CN102660645B - The specific detection of streptococcus pneumoniae and hemophilus influenza primed probe combination and test kit - Google Patents

Abstract

The present invention relates to a kind of employing Fluorescence PCR assay, the oligonucleotide sequence combination that in specific detection sample, streptococcus pneumoniae and hemophilus influenza nucleic acid exist, and include the test kit of this combination.This test kit can detect and differentiate streptococcus pneumoniae nucleic acid and hemophilus influenza nucleic acid present in sample delicately, and its Monitoring lower-cut is the 20 every reaction systems of copy, has important using value in the field such as disease surveillance, clinical diagnosis.

Description

The specific detection of streptococcus pneumoniae and hemophilus influenza with primed probe combination and Test kit

Technical field

The present invention relates to a kind of employing Fluorescence PCR assay, streptococcus pneumoniae and the bloodthirsty bar of influenza in specific detection sample The test kit that sclerotium acid exists.

Technical background

The pathogen of bacterial pneumonia has larger difference because of age, accompanying diseases with immune functional state.Just obtain For mode, common for streptococcus pneumoniae and hemophilus influenza in community acquired pneumonia pathogen.

Streptococcus pneumoniae is gram positive coccus, is the main pathogen causing bacterial pneumonia, accounts for its 2/3 left side The right side, all can fall ill the whole year, and winter-spring season is occurred frequently.Hemophilus influenza is Grain-negative dialister bacterium, high in the child at 4-18 monthly age Send out, bacterial pneumonia and bacterial meningitis can be caused.According to WTO statistical data, have more than 300,000 people every year and die from influenza Pneumonia that haemophilus causes and meningitis.The laboratory diagnosis of streptococcus pneumoniae relies primarily on Bacteria culturing.Traditional inspection Survey method needs culture of isolated could identify after going out bacterial strain, requires time for longer.And hemophilus influenza growth conditions is harsh, battalion Foster requirement is the highest, wastes time and energy, and is unfavorable for quickly detecting, and possesses certain danger for operator.In recent years PCR reaction Fast Detection Technique for pathogen starts appearance.For PCR detects, the selection of primer is most important, directly Connect the specificity of impact detection.If primer specificity is inadequate, then may cause false positive results or the false negative knot of detection The appearance of fruit.

The cause of disease is found out for the detection of streptococcus pneumoniae and hemophilus influenza in patients with pneumonia respiratory secretions Important step, selects significant for clinical diagnosis and medicine.

The present invention is directed to streptococcus pneumoniae and the special target sequence design primer and Taqman probe of hemophilus influenza, profit By the method for real-time PCR, it is used for differentiating to detect streptococcus pneumoniae and hemophilus influenza nucleic acid in sample, can be quick Obtain specific detection result.

Summary of the invention

Existing to more accurately detect sample streptococcus pneumoniae and hemophilus influenza nucleic acid, the present invention provides a kind of The reagent that in specific detection sample, the oligonucleotide sequence of streptococcus pneumoniae and hemophilus influenza combines and comprises this combination Box, it is characterised in that in the PCR reactant liquor of combined sequence or test kit, the primer for nucleic acid amplification is:

P1:5`-AGTCGTTCCAAGGTAACAAGT-3`,

P2:5`-CACGCACCGACTACCTAAACC-3`,

P3:5`-TGCAACTCCAGCTGCTAAAGTATT-3`,

P4:5`-TCTTCACCGTAAGATACTGTGCC-3`.

P1 and P2 is to draw for streptococcus pneumoniae genome specificity sequential design the specificity that filters out through preliminary experiment Thing, P3 and P4 is for hemophilus influenza genome specificity sequential design the specific primer that filters out through preliminary experiment.

Inventive feature also resides in, for the widow of fluorescence signal detection in the PCR reactant liquor of combined sequence or test kit Nucleotide probe is:

Probe1:5`-X<sub>1</sub>-GATCAGATTGAAGCTGATAAAACGATACA-Y<sub>1</sub>-3`,

Probe2:5`-X<sub>2</sub>-TAGGTCAACGTCGTGCAGATGCAGTT-Y<sub>2</sub>-3`。

X₁And X₂For fluorescent reporter group, Y₁And Y₂For fluorescent quenching group.Probe1 is for streptococcus pneumoniae genome Specific sequence design the specific probe filtered out through preliminary experiment, Probe2 is for hemophilus influenza genome specific Property sequential design the specific probe filtered out through preliminary experiment.

Inventive feature also resides in, and test kit includes PCR reactant liquor, enzyme mixation, negative quality-control product and positive quality control Product.Wherein PCR reactant liquor mainly contains above-mentioned primer and probe, reaction buffer, Mg²⁺With dNTP etc., enzyme mixation is main Containing hot start Taq polymerase, negative quality-control product is deionized water, and positive quality control product is the nucleic acid containing detection target sequence.

Another preferred embodiment of the present invention is: for fluorescence signal in the PCR reactant liquor of combined sequence or test kit The fluorophor of the oligonucleotide probe of detection, wherein Probe1 fluorescent reporter group X₁For Fam, fluorescent quenching group Y₁For Eclipse, Probe2 fluorescent reporter group X₂For Hex, fluorescent quenching group Y₂For Eclipse.

Another preferred embodiment of the present invention is: test kit comprise above-mentioned two pairs of specific primers and two specificitys Probe, belongs to the detection of real-time fluorescence double PCR, can be to streptococcus pneumoniae and hemophilus influenza core in same reaction system Acid detects and differentiates.

Another preferred embodiment of the present invention is: the PCR reaction cycle parameter of test kit is 95 DEG C, 2min；Entrance follows Loop order section: 95 DEG C of degeneration 10s, 60 DEG C of annealing extend 1min, coreaction 40 circulation.

Another preferred embodiment of the present invention, the PCR reaction system that test kit is selected is 20 μ l, buffers including 2 × PCR The each 0.5 μ l of liquid 10 μ l, 10mmol/L dNTP 1.0 μ l, 25 μm ol/L primers, each 0.2 μ l of 10 μm ol/L probes, thermal starting Taq Enzyme mixation 0.2 μ l, sample DNA 2 μ l, add aquesterilisa to whole system 20 μ l.

The test kit that the present invention provides to the Monitoring lower-cut of streptococcus pneumoniae nucleic acid is 20 and copies every reaction system；Convection current The Monitoring lower-cut of haemophilus influenza nucleic acid is 20 and copies every reaction system.

The present invention separately designs specific primer and spy according to streptococcus pneumoniae or hemophilus influenza genome conserved region Pin, it is provided that test kit can detect the nucleic acid of streptococcus pneumoniae or hemophilus influenza, but non-streptococcus pneumoniae can not be detected Or the nucleic acid of hemophilus influenza pathogen, illustrate that test kit has good specificity.

The test kit that the present invention provides can complete detection in 3 hours, double check can save the testing cost of 50%, can Experimental basis is provided for streptococcus pneumoniae or the disease surveillance of hemophilus influenza and clinical diagnosis.

Accompanying drawing explanation

Fig. 1 is the amplification curve diagram of dual real-time fluorescence PCR detection streptococcus pneumoniae nucleic acid sensitivity.5 from left to right Bar curve is respectively streptococcus pneumoniae Specific PCR fragments and builds plasmid gradient dilution (10⁵-10¹) amplification afterwards.Abscissa For reaction cycle number, vertical coordinate is the Δ Rn value of different period fluorescent assay signal.

Fig. 2 is the amplification curve diagram of dual real-time fluorescence PCR detection hemophilus influenza nucleic acid sensitivity.From left to right Article 5, curve is respectively hemophilus influenza Specific PCR fragments and builds plasmid gradient dilution (10⁵-10¹) amplification afterwards.Horizontal seat Being designated as reaction cycle number, vertical coordinate is the Δ Rn value of different period fluorescent assay signal.

Fig. 3 is that real-time fluorescence double PCR detection system is to streptococcus pneumoniae and hemophilus influenza detection of nucleic acids specificity Amplification curve diagram.All there is S type amplification curve in streptococcus pneumoniae and hemophilus influenza, and other pathogenic microorganism Quality Control bacterium are equal S type amplification curve does not occurs.Abscissa is reaction cycle number, and vertical coordinate is the Δ Rn value of different period fluorescent assay signal.

Detailed description of the invention

The preferred embodiment of the present invention is described in detail below in conjunction with specific embodiment.It is pointed out that and list here Embodiment be merely exemplary descriptive purpose, and it should not be constructed as any limitation on the scope of the present invention.Wherein make Test kit, the reagent such as buffer be only reagent specifically chosen in this specific embodiment, it should be appreciated that people in the art Member can select the corresponding reagent of other companies to realize the purpose of the present invention as required.

1, primer and the design of TaqMan probe and synthesis

Utilize Blast instrument to all of streptococcus pneumoniae in Genbank and domestic and foreign literature and hemophilus influenza Genome sequence is analyzed, select respectively its stable conservative region as detection target sequence, and for the two detect target Sequential design and synthesis overlap primer and probe more.Primer and probe are by the synthesis of TaKaRa Dalian treasured biotech firm of Japan, wherein Detection probe 5 ' the end flag F AM fluorophor of streptococcus pneumoniae, 3 ' end labelling Eclipse fluorescent quenching groups, influenza is bloodthirsty Detection probe 5 ' the end labelling Hex fluorophor of bacillus, 3 ' end labelling Eclipse fluorescent quenching groups.

2, the preparation of strain is detected

Streptococcus pneumoniae, hemophilus influenza and other negative control bacterial strain (pertussis used in the present embodiment Bordetella, Bordetella parapertussis, A group meningitis Neisseria, B group meningitis Neisseria, C group meningitis are how Plucked instrument Salmonella, mycoplasma pneumoniae, legionella pneumophilia, Chlamydia pneumoniae and moraxelle catarrhalis) all buy in China's pharmaceutical biological product Calibrating institute.

3, the extracting of bacterial strain DNA

Qiagen company QIAamp DNA Mini Kit (article No.: 51306) is selected to extract bacterial strain DNA.Concrete steps reagent Box is with reference to operating instruction.

4, primer and the screening of probe

The many sets primer and the probe streptococcus pneumoniae of Detection and Extraction, hemophilus influenza and the feminine gender respectively that use design are right According to the genomic DNA of bacterial strain antibacterial, through repeatedly testing, filter out the primed probe group that sensitivity, specificity and repeatability are optimal Close.(see sequence table, streptococcus pneumoniae forward primer p1, reverse primer p2 and probe probe1；Hemophilus influenza forward primer P3, reverse primer p4 and probe probe2)

5, the structure of standard substance and preparation

It is utilized respectively p1, p2 and p3, p4 primer, PCR amplification streptococcus pneumoniae and hemophilus influenza specific gene sheet Section, is cloned into pMD-18T carrier by PCR primer, converts DH5 α escherichia coli, utilizes alkaline lysis method of extracting positive colony plasmid, Utilize ultraviolet-uisible spectrophotometer to measure the absorptance of DNA at wavelength 260nm, at 280nm respectively, then calculate plasmid dense Degree and purity.Then 10 times of gradient dilutions copy every microlitre to 10.

6, reaction condition optimization

Optimizing the key elements such as primer, probe, enzyme one by one, the reaction system determined is: 2 × PCR buffer 10 μ l, The each 0.5 μ l of 10mmol/L dNTP 1.0 μ l, 25 μm ol/L primers, each 0.2 μ l of 10 μm ol/L probes, hot start Taq polymerase 0.2 μ l, Template 2ul, adds aquesterilisa to whole system 20 μ l.

According to amplified fragments, primer and the annealing temperature of probe and enzyme viability, mainly to reacting annealing temperature and prolonging The time of stretching is optimized, and the loop parameter finally determined is: denaturation 95 DEG C, 2min；Amplification and detection: 95 DEG C of degeneration 10s, 60 DEG C of annealing extend 1min, 40 circulations, and each circulating in is annealed and extend phase acquisition fluorescence signal.

By identical conditions analytical data after amplification terminates, determine the Ct value of each sample.

The evaluation of 7, detection limit

Judge the detection limit of the test kit that the valency present invention provides with the positive criteria in above-mentioned 5, positive criteria product concentration is: 1×10⁵Copies/ μ l, 1 × 10⁴Copies/ μ l, 1 × 10³Copies/ μ l, 1 × 10²Copies/ μ l, 1 × 10¹copies/μ L, the test kit that the present invention provides to the Monitoring lower-cut of streptococcus pneumoniae nucleic acid is 20 and copies every reaction system；Bloodthirsty to influenza The Monitoring lower-cut of bacillus nucleic acid is 20 and copies every reaction system.

8, specific evaluation is detected

The specificity of this test kit is have rated with the bacterial strain DNA in above-mentioned 2 for template.Bloodthirsty to streptococcus pneumoniae and influenza All visible clear and definite amplification curve when bacillus DNA detects, to above-mentioned 9 kinds of other pharyngeal encountered pathogenic microbial DNAs detection Time, do not produce positive amplification curve, illustrate not exist between probe and primer and other bacterial strains that we select that we use Cross reaction.

Although the most in a preferred manner, illustrated some embodiment party of the present invention by specific embodiment Formula, but it will be understood by a person skilled in the art that, and the present invention is not limited to embodiment disclosed above, but can be according to this It is modified by the knowledge of technical field that the present invention belongs to, is made an amendment without departing from the scope of protection of present invention.Such as, Fluorescent real time PCR used in the present invention can also use the fluorescence pointed out in embodiment listed in description as required Mark substance beyond group and fluorescent quenching group, such as labellings such as Tet, FAM, HEX, TAMRA, ROX, Cy3, TxRd, JOE Thing；Or use other labelling systems outside Taqman technology, such as MGB probe, molecular beacon MB probe, scorpion shape probe, glimmering The fluorescent probe labelling techniques such as light double cross probe；Or use dyestuff to be fitted together to unsaturated dyestuff and the LC such as method such as SYBR Green I The saturable dyes such as Green, as long as it using specific primer sequence of the present invention, can qualitative or quantitative testing goal The existence of gene, and then specifically detection streptococcus pneumoniae and the existence of hemophilus influenza.So, these art technology Change and the replacement of customary means that personnel are understood also fall within the scope of the present invention.Protection scope of the present invention should be by Appending claims is limited.

Claims (3)

1. detect streptococcus pneumoniae and a test kit for hemophilus influenza nucleic acid, including PCR reactant liquor, enzyme mixation, the moon Property quality-control product and positive quality control product, in PCR reactant liquor, the primer sequence for nucleic acid amplification reaction is as follows:

P1:5`-AGTCGTTCCAAGGTAACAAGT-3`,

P2:5`-CACGCACCGACTACCTAAACC-3`,

P3:5`-TGCAACTCCAGCTGCTAAAGTATT-3`,

P4:5`-TCTTCACCGTAAGATACTGTGCC-3`,

In PCR reactant liquor, the sequence for the oligonucleotide probe of fluorescence signal detection is as follows:

Probe1:5`-X<sub>1</sub>-GATCAGATTGAAGCTGATAAAACGATACA-Y<sub>1</sub>-3`,

Probe2:5`-X<sub>2</sub>-TAGGTCAACGTCGTGCAGATGCAGTT-Y<sub>2</sub>-3`,

Probe1 fluorescent reporter group X₁For Fam, fluorescent quenching group Y₁For Eclipse, Probe2 fluorescent reporter group X₂For Hex, fluorescent quenching group Y₂For Eclipse.

2. test kit as claimed in claim 1, is further characterized in that PCR reaction system is 20 μ l, including 2 × PCR buffer The each 0.5 μ l of 10 μ l, 10mmol/L dNTP 1.0 μ l, 25 μm ol/L primers, each 0.2 μ l of 10 μm ol/L probes, hot start Taq polymerase Mixed liquor 0.2 μ l, sample DNA 2 μ l, add aquesterilisa to whole system 20 μ l.

3. test kit as claimed in claim 1, is further characterized in that PCR reaction cycle parameter is:

94 DEG C, 2min；Enter the cycle stage: 94 DEG C of degeneration 10s, 60 DEG C of annealing extend 1min, coreaction 40 circulation.