CN111876501A - Real-time fluorescence PCR detection method for enterobacter aerogenes PSgc4 type strain and application - Google Patents
Real-time fluorescence PCR detection method for enterobacter aerogenes PSgc4 type strain and application Download PDFInfo
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Abstract
The invention relates to a real-time fluorescence PCR detection method for enterobacter aerogenes PSgc4 type strains and application thereof. The invention uses specific genes in enterobacter aerogenes type 4 polysaccharide antigen gene cluster, namelyorf8For target genes, a Taqman specific primer and an MGB probe for enterobacter aerogenes serotype PSgc4 strain are designed, and a real-time fluorescence PCR detection method for the enterobacter aerogenes serotype PSgc4 strain is established. The method provided by the invention can be used for accurately, quickly and sensitively carrying out molecular biological detection on the Enterobacter aerogenes PSgc4 type strain.
Description
Technical Field
The invention belongs to the technical field of bacteria detection methods, and relates to a real-time fluorescence PCR detection method for detecting the most common serotype PSgc4 type bacterial strain in enterobacter aerogenes and application thereof.
Background
Enterobacter aerogenes (A)Enterobacter aerogenes) Can cause opportunistic infection and nosocomial infection, can be detected in respiratory tract, urogenital tract, pus, blood and other specimens of patients, and is one of the most common clinical isolates in enterobacter of enterobacteriaceae. In recent years, the isolation rate of enterobacter aerogenes is obviously increased, and particularly in ICU, burn department, respiratory medicine, urology department and the like, the enterobacter aerogenes is widely existed in the disease area environment and becomes one of the important pathogenic bacteria of nosocomial infection. Research has been conducted to classify enterobacter aerogenes into 8 serotypes (PSgc 1-8) based on the gene cluster and specific gene encoding the surface polysaccharide antigen of enterobacter aerogenes, wherein PSgc4 is the most common isolated strain in clinic.
The TaqMan-MGB Real-time fluorescence PCR (Real-time fluorescence PCR) technology is characterized in that a Taqman probe marked with fluorescein is mixed with template DNA to complete thermal cycle of high-temperature denaturation, low-temperature renaturation and proper-temperature extension, the polymerase chain reaction rule is followed, the Taqman probe which is complementary and matched with the template DNA is cut off, and the fluorescein is dissociated in a reaction system and emits fluorescence under the excitation of specific light. Along with the increase of the cycle number, the amplified target gene segment grows exponentially, and the Ct value is obtained by detecting the intensity of the fluorescence signal which corresponds to the amplified target gene segment and changes along with the amplification in real time, so that the type and the content of the detection sample can be represented.
The TaqMan fluorescent probe is an oligonucleotide probe, the 5 'end of the TaqMan fluorescent probe carries a fluorescent group, such as FAM, TET, VIC, HEX and the like, and the 3' end of the TaqMan fluorescent probe carries a quenching group, such as TAMRA, BHQ and the like. During PCR amplification, a pair of primers is added, and a specific fluorescent probe is added at the same time, when the probe is complete, a fluorescent signal emitted by a reporter group is absorbed by a quenching group; during PCR amplification, the 5 '-3' exonuclease activity of Taq enzyme cuts and degrades the probe, so that the reporter fluorescent group and the quenching fluorescent group are separated, a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescent molecule is formed when one DNA chain is amplified, and the accumulation of the fluorescence signal and the formation of a PCR product are completely synchronous.
The TaqMan-MGB probe is a new technology improved on the basis of the TaqMan probe in recent years, and MGB molecules are added at the 3' end of the probe, so that the MGB probe can increase the annealing temperature (Tm value) of the probe, and the TaqMan-MGB probe can distinguish the difference of 1 base, is completely paired and has a fluorescent signal; as long as there is a base mismatch, there is no signal. The probe was designed to be 15-30 nt long first, with a Tm of 68-70 ℃. The probes were synthesized by AuGCT Biotechnology Corporation (Beijing, China) and have 6-carboxyfluorescein (FAM) and Minor Groove Binding (MGB) moieties at the 5 'and 3' ends, respectively. The length of the primer is about 25 nt, and the Tm is between 55 and 60 ℃. The PCR product produced was between 50-200 bp. Primers were synthesized by Invitrogen (shanghai, china). It may be better if the 3 'end of the forward primer is 1-20 nt (typically 10nt or less) from the 5' end of the probe.
When using Taqman MGB PCR assays, amplification reactions were performed in a total volume of 25. mu.L, including 12.5. mu.L LPremix Ex TaqTM(Takara), 0.3. mu.L of each of 10. mu.M forward and reverse primers, 0.3. mu.L of 10. mu.M probe, 0.2. mu.LROXII, 0.3. mu.L of nucleic acid DNA of the sample to be detected and 11. mu.L of ddH2And O. One protocol included an initial denaturation step at 95 ℃ for 3 minutes followed by denaturation at 95 ℃ for 15 seconds and annealing at 60 ℃ for 45 seconds for 40 cycles in triplicate in a 7500 real-time PCR system (Applied Biosystems, Foster City, Calif., USA), followed by detection and analysis of results using instrumentation such as ABI 7500. Ct value<26, if an amplification curve appears, judging the sample to be positive, otherwise, judging the sample to be negative.
Disclosure of Invention
The invention aims to provide a real-time fluorescence PCR detection technology for enterobacter aerogenes PSgc4 type strains, and provides an effective technical means for the rapid detection of the pathogenic bacteria.
The invention relates to a Taqman primer sequence, an MGB probe sequence and a real-time fluorescence PCR detection method for detecting an enterobacter aerogenes PSgc4 type strain. Wherein:
the Taqman primer is derived from Enterobacter aerogenes PSgc4orf8The sequence of 2 DNA fragments selected from the gene is shown in SEQ ID NO 1-SEQ ID NO 2.
SEQ ID (5'-3')
The MGB probe is selected fromorf8Genes, screened inside P1 and P2, and containing a 5' reporter dye (FAM) and a 3' non-fluorescent quencher (NFQ), conjugated at the 3' end with a Minor Groove Binder (MGB), the sequence of which is shown in SEQ ID NO: 3.
SEQ ID (5'-3')
Specific detection of PSgc4 type Strain with NO 3 CAGCAAGAAATTTG
The invention further discloses a real-time fluorescence PCR detection system for detecting the Enterobacter aerogenes PSgc4 type strain in the sample and application thereof. The experimental results show that:
(1) the technology provided by the invention has good accuracy, and can realize accurate detection of the Enterobacter aerogenes PSgc4 type strain. The real-time fluorescent PCR detection result of enterobacter aerogenes PSgc4 shows that Ct = 17.95;
(2) the technology provided by the invention has good specificity, and except the PSgc4 type strain, other serotypes of enterobacter aerogenes can not be detected by the technology provided by the invention. Wherein for the PSgc1 type strain: ct = 33; for the PSgc2 type strain: ct = 30; for the PSgc3 type strain: ct = 31; for the PSgc5 type strain: no Ct value and no amplification curve; for the PSgc6 type strain: ct = 37; for the PSgc7 type strain: ct = 33; for the PSgc8 type strain: ct = 31.
(3) The technology provided by the invention has high sensitivity, and can realize accurate detection of 10pg enterobacter aerogenes PSgc4 type strain genome DNA.
The invention mainly provides a technical means for detecting the most frequently clinically separated enterobacter aerogenes PSgc4 type strain by using a molecular biological method, and the main difficulty lies in the screening and determination of specific Taqman primers and MGB probes. The positive effects are as follows:
(1) the technical means for detecting the Enterobacter aerogenes PSgc4 type strain by utilizing a molecular biological means is disclosed for the first time, and an effective method is provided for the clinical detection and the epidemiological monitoring of the strain.
(2) The detection sensitivity is high: the detection sensitivity of the kit to the genomic DNA can reach 10 pg.
(3) The detection time is short: by using the technical means, the detection can be completed within 3 hours from the obtained genome DNA or pure culture colony meter.
Drawings
FIG. 1 shows the self-detection results of the Enterobacter aerogenes PSgc4 type real-time fluorescent PCR detection system: PSgc 4: ct = 17.95.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available.
The source of the Enterobacter aerogenes strain used in the present invention is as follows.
TABLE 1 bacterial species to which the invention relates
Example 1
Design of primers and probes
1. Screening for specific genes
Overall, in the cluster of genes for polysaccharide antigen synthesis on the surface of almost enterobacter aerogenes, there are 3 types of genes, which are: monosaccharide synthase gene, glycosyltransferase gene, and oligosaccharide unit processing enzyme gene. Among them, the oligosaccharide unit treating enzyme gene has the most type specificity, the glycosyltransferase gene is inferior, and the monosaccharide synthetase gene has poor specificity. Because the gene cluster of the PSgc4 type enterobacter aerogenes is special and the oligosaccharide unit processing enzyme gene does not exist, only the relative gene can be selectedSpecific glycosyltransferase genesorf8。
2. Design of primers and probes
Selected Enterobacter aerogenes PSgc4 typeorf8The gene is a specific gene and is used as a template to design a primer and a probe of Taqman-MGB. Using Primer Express 3.0 baseorf8The sequence of the gene was designed as TaqMan MGB probe and primer. The GC% content of the primers and probes is between 40 and 70%, and the formation of a ring hair clamp structure, a self-dimer and a cross-dimer is avoided. The specificity of primers and probes was verified by comparing their sequences to all sequences in GenBank using BLAST search.
The probe was designed to be 15-30 nt long first, with a Tm of 68-70 ℃. At the 5' end of the probe, the presence of G, which may cause fluorescence quenching, is avoided. The probes were synthesized by AuGCT Biotechnology Corporation (Beijing, China) and have 6-carboxyfluorescein (FAM) and Minor Groove Binding (MGB) moieties at the 5 'and 3' ends, respectively.
The length of the primer is about 25 nt, and the Tm is between 55 and 60 ℃. The PCR product produced was between 50-200 bp. Primers were synthesized by Invitrogen (shanghai, china). It may be better if the 3 'end of the forward primer is 1-20 nt (typically 10nt or less) from the 5' end of the probe.
Example 2
Extraction of sample nucleic acid
1. The obtained pure cultured bacteria were treated in the following manner:
(1) and picking a single bacterial colony of the bacteria into 10 muL of deionized water or 10 muL of bacteria liquid cultured overnight, and treating for 15 min in a boiling water bath.
(2) After being placed on ice for 1 min, the mixture was centrifuged at 8000 rpm for 1 min.
(3) And taking 3 mu L of supernatant as a template for the next real-time fluorescent PCR reaction.
2. The obtained clinical samples (blood, sputum or urine) were treated in the following manner:
(1) the sample is placed in 20 mL LB culture medium and shake cultured for 3 h at 37 ℃.
(2) 1 mL of the culture of (1) was centrifuged at 8000 rpm for 5min, and the supernatant was discarded.
(3) Adding 500 mu L of deionized water, re-suspending and uniformly mixing, centrifuging at 8000 rpm for 5min, and discarding the supernatant.
(4) And adding 100 mu L of deionized water, and treating for 15 min in a boiling water bath.
(5) After being placed on ice for 1 min, the mixture was centrifuged at 8000 rpm for 1 min.
(6) And taking 3 mu L of supernatant as a template for the next real-time fluorescent PCR reaction.
Example 3
Real-time fluorescent PCR amplification detection
The nucleic acid solution extracted in example 2 was used as a template for real-time fluorescent PCR reaction, and the PCR reaction system and reaction conditions were as follows:
reaction system (25 μ L):
reaction conditions are as follows:
as shown in FIG. 1, the Ct value of the Enterobacter aerogenes PSgc4 type was 17.95, and it was judged to be positive.
Example 4
Specificity detection
Genomic DNA templates of Enterobacter aerogenes PSgc1-8 type strains were prepared according to the procedure of example 2, and the detection was performed according to the reaction system and procedure of example 3.
The results of the experiment are shown in table 2. As can be seen, except that PSgc4 shows a positive result, the CT values of the detection results of other strains are all judged to be negative results, which is higher than the criterion of 26.
TABLE 2 specific reaction results of Enterobacter aerogenes PSgc4 type real-time fluorescent PCR detection system
Example 5
Sensitivity detection
The method for carrying out sensitivity detection experiment on the enterobacter aerogenes PSgc4 type real-time fluorescent PCR detection system comprises the following steps:
(1) the Enterobacter aerogenes PSgc4 type strain is inoculated into 5 mL of LB culture medium, cultured in a shaking table at 37 ℃ and 180 r/min overnight, and 2 mL of thallus is collected.
(2) Bacterial genomic DNA was extracted according to the procedure using a bacterial genomic DNA extraction kit (product No. DP 302) from Tiangen Biochemical technology, Inc. (Beijing).
(3) The concentration of the extracted genome was measured by a NanoDrop OD apparatus, adjusted to 3300 ng/. mu.L, and gradually diluted in order. Thus, the amounts of genomic DNA added to the reaction system in accordance with example 3 were 100ng, 10ng, 1ng, 100pg, 10pg and 1pg, respectively. 0.01 ng.
(4) The real-time fluorescent PCR reaction was carried out according to the reaction procedure of example 2, and the results showed that: the detection sensitivity for genomic DNA was 10 pg.
SEQUENCE LISTING
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Claims (5)
1. A Taqman primer specific for Enterobacter aerogenes PSgc4 type strain is characterized in that the Taqman primer is used for the Enterobacter aerogenes PSgc4orf8The nucleotide sequence of 2 DNA fragments selected from the gene is shown in SEQ ID NO. 1-SEQ ID NO. 2.
2. An MGB probe specific to Enterobacter aerogenes PSgc4 type strain, characterized in that it is located inside 2 DNA fragments of claim 1 and comprises a 5' reporter dye (FAM) and a 3' non-fluorescent quencher (NFQ), conjugated at the 3' end to a Minor Groove Binder (MGB), and its nucleotide sequence is shown in SEQ ID NO. 3.
3. The components and concentrations of the Taqman primer and MGB probe real-time fluorescence PCR reaction system specific to Enterobacter aerogenes PSgc4 strain according to claims 1 and 2 are as follows:
。
4. the use of the Taqman primers and MGB probes specific to different O antigen serological Enterobacter sakazakii of claims 1 and 2 in a real-time fluorescent PCR detection system.
5. The use of claim 4 wherein the serotype is Enterobacter sakazakii and refers to a pure culture or a crude extract of nucleic acid DNA of the bacterium isolated from blood, urine or sputum of the bacterium.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107586861A (en) * | 2017-09-20 | 2018-01-16 | 南开大学 | For detecting suspension chip and the application of serotype clostridium perfringen parting |
| CN108866215A (en) * | 2017-12-01 | 2018-11-23 | 苏州百源基因技术有限公司 | A kind of real-time fluorescence quantitative PCR kit for clostridium perfringen detection |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107586861A (en) * | 2017-09-20 | 2018-01-16 | 南开大学 | For detecting suspension chip and the application of serotype clostridium perfringen parting |
| CN108866215A (en) * | 2017-12-01 | 2018-11-23 | 苏州百源基因技术有限公司 | A kind of real-time fluorescence quantitative PCR kit for clostridium perfringen detection |
Non-Patent Citations (1)
| Title |
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| 李伟等: "《分子诊断学》", 30 September 2015, 中国医药科技出版社 * |
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