CN114854888A - Primer probe set for detecting streptococcus pneumoniae based on fluorescent quantitative PCR technology, kit and application thereof - Google Patents
Primer probe set for detecting streptococcus pneumoniae based on fluorescent quantitative PCR technology, kit and application thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting streptococcus pneumoniae by TaqMan real-time fluorescent quantitative PCR, which comprises the steps of selecting a section of gene sequence specific to streptococcus pneumoniae, and designing a primer and a TaqMan probe based on the specific gene sequence; the real-time fluorescent probe quantitative PCR detection method of the streptococcus pneumoniae is established by using the target gene clone plasmid as a standard substance and detecting indexes such as sensitivity, amplification efficiency and the like; except for the target bacteria, the genome DNA of other species of bacteria has no amplification signal, which indicates that the method has good specificity and can meet the detection requirement of a common sample; the designed probe has extremely high sensitivity, and the minimum detection limit can reach 10 2 CFU/ml; ten times ofCompared with a diluted standard product, the method has good repeatability in 3 repeated experiments, and shows that the method is stable and has reliable data; therefore, the real-time fluorescent probe quantitative PCR method established by the invention is a method for quickly and accurately detecting streptococcus pneumoniae.
Description
Technical Field
The invention relates to the technical field of biological detection, and particularly relates to a primer, a fluorescent probe and a kit for quantitative detection of streptococcus pneumoniae nucleic acid.
Background
Streptococcus genus (Streptococcus pneumoniae, SP) is gram-positive. It does not produce endo-and exotoxins, and its pathogenicity is mainly the invasion of the capsule: the variation strain without capsule is avirulent, and the pneumococcus with capsule can resist phagocytosis of phagocyte, and is favorable for the colonization and propagation of bacteria in host. Streptococcus pneumoniae can be divided into more than 90 serotypes according to the antigenicity of the polysaccharides of the bacterial capsules, and different serotypes have different pathogenicity.
The rapid detection method for streptococcus pneumoniae in the prior art comprises the following steps: 1. traditional culture methods (national standard methods), i.e. obtaining SP from blood or pleural effusion culture isolation still serves as the gold standard for diagnosis; 2. a latex agglutination method, which is an immunological agglutination method against a pneumococcal capsular antigen; 3. the automated method, the automated instrument for detecting the microorganism is commonly used at present, and the common methods for detecting streptococcus pneumoniae include: API20Strep test strips, the VlTEK AMS system, the PHOENIX SMIC/ID panel, and MICRO-SAN Walk Away, etc.; 4. immunochromatography (ICT), a rapid method currently widely used for diagnosis of SP, detects the antigenic C polysaccharide common to each serotype of SP. The method can be used for detecting urine, pleural effusion, cerebrospinal fluid and bronchial lavage; 5. the nucleic acid amplification technology, PCR (Polymerase Chain Reaction), belongs to a nucleic acid in vitro amplification technology, realizes the amplification of a target nucleic acid sequence by means of oligonucleotide-directed repeated cycles of DNA synthesis, and detects the amplified DNA by gel electrophoresis or probe hybridization; 6. real-time fluorescent quantitative PCR technology.
Fluorogenic quantitative PCR targeting lytA (major autolysin gene) and piaB (permeant gene transporter from pia-ABC) are currently used as gold standards for fluorogenic quantitative PCR detection of Streptococcus pneumoniae. The prior art CN102876774A discloses primers and probes for detecting streptococcus pneumoniae against lytA gene, which have high performance, but no experiment for sensitivity confirmation is performed. Meanwhile, the prior art also has a method for detecting Streptococcus pneumoniae by real-time fluorescent quantitative PCR aiming at SP2020 gene (Tavares et al, identification of Streptococcus pneumoniae by a real-time PCR assay SP2020, Nature, (2019) 9:3285), which indicates that when SP2020, lytA gene and piaB gene are used as target genes, the sensitivity is respectively 100%, 100% and 95.3%, and the specificity is respectively 99.8%,99.5% and 99.5%, and the detection of one target gene alone cannot necessarily obtain higher sensitivity and specificity, and the detection of two genes, namely lytA and SP2020 can realize the best effect. The detection sensitivity of other commonly used target genes such as the pneumolysin gene ply, the pneumococcal surface adhesion A gene psaA, the topoisomerase gene parE, and the like is not high enough. Thus, there is still a need for a method for detecting streptococcus pneumoniae that improves the sensitivity and specificity of fluorescent quantitative PCR detection.
Disclosure of Invention
The invention aims to solve the technical problem of providing a fluorescent quantitative PCR detection kit and a detection method capable of quickly and specifically detecting streptococcus pneumoniae in a sample.
The inventor of the invention finds that the gene HKM25 of the coding RNA polymerase sigma factor (sigma-70 family RNA polymerase sigma factor) in the pneumococcal genome is highly conserved in the pneumococcal genomes of different serotypes, and a pair of primers and a fluorescent probe are designed by taking the gene HKM25 as a target gene, so that the pneumococcus and other bacteria can be effectively distinguished, the time consumption is low, the specificity and the sensitivity are high, and the rapid diagnosis and the epidemiological investigation of the pneumococcal infection are facilitated.
Firstly, the invention provides a primer and fluorescent probe combination for detecting streptococcus pneumoniae based on a fluorescent quantitative PCR technology, wherein the primer comprises a forward primer and a reverse primer, and the primer comprises the following components in parts by weight:
the forward primer is: 5 ʹ -GGTCTCTGGCTAGATGATTATTATTCTCTT-3 ʹ (SEQ ID: 1);
the reverse primer is: 5 ʹ -ATAGTAAACTCCTTAAACACAATGCGTAA-3 ʹ (SEQ ID: 2);
the fluorescent probe is as follows: 5 ʹ -CGCCCTCGAAATCGTTCATTGCTTAAGA-3 ʹ (SEQ ID NO: 3);
the 5 ʹ end of the probe is marked with a fluorescent group, and the 3 ʹ end of the probe is marked with a quenching group; the fluorescent group comprises FAM, and the quenching group comprises BHQ 1.
Secondly, the invention provides a real-time fluorescent quantitative PCR detection kit for streptococcus pneumoniae nucleic acid, which comprises the primer and a fluorescent probe.
The kit also comprises PCR buffer solution, Taq enzyme, dNTPs and Mg 2+ And the like.
Preferably, the kit further comprises a standard positive template and a negative template, wherein the positive template is a constructed standard substance positive plasmid, and the negative template is sterilized double distilled water.
The invention also provides a TaqMan real-time fluorescence quantitative PCR method for detecting the streptococcus pneumoniae, in particular to TaqMan real-time fluorescence quantitative PCR detection for the streptococcus pneumoniae by utilizing the primer and the probe or the kit.
The method comprises the following steps:
A. extracting total DNA of bacteria in the purified strain or sample tissue;
B. taking the total DNA in the step A as a template, and carrying out PCR amplification reaction by using the primer and the probe;
C. preparing a standard substance;
D. selecting a reaction system and reaction parameters;
E. drawing a standard curve;
F. the specific sample is tested.
The technical scheme provided by the embodiment of the invention has the following beneficial effects: the primer, the fluorescent probe, the kit and the detection method for detecting the streptococcus pneumoniae nucleic acid by the real-time TaqMan fluorescent quantitative PCR have high specificity and high sensitivity, both the specificity of 100% and the positive detectable rate of 100% can be achieved, the kit can accurately and quantitatively determine the amount of the nucleic acid, false negative and false positive results can be effectively prevented, and the detection method can quickly and accurately directly detect pathogens.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows the result of agarose gel electrophoresis of the fluorescent quantitative PCR product of example 2 of the present invention.
FIG. 2 is a sensitivity detection standard curve of example 3 of the present invention.
FIG. 3 is an amplification curve of the standard and reference in example 4 of the invention.
Detailed Description
Example 1 primer and fluorescent probe design and synthesis:
genomic sequences of 90 different strains of Streptococcus pneumoniae were downloaded using the NCBI website (http:// blast. NCBI. nlm. nih. gov) and compared for repeats of their genomic sequences, where the coding region of gene HKM25_29 was conserved in sequence (SEQ ID NO:4) and there was one repeat in the genome in inverted order. blast search showed that the sequence of this region was specific for pneumococci, and similar sequences were not present in other species.
Based on the sequence, design of a primer and a probe marked by a fluorescent dye is carried out. The primers and probes are designed according to TaqMan guidelines, and are predicted by software to avoid secondary structures or primer dimers from forming. Preferred primers and probes are as follows:
primer F1: 5 ʹ -GGTCTCTGGCTAGATGATTATTATTCTCTT-3 ʹ,
primer R1: 5 ʹ -ATAGTAAACTCCTTAAACACAATGCGTAA-3 ʹ,
the fluorescent probe is 5 ʹ -FAM-CGCCCTCGAAATCGTTCATTGCTTAAGA-BHQ1-3 ʹ.
Example 2 detection of the correctness of the designed primers was achieved by cloning the Streptococcus pneumoniae ATCC23340 HKM25 gene sequence
Total DNA of Streptococcus pneumoniae ATCC23340 was extracted using a bacterial genomic DNA extraction kit (Tiangen, DP 302) and PCR amplified using the primers sequence 1 and sequence 2 described above.
The PCR amplification reaction system is 20 mu L: the forward and reverse primer concentrations were 200nM each (0.5. mu.L each) and the probe concentration was 200nM (0.5. mu.L). Taq enzyme was used 1U (0.2. mu.L), magnesium ion concentration was 2mM (2. mu.L), dNTPs concentration was 200. mu.M (2. mu.L), PCR buffer concentration was 10X (2. mu.L), cDNA template concentration was 10. mu.L, ddH 2 O addition (2.3. mu.L).
The PCR reaction conditions are as follows: 30s at 95 ℃; 5s at 95 ℃, 34s at 55 ℃ and 40 cycles; annealing at 55 ℃.
The PCR products obtained were analyzed by electrophoresis on a 1% agarose gel, and the results (see FIG. 1) revealed that a partial fragment 168bp of HKM25 gene of Streptococcus pneumoniae was amplified. The target band was recovered and ligated with pUCm-T, transformed into JM109, and subjected to Shanghai's sequencing after the identification. The sequence analysis shows that the sequence of the gene is consistent with that of the pseudoclone, which indicates that the streptococcus pneumoniae ATCC23340 HKM25 gene is amplified correctly.
Example 3. drawing of fluorescent quantitative PCR standard curve:
a) streptococcus pneumoniae ATCC23340 was used as a model bacterium for the generation of a standard curve for fluorescent quantitative PCR, which may represent other Streptococcus pneumoniae. Culturing streptococcus pneumoniae ATCC23340 in a brain heart infusion medium (BHI) overnight, and diluting by a 10-fold gradient to obtain bacterial liquids with different concentrations: 5.0X 10 1 、5.0×10 2 、5.0×10 3 、5.0×10 4 、5.0×10 5 、5.0×10 6 、5.0×10 7 cfu/ml。
b) Extracting genome DNA of bacterial liquids with different concentrations by adopting a rhizobacteria genome DNA extraction kit;
c) performing PCR amplification by using a medical real-time fluorescent quantitative PCR instrument of the Kunpeng gene Archimed X4, wherein the amplification system and the reaction conditions are as described in the above example 2; in addition, fluorescence values, namely Ct standard values, are collected at the annealing stage of 55 ℃, and melting curve analysis at 60-95 ℃ is added after the amplification conditions are carried out, wherein 3 samples are parallel;
d) obtaining Ct standard value of each concentration after fluorescent quantitative PCR, wherein the value corresponds to the cycle number of the fluorescent signal when being detected for the first time, can reflect the initial amount of the template, and uses lg standard value and Ct standard value of the bacterial numberThe standard curve y of the bacterial suspension is-3.211 x +37.052, and the correlation coefficient is 1 (figure 2), which shows that the detection method established by the invention can accurately and quantitatively detect the streptococcus pneumoniae in the sample; and its detection limit is 1x 10 2 cfu/mL。
Example 4 specificity experiments
The DNA of streptococcus pneumoniae is used as a positive control, the DNA of staphylococcus aureus, enterococcus faecalis, enterococcus faecium, streptococcus pyogenes and candida albicans is used as a negative control, double distilled water is used as a blank control, the reaction system and the procedure are the same, and the DNA of the bacteria is detected by real-time fluorescence quantitative PCR. As can be seen from FIG. 3, the detection is carried out by using the established real-time fluorescent quantitative PCR method, and a blank control is set, so that only specific amplification signals are detected in the Streptococcus pneumoniae reaction tube, and no fluorescent signals are accumulated in other reaction tubes, which indicates that the method has good specificity (FIG. 3).
EXAMPLE 5 repeatability experiments
Optionally, three copies of the target gene were amplified three times in 5 replicates each, as shown in Table 1.
Through calculation, the variation coefficients of different template concentrations are lower than 2%, and the result is satisfactory.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<110> Kunpeng Gene (Beijing) science and technology Limited liability company
<120> primer probe group for detecting streptococcus pneumoniae based on fluorescent quantitative PCR technology, kit and application thereof
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Claims (5)
1. A primer and fluorescent probe combination for detecting streptococcus pneumoniae based on a fluorescent quantitative PCR technology, wherein the primer comprises a forward primer and a reverse primer, and the method is characterized in that: the forward primer is shown as SEQ ID NO.1 in the sequence table; the reverse primer is shown as SEQ ID NO.2 in the sequence table; the fluorescent probe is shown as SEQ ID NO.3 in the sequence table.
2. The primer and fluorescent probe combination of claim 1, wherein said probe is labeled with a fluorescent group at the 5 ʹ end and a quencher group at the 3 ʹ end; the fluorescent group comprises FAM, and the quenching group comprises BHQ 1.
3. A kit for quantitative detection of streptococcus pneumoniae nucleic acid, comprising the primer of claim 1 or 2 and a fluorescent probe in combination.
4. The kit for quantitative detection of Streptococcus pneumoniae nucleic acid according to claim 3, wherein the kit further comprises a PCR buffer, a standard positive template and a standard negative template.
5. Use of the primer and fluorescent probe combination of claim 1 in the preparation of a streptococcus pneumoniae detection kit.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876774A (en) * | 2012-08-31 | 2013-01-16 | 武汉百泰基因工程有限公司 | Primer, fluorescence probe and kit for quantitative detection of streptococcus pneumonia nucleic acid and detection method of streptococcus pneumonia nucleic acid |
| CN104164508A (en) * | 2014-08-18 | 2014-11-26 | 广州医科大学附属第三医院 | LAMP (Loop-Mediated Isothermal Amplification) primer group, kit and method for detecting streptococcus pneumoniae |
| US20170349937A1 (en) * | 2014-07-25 | 2017-12-07 | Microbiome Limited | Test for Microbial Blood Infections |
| CN109402296A (en) * | 2018-04-17 | 2019-03-01 | 南京岚煜生物科技有限公司 | For detecting the kit and its application method of Respiratory Tract Adenovirus, streptococcus pneumonia and Bordetella pertussis simultaneously |
| CN112481401A (en) * | 2020-12-25 | 2021-03-12 | 郑州安图生物工程股份有限公司 | Kit for simultaneously detecting streptococcus pneumoniae, legionella pneumophila and moraxella catarrhalis |
-
2022
- 2022-06-30 CN CN202210757568.2A patent/CN114854888A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876774A (en) * | 2012-08-31 | 2013-01-16 | 武汉百泰基因工程有限公司 | Primer, fluorescence probe and kit for quantitative detection of streptococcus pneumonia nucleic acid and detection method of streptococcus pneumonia nucleic acid |
| US20170349937A1 (en) * | 2014-07-25 | 2017-12-07 | Microbiome Limited | Test for Microbial Blood Infections |
| CN104164508A (en) * | 2014-08-18 | 2014-11-26 | 广州医科大学附属第三医院 | LAMP (Loop-Mediated Isothermal Amplification) primer group, kit and method for detecting streptococcus pneumoniae |
| CN109402296A (en) * | 2018-04-17 | 2019-03-01 | 南京岚煜生物科技有限公司 | For detecting the kit and its application method of Respiratory Tract Adenovirus, streptococcus pneumonia and Bordetella pertussis simultaneously |
| CN112481401A (en) * | 2020-12-25 | 2021-03-12 | 郑州安图生物工程股份有限公司 | Kit for simultaneously detecting streptococcus pneumoniae, legionella pneumophila and moraxella catarrhalis |
Non-Patent Citations (5)
| Title |
|---|
| DÉBORA A TAVARES等: "Identification of Streptococcus pneumoniae by a real-time PCR assay targeting SP2020", 《SCI REP.》 * |
| JAMES C. MCAVIN,等: "Sensitive and Specific Method for Rapid Identification of Streptococcus pneumoniae Using Real-Time Fluorescence PCR", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| MARRIT N HABETS ET AL: "A novel quantitative PCR assay for the detection of Streptococcus pneumoniae using the competence regulator gene target comX", 《J MED MICROBIOL.》 * |
| 唐婕等: "林麝源化脓隐秘杆菌、溶血性曼氏杆菌和肺炎链球菌三重实时荧光定量PCR检测方法的建立", 《动物医学进展》 * |
| 王良玉等: "荧光定量PCR检测肺炎链球菌感染方法的建立", 《中国妇女健康研究》 * |
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