CN116536436A - Primer set and kit for detecting multiple targets of blood flow infection - Google Patents

Primer set and kit for detecting multiple targets of blood flow infection Download PDF

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CN116536436A
CN116536436A CN202310538072.0A CN202310538072A CN116536436A CN 116536436 A CN116536436 A CN 116536436A CN 202310538072 A CN202310538072 A CN 202310538072A CN 116536436 A CN116536436 A CN 116536436A
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seq
nucleotide sequence
primer
reverse primer
forward primer
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夏涵
杨启文
官远林
张烨
李长诚
佟斯垚
张栋
喻玮
朱盈
贾沛瑶
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Yuguo Biotechnology Beijing Co ltd
Peking Union Medical College Hospital Chinese Academy of Medical Sciences
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Yuguo Biotechnology Beijing Co ltd
Peking Union Medical College Hospital Chinese Academy of Medical Sciences
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Abstract

The invention belongs to the technical field of biological detection, and relates to a primer set and a kit for detecting multiple targets of blood flow infection. The invention also provides a kit, which comprises a primer group and a PCR buffer solution, wherein the combination of the primer group and the PCR buffer solution can further improve the detection limit, reduce the loss of sample DNA when the microorganism is detected by subsequent sequencing, and improve the yield of the sample DNA.

Description

Primer set and kit for detecting multiple targets of blood flow infection
Technical Field
The invention belongs to the technical field of biological detection, and relates to a primer set and a kit for detecting multiple targets of blood flow infection.
Background
Blood flow infection is the main cause of septicemia and infectious shock, and proper antibacterial agents can be selected to cope with the harm caused by blood flow infection, and with the increasing drug resistance of microorganisms to the antibacterial agents, the method of Blood Culture (BC) of collected samples under laboratory conditions is adopted at present, and the proper antibacterial agents are selected by identifying positive pathogenic microorganisms, but the method is high in time consumption and low in positive accuracy.
Blood culture is a gold standard for detecting blood flow infection pathogens, but has obvious defects of slow bacterial growth, long period, high nutrition requirement, cell growth, low blood culture positive rate and the like. The advent of molecular diagnostic techniques, typified by fluorescent qPCR techniques, has solved the problem of blood culture to some extent, but has also suffered from problems such as failure to supply pathogenic gene sequences, and low throughput. Metagenomic sequencing (mNGS) solves the short plates of pathogen detection flux, but the problems of high host nucleic acid ratio, low detection sensitivity, long cycle, high cost, etc. remain to be solved. In view of the above technical limitations, there is a need to develop a new detection method for blood flow infection.
The multiplex PCR targeted high-throughput sequencing is a method which can potentially replace or solve the detection problem of the blood pathogen infection, and has the advantages of low cost, short time, high accuracy and the like, but because the number of primers in a single reaction of multiplex PCR amplification is often hundreds or even thousands, the technical difficulty is high; meanwhile, the sequence similarity of pathogenic microorganisms is high, and the difficulty of multiplex PCR library establishment is further increased. In recent years, detection of pathogenic microorganisms by using a multiplex PCR library kit has been reported, but most of them are detected against only a part of pathogens in a certain type of sample. In addition, the existing pathogenic microorganism multiplex PCR French library construction kit has the following common problems because hundreds or even thousands of primers exist in the same reaction tube, and the primer group and the reaction system of the whole kit are the results of whole analysis optimization: (1) The primers interfere with each other to form a large number of dimers, so that the performance of the kit is reduced; (2) Non-specific amplification, producing a large number of non-target amplicons, affecting subsequent sequencing; (3) accurate quantification cannot be performed; (4) the amplification uniformity cannot be guaranteed; (5) expansibility and flexibility are poor.
Disclosure of Invention
The invention aims to solve the problems and provide a primer group, a kit and a method for detecting blood flow infection based on multi-target high-throughput sequencing of multiple amplification.
Based on the above objects, the present invention provides a primer set and a kit for detecting multiple targets of blood flow infection, which are used for solving the technical problems in the art.
In one aspect, the invention relates to a kit for multi-target detection of blood flow infection, wherein a detection sample is blood, and the kit comprises a primer set, wherein the primer set comprises primer pairs respectively aiming at high-altitude microorganisms such as escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, proteus mirabilis, enterobacter cloacae, brucella, salmonella, streptococcus pneumoniae, staphylococcus aureus, candida albicans and group B streptococcus;
primer pairs respectively aiming at drug-resistant genes mecA, mecC, tetK, CRKP, OXA-51;
primer pairs respectively aiming at virulence genes PVL and asal;
a primer pair for internal reference;
the nucleotide sequence of the forward primer for the escherichia coli is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer for the escherichia coli is shown as SEQ ID No. 2;
the nucleotide sequence of the forward primer for klebsiella pneumoniae is shown as SEQ ID No.3, and the nucleotide sequence of the reverse primer for klebsiella pneumoniae is shown as SEQ ID No. 4;
the nucleotide sequence of the forward primer for pseudomonas aeruginosa is shown as SEQ ID No.5, and the nucleotide sequence of the reverse primer for pseudomonas aeruginosa is shown as SEQ ID No. 6;
the nucleotide sequence of the forward primer for the Proteus mirabilis is shown as SEQ ID No.7, and the nucleotide sequence of the reverse primer for the Escherichia coli is shown as SEQ ID No. 8;
the nucleotide sequence of the forward primer for enterobacter cloacae is shown as SEQ ID No.9, and the nucleotide sequence of the reverse primer for escherichia coli is shown as SEQ ID No. 10;
the nucleotide sequence of the forward primer for the brucella is shown as SEQ ID No.11, and the nucleotide sequence of the reverse primer for the brucella is shown as SEQ ID No. 12;
the nucleotide sequence of the forward primer for salmonella is shown as SEQ ID No.13, and the nucleotide sequence of the reverse primer for salmonella is shown as SEQ ID No. 14;
the nucleotide sequence of the forward primer for streptococcus pneumoniae is shown as SEQ ID No.15, and the nucleotide sequence of the reverse primer for streptococcus pneumoniae is shown as SEQ ID No. 16;
the nucleotide sequence of the forward primer for staphylococcus aureus is shown as SEQ ID No.17, and the nucleotide sequence of the reverse primer for staphylococcus aureus is shown as SEQ ID No. 18;
the nucleotide sequence of the forward primer for candida albicans is shown as SEQ ID No.19, and the nucleotide sequence of the reverse primer for candida albicans is shown as SEQ ID No. 20;
the nucleotide sequence of the forward primer for the group B streptococcus is shown as SEQ ID No.21, and the nucleotide sequence of the reverse primer for the group B streptococcus is shown as SEQ ID No. 22;
the nucleotide sequence of the forward primer for mecA is shown as SEQ ID No.23, and the nucleotide sequence of the reverse primer for mecA is shown as SEQ ID No. 24;
the nucleotide sequence of the forward primer for mecC is shown as SEQ ID No.25, and the nucleotide sequence of the reverse primer for mecC is shown as SEQ ID No. 26;
the nucleotide sequence of the forward primer for tetK is shown as SEQ ID No.27, and the nucleotide sequence of the reverse primer for tetK is shown as SEQ ID No. 28;
the nucleotide sequence of the forward primer for CRKP is shown as SEQ ID No.29, and the nucleotide sequence of the reverse primer for CRKP is shown as SEQ ID No. 30;
the nucleotide sequence of the forward primer for OXA-51 is shown as SEQ ID No.31, and the nucleotide sequence of the reverse primer for OXA-51 is shown as SEQ ID No. 32;
the nucleotide sequence of the forward primer for PVL is shown as SEQ ID No.33, and the nucleotide sequence of the reverse primer for PVL is shown as SEQ ID No. 34;
the nucleotide sequence of the forward primer for asal is shown as SEQ ID No.35, and the nucleotide sequence of the reverse primer for asal is shown as SEQ ID No. 36;
the nucleotide sequence of the primer pair for internal reference is shown as SEQ ID No.37, and the nucleotide sequence of the primer pair for internal reference is shown as SEQ ID No. 38.
Further, the kit for detecting multiple targets of blood flow infection provided by the invention further comprises a buffer solution, wherein the buffer solution comprises the following components: beta-mercaptoethanol, tetrahydropyrimidine carboxylic acid, PCR premix and PCR enzyme solution;
the PCR premix comprises 10 XPCR buffer and MgCl 2 And dNTPs;
the PCR enzyme solution comprises a hot start DNA polymerase and a UNG enzyme.
Furthermore, in the kit for detecting blood flow infection multiple targets, when DNA of a blood sample is extracted, chitinase and chondroitin sulfate AC lyase are pre-added into the blood sample, and then DNA extraction is carried out.
Compared with the prior art, the technical scheme provided by the invention has the following beneficial effects or advantages:
the blood flow infection pathogen multi-target gene detection product can be used for detecting blood after enrichment culture, identifying 11 blood flow infection pathogens, mixing copy numbers of plasmids and the like of all target genes together, and adjusting primer concentration of each pathogen to ensure that peak heights of each target point are equivalent, thereby achieving the purpose of equivalently amplifying all target genes. The method can detect blood samples of common blood flow infected patients or suspected patients, provide etiology diagnosis information about common blood flow infected pathogens, help clinicians to timely determine pathogen types and take effective treatment schemes, reduce the use of empirical antibiotics, and reduce medical cost. When the DNA of the blood sample is extracted, chitinase and chondroitin sulfate AC lyase are pre-added into the blood sample, and then the DNA extraction is carried out, so that the DNA yield of the blood sample is improved. According to the invention, beta-mercaptoethanol and tetrahydropyrimidine carboxylic acid are added into the buffer solution component, so that the DNA of a blood sample is protected, the loss of the DNA of the sample during subsequent sequencing detection of microorganisms is reduced, the high specificity is shown, and pathogens as low as 10 copies/mu L can be detected, and the sensitivity is high.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention.
FIG. 1 shows the detection result of sample No. 6;
FIG. 2 shows the result of sample number 15;
FIG. 3 shows the result of sample number 22;
fig. 4 shows the detection result of sample number 26 #.
Detailed Description
The following describes the technical aspects of the present invention with reference to examples, but the present invention is not limited to the following examples.
In order that those skilled in the art will better understand the technical solution of the present invention, the present invention will be further described with reference to specific examples, but the examples are not intended to limit the present invention.
The experimental methods and the detection methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.
Example 1
This example provides an experiment for DNA extraction of blood samples.
Healthy human blood samples were obtained, 10CFU/mL of Staphylococcus aureus (standard strain ATCC 25923) was incorporated, and pathogen DNA was extracted using a magnetic bead method blood stream infection pathogen DNA extraction kit (purchased from Beijing source microorganism technologies Co., ltd.). Test set 6 groups, 1#, respectively: 15 mg/mu L of chitinase and 10 mg/mu L of chondroitin sulfate AC lyase are added into a sample before DNA extraction, and the sample is subjected to water bath at 50 ℃ for 15min, shaking and vibrating for 2 times; 2#: adding 12.5 mg/mu L of chitinase and 12.5 mg/mu L of chondroitin sulfate AC lyase into a sample before DNA extraction, and shaking for 2 times in a water bath at 50 ℃ for 15 min; 3#: before DNA extraction, 10 mg/mu L of chitinase and 15 mg/mu L of chondroitin sulfate AC lyase are added into a sample, and water bath is carried out for 15min at 50 ℃ for shaking for 2 times; 4#: adding 25 mg/mu L of chitinase into a sample before DNA extraction, and shaking for 2 times in a water bath at 50 ℃ for 15 min; 5#: before DNA extraction, 25 mg/mu L of chondroitin sulfate AC lyase is added into the sample, and the mixture is subjected to water bath at 50 ℃ for 15min, shaking and vibrating for 2 times; 6#: no addition was made to the sample prior to DNA extraction, with a 50 ℃ water bath for 15min, during which shaking was performed 2 times. The detection was performed using a micro-droplet digital pcr, the detection instrument was purchased from beijing shilian boyance technologies, ltd, and 9 tests were performed for each group, and the test results are shown in table 1.
Table 1: detection result of positive point number of sample
Group number 1 2 3 4 5 6 7 8 9
1# 47 52 47 50 57 47 51 47 45
2# 52 47 55 49 54 58 47 53 47
3# 49 58 59 58 48 51 48 56 47
4# 30 37 37 36 35 32 30 37 30
5# 33 30 35 31 31 34 37 35 33
6# 24 27 25 29 30 28 28 28 23
The results in Table 1 show that under the condition of the same trace cell concentration, the sample obtained by the sample treatment method can obtain more positive points through detection, which indicates that the method can ensure the positive detection rate of the trace pathogenic bacteria, namely the yield of sample DNA is improved.
Example 2
The embodiment provides a test for carrying out gene detection on a DNA sample extracted from human peripheral blood plasma suspected to be infected by blood flow by adopting the kit provided by the invention.
The kit comprises a primer group, a positive control substance, a negative control substance and a buffer solution.
The primer set includes: primer pairs for high-hairing microorganisms such as escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, proteus mirabilis, enterobacter cloacae, brucella, salmonella, streptococcus pneumoniae, staphylococcus aureus, candida albicans and group B streptococcus respectively; primer pairs respectively aiming at drug-resistant genes mecA, mecC, tetK, CRKP, OXA-51; primer pairs respectively aiming at virulence genes PVL and asal; primer pairs for internal references (ICs). The primer sequence table is shown in table 2.
The positive control was a plasmid mixture (available from Biyun biotechnology Co., ltd.) that included all the target genes of interest.
The negative control was nuclease-free ultrapure water.
PCR premix and PCR enzyme solution (available from Biyun Biotechnology Co., ltd.).
The primers were all synthesized by Beijing-Gein Biotechnology Co.
The component amount in the system is as follows10 XPCR buffer 1 volume, 10. Mu.M dNTPs 0.2 volume, beta-mercaptoethanol 0.3 volume, tetrahydropyrimidinecarboxylic acid 0.2 volume, 25mmol/L MgCl 2 0.8 volume of solution, 1 volume of primer mixture (concentration of each primer is 300 nM), 0.4 volume of 5U/. Mu.L of hot-start DNA polymerase, 0.5 volume of 1U/. Mu.L of UNG enzyme, 5 volumes of DNA template, and 1.1 volume of nuclease-free pure water. The amount of DNA template used was 50 ng/volume.
Table 2: primer sequence listing
(1) And (3) collecting human peripheral blood plasma suspected of blood flow infection, respectively extracting nucleic acid, extracting 80 mu L of positive control and negative control, and adding 5 mu L of IC (integrated circuit) into each sample involved in extraction for extraction. Nucleic acid extraction methods refer to Experimental group 1# of example 1.
(2) With reference to the above reaction system, 50. Mu.L of the reaction system was prepared, and after vortexing and mixing, centrifugation was performed with a centrifuge, and then split charging into PCR reaction tubes.
(3) The extracted nucleic acid is added into a PCR reaction tube filled with the prepared reaction system. The amplification procedure was as follows: firstly, pre-denaturing at 95 ℃ for 5min, then denaturing at 94 ℃ for 30s, annealing at 65 ℃ for 1.5min, and 5 cycles; denaturation at 94℃for 30s, annealing at 57℃for 30s, extension at 72℃for 1min for 20 cycles; finally, the extension is carried out for 5min at 72 ℃.
(4) The PCR product was purified using 0.9 XSPRI beads, eluted at 20. Mu.L, and the concentration was determined using a Qubit method. DNA library was established using the HyperPrep Kit, the initial template for library establishment was quantified 5ng, and absolute quantification was performed on the DNA library using Library Quantification Kit.
(5) Sequencing at both ends of S5 2 ×75bp base pairing read sequence, dividing the sequenced original reads sequence into forward read length and reverse read length, combining forward and reverse reads sequences by S5 software, wherein the combined parameters are 100% similarity, and the two ends are overlapped by at least 10bp.
(6) And counting the sequence by utilizing Ion report software, and firstly modifying a configuration file of the software, wherein the configuration file comprises typing statistics on the sequence.
(7) The similarity between the length coverage and alignment was set to 95% by BLAST for reads to the template sequence, and bacterial reads with a reading above 10reads were considered positive, and positive results were confirmed by blood culture.
The blood plasma infected by staphylococcus aureus blood stream provided in the example is adopted for testing for 50 times in parallel to examine the stability and effect of the mixed amplification system of the kit provided by the application. The test shows that: in 50 tests, the target gene locus of the target gene is effectively amplified by the mixed amplification system primer, and the target gene is not deleted, which indicates that the kit relates to the stability and effectiveness of the primer.
The peripheral blood of 50 suspected blood flow infected patients is taken, the detection result is shown in table 3, and the schematic diagrams of the detection result of partial samples and the detection result of positive control are shown in fig. 1-4.
Table 3: detection result
The number of positive and negative results obtained by blood culture were counted, and the results are shown in Table 4.
Table 4: detecting statistical results
As shown in Table 4, the kit and the kit provided by the application adopt a multi-target gene high-throughput sequencing method with higher sensitivity and specificity, the specificity and sensitivity of the detection result are obviously improved, the detection time is about 12 hours, the time consumption is short, and the detection time of the conventional culture and identification method is generally more than 24 hours.
The present invention may be better implemented as described above, and the above examples are merely illustrative of preferred embodiments of the present invention and not intended to limit the scope of the present invention, and various changes and modifications made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the present invention without departing from the spirit of the design of the present invention.

Claims (3)

1. The kit for detecting the blood flow infection multi-target is characterized by comprising a primer set, wherein the primer set comprises primer pairs respectively aiming at high-fever microorganisms such as escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, proteus mirabilis, enterobacter cloacae, brucella, salmonella, streptococcus pneumoniae, staphylococcus aureus, candida albicans and group B streptococcus;
primer pairs respectively aiming at drug-resistant genes mecA, mecC, tetK, CRKP, OXA-51;
primer pairs respectively aiming at virulence genes PVL and asal;
a primer pair for internal reference;
the nucleotide sequence of the forward primer for the escherichia coli is shown as SEQ ID No.1, and the nucleotide sequence of the reverse primer for the escherichia coli is shown as SEQ ID No. 2;
the nucleotide sequence of the forward primer for klebsiella pneumoniae is shown as SEQ ID No.3, and the nucleotide sequence of the reverse primer for klebsiella pneumoniae is shown as SEQ ID No. 4;
the nucleotide sequence of the forward primer for pseudomonas aeruginosa is shown as SEQ ID No.5, and the nucleotide sequence of the reverse primer for pseudomonas aeruginosa is shown as SEQ ID No. 6;
the nucleotide sequence of the forward primer for the Proteus mirabilis is shown as SEQ ID No.7, and the nucleotide sequence of the reverse primer for the Escherichia coli is shown as SEQ ID No. 8;
the nucleotide sequence of the forward primer for enterobacter cloacae is shown as SEQ ID No.9, and the nucleotide sequence of the reverse primer for escherichia coli is shown as SEQ ID No. 10;
the nucleotide sequence of the forward primer for the brucella is shown as SEQ ID No.11, and the nucleotide sequence of the reverse primer for the brucella is shown as SEQ ID No. 12;
the nucleotide sequence of the forward primer for salmonella is shown as SEQ ID No.13, and the nucleotide sequence of the reverse primer for salmonella is shown as SEQ ID No. 14;
the nucleotide sequence of the forward primer for streptococcus pneumoniae is shown as SEQ ID No.15, and the nucleotide sequence of the reverse primer for streptococcus pneumoniae is shown as SEQ ID No. 16;
the nucleotide sequence of the forward primer for staphylococcus aureus is shown as SEQ ID No.17, and the nucleotide sequence of the reverse primer for staphylococcus aureus is shown as SEQ ID No. 18;
the nucleotide sequence of the forward primer for candida albicans is shown as SEQ ID No.19, and the nucleotide sequence of the reverse primer for candida albicans is shown as SEQ ID No. 20;
the nucleotide sequence of the forward primer for the group B streptococcus is shown as SEQ ID No.21, and the nucleotide sequence of the reverse primer for the group B streptococcus is shown as SEQ ID No. 22;
the nucleotide sequence of the forward primer for mecA is shown as SEQ ID No.23, and the nucleotide sequence of the reverse primer for mecA is shown as SEQ ID No. 24;
the nucleotide sequence of the forward primer for mecC is shown as SEQ ID No.25, and the nucleotide sequence of the reverse primer for mecC is shown as SEQ ID No. 26;
the nucleotide sequence of the forward primer for tetK is shown as SEQ ID No.27, and the nucleotide sequence of the reverse primer for tetK is shown as SEQ ID No. 28;
the nucleotide sequence of the forward primer for CRKP is shown as SEQ ID No.29, and the nucleotide sequence of the reverse primer for CRKP is shown as SEQ ID No. 30;
the nucleotide sequence of the forward primer for OXA-51 is shown as SEQ ID No.31, and the nucleotide sequence of the reverse primer for OXA-51 is shown as SEQ ID No. 32;
the nucleotide sequence of the forward primer for PVL is shown as SEQ ID No.33, and the nucleotide sequence of the reverse primer for PVL is shown as SEQ ID No. 34;
the nucleotide sequence of the forward primer for asal is shown as SEQ ID No.35, and the nucleotide sequence of the reverse primer for asal is shown as SEQ ID No. 36;
the nucleotide sequence of the primer pair for internal reference is shown as SEQ ID No.37, and the nucleotide sequence of the primer pair for internal reference is shown as SEQ ID No. 38.
2. The kit for multi-target detection of blood flow infection of claim 1, further comprising a buffer comprising: beta-mercaptoethanol, tetrahydropyrimidine carboxylic acid, PCR premix and PCR enzyme solution;
the PCR premix comprises 10 XPCR buffer and MgCl 2 And dNTPs;
the PCR enzyme solution comprises a hot start DNA polymerase and a UNG enzyme.
3. The kit for multi-target detection of blood flow infection according to claim 1, wherein chitinase and chondroitin sulfate AC lyase are pre-added to the blood sample for DNA extraction, and then DNA extraction is performed.
CN202310538072.0A 2023-05-09 2023-05-12 Primer set and kit for detecting multiple targets of blood flow infection Pending CN116536436A (en)

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CN202310518659 2023-05-09
CN2023105186595 2023-05-09

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CN116536436A true CN116536436A (en) 2023-08-04

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Country Link
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