitivity, good repeatability, low false negative and false positive, and can effectively identify respiratory tract infection caused by new coronavirus infection and common viral influenza or bacteria, thereby realizing accurate diagnosis and subsequent accurate treatment of patients.
The respiratory tract pathogenic bacteria detection method based on targeted sequencing provided by the invention can enrich target pathogenic microorganisms without removing human genome DNA. The detection kit can realize simultaneous detection of staphylococcus aureus, acinetobacter baumannii, streptococcus pneumoniae, pseudomonas aeruginosa and streptococcus pyogenes, can quickly obtain detection results by matching with a rapid sequencing technology, realizes rapid and instant detection, and is applied to diagnosis of respiratory tract infectious diseases.
Disclosure of Invention
Terminology and statement of the invention:
in the present invention, the term "nucleotide sequence" refers to the arrangement order of bases in DNA or RNA.
In the present invention, the term "primer" refers to two oligonucleotide sequences synthesized artificially, one primer being complementary to one DNA template strand at one end of the target gene and the other primer being complementary to the other DNA template strand at the other end of the target gene.
As used herein, the term "pathogenic bacteria" refers to microorganisms, including bacteria, fungi, viruses, etc., which are capable of invading a host to cause infection, and which are capable of producing pathogenic agents that cause infection of the host.
In the present invention, the term "Pseudomonas aeruginosaPseudomonas aeruginosa，P.aeruginosa) "is the most common conditional pathogen of Pseudomonas species responsible for respiratory tract infection, wound infection.
In the invention, the term Acinetobacter baumanniiAcinetobacter baumannii，A.baumannii) "is a gram-negative, nonfermented Brevibacterium.
In the present invention, the term Streptococcus pneumoniaeStreptococcus pneumoniae，S.pneumoniae) "is a conditionally pathogenic gram-positive coccus.
In the present invention, the term "Staphylococcus aureusStaphylococcus aureus，S.aureus) "is a gram-positive bacterium belonging to the genus Staphylococcus.
In the present invention, the term Streptococcus pyogenesStreptococcus pyogenes，S.pyogenes) "is a group of human-host streptococci, which are widely present in the faeces and nasopharyngeal sites of humans and cause common human-specific pathogenic bacteria ranging from common non-invasive diseases to invasive and severely invasive diseases.
In order to effectively detect the respiratory tract pathogenic bacteria, the invention provides a kit for detecting the respiratory tract pathogenic bacteria and application thereof, and the kit can realize semi-quantitative detection of different detection targets by introducing a single molecule identification tag sequence, has stronger specificity and higher accuracy, and simultaneously has a great application value in the aspect of respiratory tract pathogenic bacteria detection, and the result is easy to interpret.
The technical scheme of the invention comprises the following steps:
in a first aspect, the invention provides a primer combination for detecting respiratory pathogens, the primer combination comprising an upstream primer of pseudomonas aeruginosa, a downstream primer of pseudomonas aeruginosa, an upstream primer of acinetobacter baumannii, a downstream primer of acinetobacter baumannii, an upstream primer of streptococcus pneumoniae, a downstream primer of streptococcus pneumoniae, an upstream primer of staphylococcus aureus, a downstream primer of staphylococcus aureus, an upstream primer of streptococcus pyogenes, and a downstream primer of streptococcus pyogenes;
the nucleotide sequence of the upstream primer of the pseudomonas aeruginosa is shown as SEQ ID NO. 1;
the nucleotide sequence of the downstream primer of the pseudomonas aeruginosa is shown as SEQ ID NO. 2;
the nucleotide sequence of the upstream primer of the Acinetobacter baumannii is shown as SEQ ID NO. 3;
the nucleotide sequence of the downstream primer of the Acinetobacter baumannii is shown as SEQ ID NO. 4;
the nucleotide sequence of the upstream primer of the streptococcus pneumoniae is shown as SEQ ID NO. 5;
the nucleotide sequence of the downstream primer of the streptococcus pneumoniae is shown as SEQ ID NO. 6;
the nucleotide sequence of the upstream primer of staphylococcus aureus is shown as SEQ ID NO. 7;
the nucleotide sequence of the downstream primer of staphylococcus aureus is shown as SEQ ID NO. 8;
the nucleotide sequence of the upstream primer of the streptococcus pyogenes is shown as SEQ ID NO. 9;
the nucleotide sequence of the downstream primer of the streptococcus pyogenes is shown as SEQ ID NO. 10.
In yet another aspect, the invention provides the use of a primer combination as described above for the preparation of a kit for the detection of respiratory pathogens.
In yet another aspect, the invention provides a kit for detecting a respiratory pathogen, said kit comprising a primer combination according to the first aspect of the invention.
Preferably, the respiratory pathogenic bacteria include staphylococcus aureus, acinetobacter baumannii, streptococcus pneumoniae, pseudomonas aeruginosa and streptococcus pyogenes.
Preferably, the kit comprises a forward primer F1 and a reverse primer R1;
further preferably, the forward primer F1 comprises a connecting sequence Read 1, a single molecule identification tag sequence SMB and the upstream primer sequence, which are connected end to end in the 5'-3' direction.
Further preferably, the nucleotide sequence of the connecting sequence Read 1 is shown as SEQ ID NO. 11;
further preferably, the sequence of the single molecule recognition tag SMB is NNNNNNNN, wherein N represents any one of A, T, C, G.
Preferably, the forward primer F1 has the following (5 '-3') sequence base composition:
F1：CTACACGACGCTCTTCCGATCT（SEQ ID NO:11）+ SMB +Primer F。
preferably, the reverse primer R1 comprises a ligation sequence Read 2 and the above downstream primer sequence, which are ligated end to end in the 5'-3' direction.
Further preferably, the nucleotide sequence of the connecting sequence Read 2 is shown as SEQ ID NO. 12.
Preferably, the reverse primer R1 has the following (5 '-3') sequence base composition:
R1：GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC（SEQ ID NO:12）+Primer R。
preferably, the kit comprises a second round of PCR amplification primers, wherein the second round of PCR amplification primers comprise a forward primer F2 and a reverse primer R2; the nucleotide sequence of the forward primer F2 is shown as SEQ ID NO. 13, and the nucleotide sequence of the reverse primer R2 is shown as SEQ ID NO. 14.
Specifically, the sequence base composition of the reverse primer R2 is as follows (5 '-3'):
CAAGCAGAAGACGGCATACGAGAT+Index+GTGACTGGAGTTCAGACGTGT（SEQ ID NO:14）；
specifically, the Index identifies the barcode sequence for the sample. The nucleotide sequence of the Index is any Index suitable for an Illumina sequencer.
Optionally, the kit can also comprise Phanta Flash Super-Fidelity DNA Polymerase, dNTP and MgCl 2 An optimized buffer system.
Specifically, the buffer solution is at least one selected from Tris buffer solution, borax buffer solution, phosphate buffer solution, HEPES buffer solution and MOPS buffer solution.
Preferably, the reaction system for detecting respiratory tract pathogenic bacteria comprises the following components in parts by volume: 1-5 parts of the primer combination and 20-30 parts of high-fidelity enzyme buffer solution; the reaction system for detecting respiratory tract pathogenic bacteria comprises 1-5 parts of templates according to parts by weight.
Further preferably, the reaction system for detecting respiratory tract pathogenic bacteria comprises the following components in parts by volume: 2 parts of primer combination and 25 parts of high-fidelity enzyme buffer solution; the reaction system for detecting respiratory tract pathogenic bacteria comprises 2 parts of templates according to parts by weight.
Preferably, the final concentration of the system of the upstream primer is 10 mu M;
preferably, the final system concentration of the downstream primer is 10. Mu.M;
preferably, the total volume of the reaction system for detecting respiratory pathogens is 50. Mu.L.
Preferably, the first round PCR amplification reaction procedure of the kit is: pre-denaturation at 98 ℃ for 2 min, and circulation for 1 time; denaturation at 98℃for 10 sec, annealing at 58℃for 15 sec, elongation at 72℃for 30 sec, cycling 20 times; extending for 1 min at 72deg.C, circulating for 1 time, and preserving at 4deg.C.
Further preferably, the second round PCR amplification reaction procedure of the kit is: pre-denaturation at 98 ℃ for 2 min, and circulation for 1 time; denaturation at 98℃for 10 sec, annealing at 58℃for 15 sec, elongation at 72℃for 30 sec, cycle 6 times; extending for 1 min at 72deg.C, circulating for 1 time, and preserving at 4deg.C.
Preferably, the sequencing result of the kit is used to identify respiratory pathogens in the sample to be tested by the following analytical steps:
(1) Removing low quality and over short sequences;
(2) The residual sequence is aligned with a target sequence of the pathogenic microorganism;
(3) Counting the reading number of the corresponding target on the accurate comparison;
(4) Analyzing SMB sequences corresponding to the same reading number, and if the SMB sequences are the same, considering that the SMB sequences are derived from the same mother chain template;
(5) Counting the number of SMBs corresponding to the different numbers of the SMBs respectively;
(6) The cut off value of the target sequence of the pathogenic microorganism is identified, the accurate alignment reading number is more than 3, and the SMB is more than 1.
Preferably, the invention provides a method for detecting respiratory tract pathogenic bacteria, comprising the following steps:
s1, obtaining DNA of pathogenic microorganisms;
s2, performing a first round of PCR amplification by using DNA of pathogenic microorganisms as a template and using primer combinations of SEQ ID NO. 1-10 to obtain a first round of PCR amplification product;
s3, carrying out second-round PCR amplification by using the first-round PCR amplification product obtained in the step S2 as a template and utilizing a forward primer F2 and a reverse primer R2 to obtain a second-round PCR amplification product;
s4, carrying out high-throughput sequencing on the second round of PCR amplification product obtained in the step S3, and identifying respiratory tract pathogenic bacteria in the sample to be tested according to a sequencing result.
Specifically, the first round of PCR amplification in S2 comprises a first round of PCR amplification primer;
further specifically, the first round PCR amplification primers include a forward primer F1 and a reverse primer R1.
Preferably, the template DNA used in the first round of PCR amplification in S2 has a content of 100 pg.
Preferably, the system of the first round of PCR amplification reaction in S2 comprises:
Wherein the Primer Mix is the Primer combination, the primers are mixed in equal proportion, the final concentration of each Primer is 10 mu M, the volume is 2 mu L, and the RNase-free ddH 2 O was made up to 50. Mu.L.
Specifically, the second round PCR amplification reaction system in the step S3 comprises the following steps: the amplified DNA product of the first round of PCR was used as a template, 2X Phanta Flash Master Mix. Mu.L, 10. Mu.M upstream primer 2. Mu.L, 10. Mu.M downstream primer 2. Mu.L, RNase-free ddH 2 O was made up to 50. Mu.L.
In some embodiments of the invention, the first round and the second round of PCR amplification products are purified after the first round and the second round of PCR amplification are completed, respectively.
Specifically, the purification mode includes, but is not limited to, enzymatic digestion and magnetic bead purification.
In some embodiments of the invention, the purification is performed using nupraise magnetic beads.
Specifically, in S4, the step of library quality control is included before the second round of PCR amplification products obtained in S3 are subjected to high throughput sequencing.
More specifically, the library quality control includes, but is not limited to, determining library concentration using QPCR, agilent Bioanalyzer 2100 determining library fragment size.
In some embodiments of the invention, the high throughput sequencing is performed by Illumina sequencing methods, including but not limited to Illumina MiSeq, nova Seq sequencing platforms.
In some embodiments of the invention, the method of detecting respiratory pathogens is a method based on non-diagnostic, non-therapeutic purposes.
The beneficial effects of the invention include:
(1) The accuracy is high: according to the invention, through the design of the SMB single-molecule tag sequence, each original template nucleic acid is marked, so that each original template nucleic acid only corresponds to one SMB tag, and therefore, each detection target PCR amplification error is corrected, semi-quantitative can be carried out on pathogenic microorganisms, and the detection accuracy is high.
(2) The sensitivity is high: the primer combination and the method adopted by the invention enrich pathogenic bacteria targets through multiplex PCR, can detect trace nucleic acid existing in a sample, and have high detection sensitivity.
(3) The cost is low: the invention only carries out multiplex PCR sequencing on target pathogenic bacteria in the detection range, and compared with the mNSS method widely adopted in the prior art, the invention has the advantages of no pollution of human genome and low required sequencing data volume, thus the cost of the invention is relatively low.
(4) The flux is high: the invention can detect a plurality of pathogenic bacteria at one time aiming at the same sample, and can realize the simultaneous detection of a plurality of samples in one sequencing.
Drawings
FIG. 1 is a graph of the results of the 100 pg initial Agilent Bioanalyer 2100 assay.
Fig. 2 is a graph of the detection result of 10 pg at the beginning Agilent Bioanalyer 2100.
FIG. 3 is a graph of the results of the initial Agilent Bioanalyer 2100 test of FIG. 1 pg.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Example 1 primers for detection of respiratory bacteria
1. In this example, two amplification primers, namely, forward (F) and reverse (R), were designed for pseudomonas aeruginosa, acinetobacter baumannii, streptococcus pneumoniae, staphylococcus aureus and streptococcus pyogenes, respectively, as shown in table 1 below:
TABLE 1 pathogenic microorganism amplification primers
Example 2 detection of respiratory pathogens
DNA of pathogenic bacteria was purchased from beijing north na allied biotechnology institute and tested using the primer combination of example 1.
The specific implementation steps are as follows:
(1) Performing first round amplification by taking DNA of pathogenic bacteria as a template;
in the first round of PCR amplification, the Primer comprises a forward Primer F1 and a reverse Primer R1, wherein the forward Primer F1 comprises a connecting sequence Read 1, a single molecule identification tag sequence SMB and a target gene specific Primer F, which are connected end to end in the 5'-3' direction.
The connection sequence Read 1: CTACACGACGCTCTTCCGATCT (SEQ ID NO: 11);
the base sequence composition of forward primer F1 is as follows (5 '-3'):
F1：CTACACGACGCTCTTCCGATCT（SEQ ID NO:11）+ SMB +Primer F ；
wherein SMB is a single molecule identification tag, and the sequence is NNNNNNNN, wherein N represents any one of A, T, C, G.
The reverse Primer R1 comprises a Read 2 and a target gene specific Primer R, which are connected end to end in the 5'-3' direction.
The connection sequence Read 2: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC (SEQ ID NO: 12);
the sequence base composition of the reverse primer R1 is as follows (5 '-3'):
R1：GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC（SEQ ID NO:12） +Primer R。
(2) Performing a second round of PCR amplification by taking the first round of PCR amplification product as a template; after amplification, a library meeting the sequencing requirements can be obtained.
The primers for the second round of PCR amplification include forward primer F2 and reverse primer R2.
The base sequence composition of forward primer F2 is as follows (5 '-3'):
AATGATACGGCGACCACCGAGATCTACACTCTTTCC+CTACACGACGCTC（SEQ ID NO:13）；
reverse primer R2:
the sequence base composition of R2 is as follows (5 '-3'):
CAAGCAGAAGACGGCATACGAGAT+Index+GTGACTGGAGTTCAGACGTGT（SEQ ID NO:14）；
wherein the nucleotide sequence of Index is any Index suitable for an Illumina sequencer.
(3) Multiplex PCR first round amplification of target genes;
preparing a target gene multiplex PCR amplification system by a reaction system:
pathogenic bacteria template DNA Mix was started according to 100 pg, 2X Phanta Flash Master Mix. Mu.L, 10. Mu.M upstream primer composition 2. Mu.L, 10. Mu.M downstream primer composition 2. Mu.L, RNase-free ddH 2 O was made up to 50. Mu.L.
The reaction system is specifically shown in table 2 below:
TABLE 2 first round PCR reaction System
The target gene multiplex PCR amplification procedure was set up as follows:
pre-denaturation at 98 ℃ for 2 min, and circulation for 1 time; denaturation at 98℃for 10 sec, annealing at 58℃for 15 sec, elongation at 72℃for 30 sec, cycling 20 times; extending for 1 min at 72 ℃, and circulating for 1 time; preserving at 4 ℃.
(4) Purifying target gene multiplex PCR first round amplification products;
the purification of PCR amplified products using Norflua magnetic beads comprises the following steps:
(1) taking out the magnetic beads of the North praise from the refrigerator at the temperature of 4 ℃ 30 min in advance, reversing and uniformly mixing, and standing to balance the temperature to the room temperature.
(2) After the magnetic beads of the Nuo-vozan are fully and evenly mixed, the magnetic beads of the 1X-Nuo-vozan are added into an EP tube with low adsorption of 0.2 and mL, all PCR products are transferred into the EP tube, and the mixture is evenly mixed by low-speed vortex oscillation and stands for 5 min at room temperature.
(3) The tube was placed on a magnetic rack for about 2 min until the liquid was clear, and the supernatant was carefully aspirated with a pipette without aspirating the beads.
(4) 200. Mu.L of 80% ethanol was added thereto, and the mixture was allowed to stand for 30 sec, whereupon the supernatant was carefully pipetted off without pipetting the beads.
(5) 200. Mu.L of 80% ethanol was added, left to stand for 30 sec, and the supernatant was carefully pipetted off without any magnetic beads, and centrifuged briefly. Placing on a magnetic rack for about 2 min, and sucking residual liquid at the bottom of the tube by using a 20 mu L pipette after the magnetic rack adsorbs the magnetic beads, without sucking the magnetic beads.
(6) Air-drying at room temperature for 2-5 min until the magnetic beads are not reflected, and air-drying the magnetic beads until cracks appear.
(7) The EP tube was removed from the magnet holder, 20. Mu.L of sterile water was added, mixed well by low-speed vortex shaking, allowed to stand at room temperature for 5 min, centrifuged briefly, the tube was placed on the magnet holder for 2 min until the liquid was clear, the supernatant carefully aspirated, taking care not to aspirate magnetic beads, and transferred to a new 0.2 mL sterile EP tube.
(5) Multiplex PCR second round amplification of target genes;
using the DNA product amplified by the first round of PCR as a template, 2X Phanta Flash Master Mix. Mu.L, 2. Mu.L of the upstream primer (10. Mu.M), 2. Mu.L of the downstream primer (10. Mu.M), RNase-free ddH 2 O was made up to 50. Mu.L.
The target gene multiplex PCR amplification procedure was set up as follows:
pre-denaturation at 98 ℃ for 2 min, and circulation for 1 time; denaturation at 98℃for 10 sec, annealing at 58℃for 15 sec, elongation at 72℃for 30 sec, cycle 6 times; extending for 1 min at 72 ℃, and circulating for 1 time; preserving at 4 ℃.
(6) Purifying target gene multiplex PCR second round amplification products;
the PCR amplified product was purified using nupraise magnetic beads and by double-screen purification:
(1) taking out the magnetic beads of the North praise from the refrigerator at the temperature of 4 ℃ 30 min in advance, reversing and uniformly mixing, and standing to balance the temperature to the room temperature.
(2) After the magnetic beads of the nuozheng are fully and uniformly mixed, 35 mu L of the magnetic beads of the nuozheng are added into a 0.2 mL low-adsorption EP tube, a certain PCR product is transferred into the EP tube, and the mixture is uniformly mixed by low-speed vortex oscillation and stands for 5 min at room temperature.
(3) The tube was placed on a magnetic rack for about 2 min until the liquid was clear, and the supernatant was carefully pipetted into a new 0.2 mL low adsorption EP tube without sucking up the magnetic beads.
(4) 10 mu L of nuozhen magnetic beads are added into the 0.2 mL low-adsorption EP tube in the last step, and the mixture is uniformly mixed by low-speed vortex oscillation and stands for 5 min at room temperature.
(5) The tube was placed on a magnetic rack for about 2 min until the liquid was clear, and the supernatant was carefully aspirated with a pipette without aspirating the beads.
(6) 200. Mu.L of 80% ethanol was added thereto, and the mixture was allowed to stand for 30 sec, whereupon the supernatant was carefully pipetted off without pipetting the beads.
(7) 200. Mu.L of 80% ethanol was added, left to stand for 30 sec, and the supernatant was carefully pipetted off without any magnetic beads, and centrifuged briefly. Placing on a magnetic rack for about 2 min, and sucking residual liquid at the bottom of the tube by using a 20 mu L pipette after the magnetic rack adsorbs the magnetic beads, without sucking the magnetic beads.
(8) Air-drying at room temperature for 2-5 min until the magnetic beads are not reflected, and taking care not to allow the magnetic beads to air-dry until cracks appear.
(9) The EP tube was removed from the magnet holder, 16. Mu.L of sterile water was added, mixed well by low-speed vortex shaking, allowed to stand at room temperature for 5 min, centrifuged briefly, the tube was placed on the magnet holder for 2 min until the liquid was clear, the supernatant carefully aspirated, taking care not to aspirate magnetic beads, and transferred to a new 0.2 mL sterile EP tube.
(7) Library quality control
Taking 1 mu L of the PCR amplified product after the second round of purification for QPCR quantitative detection;
the QPCR quantitative detection comprises the following steps:
absolute quantification is adopted, a standard substance is prepared, a system is prepared, an upper computer program is selected for detection, and the concentration of a library sample is calculated according to a constructed standard curve and a detection result.
The quantitative results are shown in Table 3:
TABLE 3 quantitative results of sample library QPCR
1 μl of the second round of purified PCR amplification product was used to determine library fragment size using Agilent Bioanalyzer 2100, as shown in figures 1-3.
The experimental steps are as follows:
according to Agilent Bioanalyzer 2100, a chip is prepared, 1 mu L of sample to be measured is added, the sample is uniformly mixed in a uniformly mixing instrument for 1 min, a 2100 detector is opened, a page is opened by clicking software, the chip is placed in a chip groove, and after the chip is fixed, a cover is closed. A specific program is selected and the operation is started.
All libraries were mixed into a single vial in a certain ratio to give a total library with a final concentration of 3 or more nM.
The criteria for library quality control were:
1) The concentration is more than or equal to 3 nM (QPCR quantitative concentration);
2) The residual volume after library detection is more than or equal to 10 mu L;
3) The detection result shows that no obvious adapter dimer (peaks near 130 bp);
4) The detection results showed that the library main band was concentrated, single, and close to the reference size.
(8) High throughput sequencing
Performing on-machine sequencing on the obtained library by using an Illumina sequencing platform;
the on-machine sequencing comprises the following steps:
1) Denaturation (denaturation)
The above-prepared mixed library was denatured together with Illumina sequencing reagent Phix (for balancing bases).
2) The Flow cell is placed in a small tube of saline solution, taken out, repeatedly rinsed with ultra pure water, and the saline solution is rinsed clean. And then carefully wiped clean with a piece of mirror wiping paper for later use.
3) Opening Illumina experiment manager software, selecting a sequencer and other programs, putting various reagents, flow cells, buffers and waste liquid bottles, and clicking Start Run after confirming that the solution is correct.
(9) Data analysis
The data analysis steps of the machine are as follows:
1) Removing low quality and over short sequences using FastQC;
2) Comparing the filtered residual sequence with a target sequence of the pathogenic microorganism;
3) Counting and accurately comparing the numbers of reads of the corresponding targets;
4) The SMB sequences corresponding to the same reads were analyzed and if the SMB sequences were identical, they were considered to originate from the same master template.
5) And counting the number of different reads corresponding to different SMBs respectively, namely the initial number of a certain detection target.
6) The cut off value of the target sequence of the pathogenic microorganism is accurately aligned with the number of reads being more than 3 and the SMB being more than 1.
Experimental results:
the template initial amount set in this example was 100 pg, and the detection results are shown in table 4 below;
TABLE 4 example 1 sequencing result data statistics
The detection result shows that:
accuracy:
starting with a template of 100 pg, the numbers of reads are different from the numbers of reads detected, and the number of reads detected by staphylococcus aureus is 149,962 at most; the number of reads detected by Streptococcus pyogenes was minimal, 16,940. However, from the viewpoint of detecting the number of the SMB, the number of the molecular tags corresponding to the detection of the five bacteria is not greatly different, the number of the S.pyogenes and the S.aureus detected the SMB is 3,043 and 2,412 respectively, and the number of the reads represents all the numbers of amplified products and the number of the SMB represents the amount of the templates which are input in a reaction way, so that the number of the SMB can represent the real template amount more. According to the invention, through SMB single-molecule label sequence design, each original template nucleic acid is marked to correct each detection target PCR amplification error, meanwhile, the influence of different primer amplification efficiencies is reduced, and the accuracy of a detection result is higher.
Comparative example 1
Comparative example 1 differs from example 1 described above in that the primer composition was replaced with the reported primer composition for detecting respiratory pathogens, and the other conditions were unchanged.
Primer sequences are shown in table 5 below:
table 5 reported primers for detecting respiratory pathogens
Example 3
The sensitivity of the primer combination of example 1 and the primer combination of comparative example 1 was examined using the detection procedure in example 2.
Sensitivity:
this example 3 was also provided with different template initiation amounts of 100 pg, 10 pg and 1 pg, respectively, to test the sensitivity of the primer combinations of example 1 and comparative example 1.
The test results of example 1 are shown in table 6 below;
TABLE 6 statistics of sequencing results for the primer set of example 1
The results of the experiment of comparative example 1 are shown in tables 7 to 8 below:
TABLE 7 quantitative results of library QPCR of comparative example 1
TABLE 8 statistics of sequencing results for comparative example 1 primer set
As can be seen from the sensitivity experiment result table, the pathogenic bacteria can detect more reads and SMB numbers by using the primer combination, so that the primer combination has the characteristics of better specificity and more sensitivity.
Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A primer combination for detecting respiratory pathogens, which is characterized by comprising an upstream primer of pseudomonas aeruginosa, a downstream primer of pseudomonas aeruginosa, an upstream primer of acinetobacter baumannii, a downstream primer of acinetobacter baumannii, an upstream primer of streptococcus pneumoniae, a downstream primer of streptococcus pneumoniae, an upstream primer of staphylococcus aureus, a downstream primer of staphylococcus aureus, an upstream primer of streptococcus pyogenes and a downstream primer of streptococcus pyogenes;
the nucleotide sequence of the upstream primer of the pseudomonas aeruginosa is shown as SEQ ID NO. 1;
the nucleotide sequence of the downstream primer of the pseudomonas aeruginosa is shown as SEQ ID NO. 2;
the nucleotide sequence of the upstream primer of the Acinetobacter baumannii is shown as SEQ ID NO. 3;
the nucleotide sequence of the downstream primer of the Acinetobacter baumannii is shown as SEQ ID NO. 4;
the nucleotide sequence of the upstream primer of the streptococcus pneumoniae is shown as SEQ ID NO. 5;
the nucleotide sequence of the downstream primer of the streptococcus pneumoniae is shown as SEQ ID NO. 6;
the nucleotide sequence of the upstream primer of staphylococcus aureus is shown as SEQ ID NO. 7;
the nucleotide sequence of the downstream primer of staphylococcus aureus is shown as SEQ ID NO. 8;
the nucleotide sequence of the upstream primer of the streptococcus pyogenes is shown as SEQ ID NO. 9;
the nucleotide sequence of the downstream primer of the streptococcus pyogenes is shown as SEQ ID NO. 10.
2. Use of a primer combination according to claim 1 for the preparation of a kit for the detection of respiratory pathogens.
3. A kit for detecting a respiratory pathogen, said kit comprising the primer combination of claim 1.
4. The kit of claim 3, wherein the respiratory pathogenic bacteria comprise staphylococcus aureus, acinetobacter baumannii, streptococcus pneumoniae, pseudomonas aeruginosa and streptococcus pyogenes.
5. A kit according to claim 3, wherein the kit comprises a forward primer F1 and a reverse primer R1;
the forward primer F1 comprises a connecting sequence Read 1 and a single-molecule identification tag sequence SMB;
the nucleotide sequence of the connecting sequence Read 1 is shown as SEQ ID NO. 11;
the sequence of the single-molecule recognition tag SMB is NNNNNNNN, wherein N represents any one of A, T, C, G.
6. The kit of claim 5, wherein the reverse primer R1 comprises a ligation sequence Read 2; the nucleotide sequence of the connecting sequence Read 2 is shown as SEQ ID NO. 12.
7. The kit of claim 3, wherein the kit comprises a second round of PCR amplification primers comprising a forward primer F2 and a reverse primer R2; the nucleotide sequence of the forward primer F2 is shown as SEQ ID NO. 13, and the nucleotide sequence of the reverse primer R2 is shown as SEQ ID NO. 14.
8. The kit of claim 3, wherein the first round of PCR amplification reaction procedure of the kit is: pre-denaturation at 98 ℃ for 2 min, and circulation for 1 time; denaturation at 98℃for 10 sec, annealing at 58℃for 15 sec, elongation at 72℃for 30 sec, cycling 20 times; extending for 1 min at 72 ℃, and circulating for 1 time.
9. The kit of claim 8, wherein the second round of PCR amplification reaction procedure of the kit is: pre-denaturation at 98 ℃ for 2 min, and circulation for 1 time; denaturation at 98℃for 10 sec, annealing at 58℃for 15 sec, elongation at 72℃for 30 sec, cycle 6 times; extending for 1 min at 72 ℃, and circulating for 1 time.
10. A kit according to claim 3, wherein the step of analyzing the sequencing result of the kit to identify respiratory pathogens in the sample to be tested comprises the steps of:
(1) Removing low quality and over short sequences;
(2) The residual sequence is aligned with a target sequence of the pathogenic microorganism;
(3) Counting the reading number of the corresponding target on the accurate comparison;
(4) Analyzing SMB sequences corresponding to the same reading number, and if the SMB sequences are the same, considering that the SMB sequences are derived from the same mother chain template;
(5) Counting the number of SMBs corresponding to the different numbers of the SMBs respectively;
(6) The cut off value of the target sequence of the pathogenic microorganism is identified, the accurate alignment reading number is more than 3, and the SMB is more than 1.
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