CN102660645A - Primer and probe combination and kit for specifically detecting streptococcus pneumoniae and haemophilus influenzae - Google Patents
Primer and probe combination and kit for specifically detecting streptococcus pneumoniae and haemophilus influenzae Download PDFInfo
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- CN102660645A CN102660645A CN2012101370670A CN201210137067A CN102660645A CN 102660645 A CN102660645 A CN 102660645A CN 2012101370670 A CN2012101370670 A CN 2012101370670A CN 201210137067 A CN201210137067 A CN 201210137067A CN 102660645 A CN102660645 A CN 102660645A
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Abstract
The invention relates to an oligonucleotide sequence combination for specifically detecting existence of streptococcus pneumoniae and haemophilus influenzae nucleic acids in samples by adopting fluorescent PCR (polymerase chain reaction) technology and a kit comprising the combination. The kit can sensitively detect and identify the streptococcus pneumoniae and haemophilus influenzae nucleic acids in the samples, has limits of detection of 20 copies per reaction system and has important application values in the fields such as disease surveillance and clinical diagnosis.
Description
Technical field
The present invention relates to a kind of employing fluorescent PCR technology, the oligonucleotide sequence combination that streptococcus pneumoniae and hemophilus influenzae nucleic acid exist in the specific detection sample, and the test kit that comprises this combination.
Technical background
The pathogenic agent of bacterial pneumonia has than big-difference because of host's age, accompanying diseases and immune functional state.With regard to the acquisition mode, common in the community acquired pneumonia pathogenic agent is streptococcus pneumoniae and hemophilus influenzae.
Streptococcus pneumoniae is a GPC, is the main pathogenic agent that causes bacterial pneumonia, account for its about 2/3, all can fall ill the whole year, winter-spring season is occurred frequently.Hemophilus influenzae is the gram-negative dialister bacterium, and is occurred frequently in the children at 4-18 monthly age, can cause bacterial pneumonia and bacterial meningitis.According to the WTO statistic data, there are every year 300000 people of surpassing to die from pneumonia and the meningitis that hemophilus influenzae causes.The laboratory diagnosis of streptococcus pneumoniae mainly relies on pathogenic bacteria to cultivate.Traditional detection method needs culture of isolated to go out could to identify after the bacterial strain, need the time longer.And the hemophilus influenzae growth conditions is harsh, and nutritional requirement is very high, wastes time and energy, and is unfavorable for rapid detection, and for operator, possesses certain danger.PCR reaction Fast Detection Technique to pathogenic agent begins to occur in recent years.Detect for PCR, selection of primers is most important, directly the specificity of influence detection.If primer specificity is not enough, the false positive results that then possibly cause detecting or the appearance of false negative result.
Detection for streptococcus pneumoniae and hemophilus influenzae in the pneumonia patient respiratory road secretory product is an important step of finding out the cause of disease, selects significant for clinical diagnosis and medicine.
The present invention is directed to streptococcus pneumoniae and hemophilus influenzae special target sequence design primer and Taqman probe; Utilize the method for real-time PCR; Be used for differentiating streptococcus pneumoniae and hemophilus influenzae nucleic acid in the test sample, can obtain the specific detection result fast.
Summary of the invention
For test sample streptococcus pneumoniae and hemophilus influenzae nucleic acid exist more exactly; The present invention provides the oligonucleotide sequence combination of streptococcus pneumoniae and hemophilus influenzae in a kind of specific detection sample and comprises the test kit of this combination, it is characterized in that the primer that is used for nucleic acid amplification in the PCR reaction solution of combined sequence or test kit is:
P1：5`-AGTCGTTCCAAGGTAACAAGT-3`，
P2：5`-CACGCACCGACTACCTAAACC-3`，
P3：5`-TGCAACTCCAGCTGCTAAAGTATT-3`，
P4：5`-TCTTCACCGTAAGATACTGTGCC-3`。
P1 and P2 Auele Specific Primer for being directed against streptococcus pneumoniae genome specificity sequences Design and filtering out through preliminary experiment, P3 and P4 Auele Specific Primer for being directed against hemophilus influenzae genome specificity sequences Design and filtering out through preliminary experiment.
Characteristic of the present invention is that also the oligonucleotide probe that is used for the fluorescent signal monitoring in the PCR reaction solution of combined sequence or test kit is:
Probe1：5`-X
1-GATCAGATTGAAGCTGATAAAACGATACA-Y
1-3`，
Probe2：5`-X
2-TAGGTCAACGTCGTGCAGATGCAGTT-Y
2-3`。
X
1And X
2Be fluorescence report group, Y
1And Y
2Be the fluorescent quenching group.The specific probe of Probe1 for being directed against streptococcus pneumoniae genome specificity sequences Design and filtering out through preliminary experiment, the specific probe of Probe2 for being directed against hemophilus influenzae genome specificity sequences Design and filtering out through preliminary experiment.
Characteristic of the present invention is that also test kit comprises PCR reaction solution, enzyme mixed solution, negative quality control product and positive quality control product.Wherein the PCR reaction solution mainly contains above-mentioned primer and probe, reaction buffer, Mg
2+With dNTP etc., the enzyme mixed solution mainly contains warm start Taq enzyme, and negative quality control product is a deionized water, and positive quality control product is to contain the nucleic acid that detects target sequence.
Another preferred embodiment of the present invention is: be used for the fluorophor branch of the oligonucleotide probe of fluorescent signal monitoring in the PCR reaction solution of combined sequence or test kit, wherein Probe1 fluorescence report radicals X
1Be Fam, fluorescent quenching group Y
1Be Eclipse, Probe2 fluorescence report radicals X
2Be Hex, fluorescent quenching group Y
2Be Eclipse.
Another preferred embodiment of the present invention is: test kit comprise above-mentioned two pairs of Auele Specific Primers and two specific probes; Belong to the real-time fluorescence double PCR and detect, can be in same reaction system streptococcus pneumoniae and hemophilus influenzae nucleic acid are detected and differentiates.
Another preferred embodiment of the present invention is: the PCR reaction cycle parameter of test kit is 95 ℃, 2min; Get into the cycle stage: 95 ℃ of sex change 10s, 1min, 40 circulations of coreaction are extended in 60 ℃ of annealing.
Another preferred embodiment of the present invention; The PCR reaction system that test kit is selected for use is 20 μ l; Comprise 2 * PCR damping fluid, 10 μ l, 10mmol/L dNTP 1.0 μ l, each 0.5 μ l of 25 μ mol/L primers, each 0.2 μ l of 10 μ mol/L probes, warm start Taq enzyme mixed solution 0.2 μ l, sample DNA 2 μ l, adding aqua sterilisa to end-body is 20 μ l.
Be limited to 20 every reaction systems of copy under the detection of test kit provided by the invention to streptococcus pneumoniae nucleic acid; Be limited to 20 every reaction systems of copy under the detection to hemophilus influenzae nucleic acid.
The present invention is respectively according to streptococcus pneumoniae or hemophilus influenzae genome conserved regions difference design specific primers and probe; The test kit that provides can detect the nucleic acid of streptococcus pneumoniae or hemophilus influenzae; But can not detect the nucleic acid of non-streptococcus pneumoniae or hemophilus influenzae pathogenic agent, explain that test kit has excellent specificity.
Test kit provided by the invention can be accomplished detection in 3 hours, and double check can be saved 50% testing cost, and the disease surveillance and the clinical diagnosis that can be streptococcus pneumoniae or hemophilus influenzae provide experimental basis.
Description of drawings
Fig. 1 detects the amplification curve diagram of streptococcus pneumoniae nucleic acid sensitivity for dual real-time fluorescence PCR.5 curves from left to right are respectively streptococcus pneumoniae specific PCR fragment and make up plasmid gradient dilution (10
5-10
1) the back amplification.X-coordinate is the reaction cycle number, and ordinate zou is the Δ Rn value of different cycle number fluoroscopic examination signals.
Fig. 2 detects the amplification curve diagram of hemophilus influenzae nucleic acid sensitivity for dual real-time fluorescence PCR.5 curves from left to right are respectively hemophilus influenzae specific PCR fragment and make up plasmid gradient dilution (10
5-10
1) the back amplification.X-coordinate is the reaction cycle number, and ordinate zou is the Δ Rn value of different cycle number fluoroscopic examination signals.
Fig. 3 is that real-time fluorescence double PCR detection architecture is to streptococcus pneumoniae and hemophilus influenzae detection of nucleic acids specific amplification graphic representation.S type amplification curve all appears in streptococcus pneumoniae and hemophilus influenzae, and S type amplification curve does not all appear in other pathogenic micro-organism Quality Control bacterium.X-coordinate is the reaction cycle number, and ordinate zou is the Δ Rn value of different cycle number fluoroscopic examination signals.
Embodiment
Specify preferred implementation of the present invention below in conjunction with specific embodiment.It is pointed out that the embodiment that lists only is the purpose of exemplary illustration here, and should it be interpreted as any restriction to the scope of the invention.Reagent such as use therein test kit, damping fluid only are the concrete reagent of selecting in this specific embodiment, should be understood that corresponding reagent that those skilled in the art can select other companies as required is to realize the object of the invention.
1, the design of primer and TaqMan probe is with synthetic
Utilize the Blast instrument that all streptococcus pneumoniaes in Genbank and the domestic and foreign literature and hemophilus influenzae genome sequence are analyzed; Select its stable conservative region as the detection target sequence respectively, and be directed against design of these two detection target sequences and synthetic primers and the probes (seeing table 1) of overlapping more.Primer and probe are synthetic by the precious biotech firm in Japanese TaKaRa Dalian; Wherein the detection probes 5 ' of streptococcus pneumoniae is held flag F AM fluorophor; 3 ' end mark Eclipse fluorescent quenching group; Detection probes 5 ' the end mark Hex fluorophor of hemophilus influenzae, 3 ' end mark Eclipse fluorescent quenching group.
2, detect the preparation of bacterial classification
Employed streptococcus pneumoniae, hemophilus influenzae and other negative control bacterial strains in the present embodiment (Bordetella pertussis, Bordetella parapertussis, A group meningitis Neisseria, B group meningitis Neisseria, C group meningitis Neisseria, mycoplasma pneumoniae, legionella pneumophilia, CPN and moraxelle catarrhalis) are all bought in Nat'l Pharmaceutical & Biological Products Control Institute.
3, the extracting of bacterial strain DNA
Select the QIAamp DNA Mini Kit of Qiagen company (article No.: 51306) extract bacterial strain DNA for use.The concrete steps test kit is with reference to process specifications.
4, the screening of primer and probe
Many covers primer of employing design and probe be the genomic dna of streptococcus pneumoniae, hemophilus influenzae and the negative control bacterial strain bacterium of Detection and Extraction respectively, through experiment repeatedly, filters out sensitivity, specificity and repeated best primer probe combinations.(see sequence table, streptococcus pneumoniae forward primer p1, reverse primer p2 and probe probe1; Hemophilus influenzae forward primer p3, reverse primer p4 and probe probe2)
5, the structure of standard substance and preparation
Utilize p1, p2 and p3, p4 primer respectively; Pcr amplification streptococcus pneumoniae and hemophilus influenzae specific gene fragment; The PCR product cloning to the pMD-18T carrier, is transformed DH5 α intestinal bacteria, utilize alkaline lysis method of extracting positive colony plasmid; Utilize ultraviolet-visible pectrophotometer to be determined at the light absorption ratio of wavelength 260nm place, the DNA of 280nm place respectively, calculate plasmid concentration and purity then.10 times of gradient dilutions to 10 every microlitre of copy then.
6, reaction condition optimization
Key elements such as primer, probe, enzyme are optimized one by one; The reaction system of confirming is: 2 * PCR damping fluid, 10 μ l, 10mmol/L dNTP 1.0 μ l, each 0.5 μ l of 25 μ mol/L primers, each 0.2 μ l of 10 μ mol/L probes, warm start Taq enzyme 0.2 μ l, template 2ul, adding aqua sterilisa to end-body is 20 μ l.
According to amplified fragments length, primer and the annealing temperature of probe and the characteristic of enzyme, mainly reaction annealing temperature and extension time to be optimized, the final loop parameter of confirming is: sex change is 95 ℃ in advance, 2min; Amplification and detection: 95 ℃ of sex change 10s, 1min are extended in 60 ℃ of annealing, 40 circulations, each circulates in annealing and extends the phase acquisition fluorescent signal.
Finish the back by the identical conditions analytical data in amplification, confirm the Ct value of each sample.
7, the evaluation of detectability
Estimate the detectability of the test kit that provides of the present invention with the positive criteria article in above-mentioned 5, positive criteria article concentration is: 1 * 10
5Copies/ μ l, 1 * 10
4Copies/ μ l, 1 * 10
3Copies/ μ l, 1 * 10
2Copies/ μ l, 1 * 10
5Copies/ μ l, 1 * 10
5Copies/ μ l is limited to 20 every reaction systems of copy under the detection of test kit provided by the invention to streptococcus pneumoniae nucleic acid; Detection lower limit to hemophilus influenzae nucleic acid is 20 every reaction systems of copy.
8, the evaluation of detection specificity
Bacterial strain DNA with in above-mentioned 2 is the specificity that template has been estimated this test kit.All visible clear and definite amplification curve when streptococcus pneumoniae and hemophilus influenzae DNA are detected; When above-mentioned 9 kinds of other pharyngeal encountered pathogenic microbial DNAs are detected; Do not produce positive amplification curve, explain between probe that we use and primer and other bacterial strains that we select not have cross reaction.
Although in the preceding text with preferred mode; Through the specific embodiment exemplary illustration some embodiment of the present invention; But it will be understood by a person skilled in the art that; The present invention is not limited to top disclosed embodiment, but the knowledge of technical field is made amendment to it under can be according to the present invention, make an amendment and can not exceed the scope of requirement protection of the present invention.For example; Fluorescent real time PCR used in the present invention also can adopt fluorophor and the fluorescent quenching group mark substance of pointing out among the embodiment listed in the specification sheets in addition as required, like affinity tags such as Tet, FAM, HEX, TAMRA, ROX, Cy3, TxRd, JOE; Or other mark systems outside the use Taqman technology, for example fluorescent probe labeling techniques such as MGB probe, molecular beacon MB probe, scorpion shape probe, fluorescence double cross probe; Or use saturable dyes such as unsaturated dyestuff such as chimeric method of dyestuff such as SYBR Green I and LC Green; As long as it has used specific primer sequence of the present invention; Get final product the existence of qualitative or detection by quantitative goal gene, and then detect the existence of streptococcus pneumoniae and hemophilus influenzae specifically.So the change that these those skilled in the art understood and the replacement of customary means also fall in protection scope of the present invention.Protection scope of the present invention should be limited appending claims.
Claims (9)
1. combination of primers that is used for specific detection sample streptococcus pneumoniae and hemophilus influenzae nucleic acid, this cover combined sequence is characterised in that and comprises following primer:
P1：5`-AGTCGTTCCAAGGTAACAAGT-3`，
P2：5`-CACGCACCGACTACCTAAACC-3`，
P3：5`-TGCAACTCCAGCTGCTAAAGTATT-3`，
P4：5`-TCTTCACCGTAAGATACTGTGCC-3`。
2. a primer and oligonucleotide probe combination that is used for specific detection sample streptococcus pneumoniae and hemophilus influenzae nucleic acid, wherein primer is the described primer of claim 1, oligonucleotide probe is:
Probe1：5`-X
1-GATCAGATTGAAGCTGATAAAACGATACA-Y
1-3`，
Probe2：5`-X
2-TAGGTCAACGTCGTGCAGATGCAGTT-Y
2-3`。
X
1And X
2Be fluorescence report group, Y
1And Y
2Be the fluorescent quenching group.
3. of claim 2, this cover combined sequence characteristic also is, wherein Probe1 fluorescence report radicals X
1Be Fam, fluorescent quenching group Y
1Be Eclipse, Probe2 fluorescence report radicals X
2Be Hex, fluorescent quenching group Y
2Be Eclipse.
4. of claim 2, this cover combined sequence characteristic is that also can be used for the dual real-time fluorescence polymerase chain reaction and detect, promptly a reaction system can detect and differentiate streptococcus pneumoniae and hemophilus influenzae nucleic acid simultaneously.
5. test kit that is used for specific detection sample streptococcus pneumoniae and hemophilus influenzae nucleic acid is comprising PCR reaction solution, enzyme mixed solution, negative quality control product and positive quality control product.The primer sequence that it is characterized in that being used in the PCR reaction solution nucleic acid amplification reaction is following:
P1：5`-AGTCGTTCCAAGGTAACAAGT-3`，
P2：5`-CACGCACCGACTACCTAAACC-3`，
P3：5`-TGCAACTCCAGCTGCTAAAGTATT-3`，
P4：5`-TCTTCACCGTAAGATACTGTGCC-3`。
6. test kit as claimed in claim 5, the sequence of oligonucleotide probe that it is characterized in that being used in the PCR reaction solution fluorescent signal monitoring is following:
Probe1：5`-X
1-GATCAGATTGAAGCTGATAAAACGATACA-Y
1-3`，
Probe2：5`-X
2-TAGGTCAACGTCGTGCAGATGCAGTT-Y
2-3`。
X
1And X
2Be fluorescence report group, Y
1And Y
2Be the fluorescent quenching group.
7. test kit as claimed in claim 5, its characteristic also are to be used in the PCR reaction solution oligonucleotide probe of fluorescent signal monitoring: Probe1 fluorescence report radicals X wherein
1Be Fam, fluorescent quenching group Y
1Be Eclipse, Probe2 fluorescence report radicals X
2Be Hex, fluorescent quenching group Y
2Be Eclipse.
8. test kit as claimed in claim 5; Its characteristic is that also the PCR reaction system is 20 μ l; Comprise 2 * PCR damping fluid, 10 μ l, 10mmol/L dNTP 1.0 μ l, each 0.5 μ l of 25 μ mol/L primers, each 0.2 μ l of 10 μ mol/L probes, warm start Taq enzyme mixed solution 0.2 μ l, sample DNA 2 μ l, adding aqua sterilisa to end-body is 20 μ l.
9. test kit as claimed in claim 5, its characteristic are that also PCR reaction cycle parameter is:
94 ℃, 2min; Get into the cycle stage: 94 ℃ of sex change 10s, 1min, 40 circulations of coreaction are extended in 60 ℃ of annealing.
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Cited By (7)
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| CN102888460A (en) * | 2012-10-12 | 2013-01-23 | 江苏大学 | Multi-landing PCR kit and detection method of streptococcus pneumonia |
| CN105063218A (en) * | 2015-08-20 | 2015-11-18 | 杭州市第一人民医院 | Multiple quantitative PCR (polymerase chain reaction) kit for quick combined detection of four bacteria difficult to cultivate and identify |
| CN108559790A (en) * | 2018-04-17 | 2018-09-21 | 南京岚煜生物科技有限公司 | The kit and its application method of three kinds of respiratory pathogens are detected based on micro-fluidic chip |
| WO2019161469A1 (en) * | 2018-02-20 | 2019-08-29 | Fundação Oswaldo Cruz | Oligonucleotide, set of oligonucleotides, method for simultaneous detection of neisseria meningitidis, streptococcus pneumoniae and haemophilus influenzae, and kit |
| CN110904253A (en) * | 2019-12-18 | 2020-03-24 | 上海伯杰医疗科技有限公司 | Encephalitis meningitis nucleic acid typing detection kit and detection method |
| CN112695110A (en) * | 2020-12-29 | 2021-04-23 | 复旦大学 | Primer group and kit for rapidly detecting streptococcus pneumoniae nucleic acid through polymerase helix reaction and application of primer group and kit |
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| CN102888460A (en) * | 2012-10-12 | 2013-01-23 | 江苏大学 | Multi-landing PCR kit and detection method of streptococcus pneumonia |
| CN102888460B (en) * | 2012-10-12 | 2013-10-23 | 江苏大学 | Multi-landing PCR kit and detection method of streptococcus pneumonia |
| CN105063218A (en) * | 2015-08-20 | 2015-11-18 | 杭州市第一人民医院 | Multiple quantitative PCR (polymerase chain reaction) kit for quick combined detection of four bacteria difficult to cultivate and identify |
| CN105063218B (en) * | 2015-08-20 | 2018-06-29 | 杭州市第一人民医院 | The multiple quantitative PCR reagent kit of the difficult culture identification bacterium of four kinds of fast joint inspection |
| WO2019161469A1 (en) * | 2018-02-20 | 2019-08-29 | Fundação Oswaldo Cruz | Oligonucleotide, set of oligonucleotides, method for simultaneous detection of neisseria meningitidis, streptococcus pneumoniae and haemophilus influenzae, and kit |
| CN108559790A (en) * | 2018-04-17 | 2018-09-21 | 南京岚煜生物科技有限公司 | The kit and its application method of three kinds of respiratory pathogens are detected based on micro-fluidic chip |
| CN108559790B (en) * | 2018-04-17 | 2021-04-13 | 南京岚煜生物科技有限公司 | Kit for detecting three respiratory pathogens based on microfluidic chip and use method thereof |
| CN110904253A (en) * | 2019-12-18 | 2020-03-24 | 上海伯杰医疗科技有限公司 | Encephalitis meningitis nucleic acid typing detection kit and detection method |
| CN112695110A (en) * | 2020-12-29 | 2021-04-23 | 复旦大学 | Primer group and kit for rapidly detecting streptococcus pneumoniae nucleic acid through polymerase helix reaction and application of primer group and kit |
| CN114790489A (en) * | 2021-11-04 | 2022-07-26 | 江汉大学 | MNP (MNP) marker site of haemophilus influenzae, primer composition, kit and application of MNP marker site |
| CN114790489B (en) * | 2021-11-04 | 2023-06-20 | 江汉大学 | MNP (MNP) marking site of haemophilus influenzae, primer composition, kit and application of MNP marking site |
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