CN110093434A - A kind of primer and probe composition and kit - Google Patents
A kind of primer and probe composition and kit Download PDFInfo
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Abstract
The invention discloses a kind of primer and probe composition and kit, the primer and probe composition is for detecting coagulase-negative staphylococci, staphylococcus aureus and methicillin resistance gene；The coagulase-negative staphylococci detection target is sodA gene, and the staphylococcus aureus detection target is femA gene, and the methicillin resistance gene is mecA gene；The primer and probe composition includes forward primer, reverse primer and the probe for being respectively used to detect the sodA gene, the femA gene and the mecA gene, as shown in SEQ ID NO.1~SEQ ID NO.9.Present invention application real-time fluorescence quantitative PCR detects coagulase-negative staphylococci, staphylococcus aureus and methicillin resistance gene.Detection cycle of the present invention greatly shortens, efficiency and detection flux are improved, detection demand, and specificity, sensitivity and accuracy with higher have been catered to, simultaneously because there is no the detection process after amplification in experiment, the pollution problem in experiment can effectively solve the problem that.
Description
Technical field
The present invention relates to molecular biology fields, and in particular to a kind of primer and probe composition and kit.
Background technique
Since late 1980s, the beginning of the nineties, infection caused by the gram-positive bacterias such as staphylococcus is in always
Existing ascendant trend, by taking bloodstream infection as an example, coagulase-negative staphylococci and staphylococcus aureus have become nosocomial infection weight
Want cause of disease.
Coagulase-negative staphylococci (CNS) is gained the name because not generating coagulase, up to the present, certified coagulase
The strain of negative staphylococcus, with the progress of modern medicine, gradually confirmed coagulase-negative Portugal more than more than 40
More pathogenic effects of grape coccus, and in clinical investigation further confirm this bacterial infection there are also increased trend；It is golden yellow
Color staphylococcus is a kind of coagulase-positive staphylococci, can lead to extensive clinical infectious disease, wherein most important
Infectious endocarditis and bacteremia, can threat to life and the death rate it is high.
Phase early 1940s, penicillin investment clinical application make disease caused by staphy lococcus infection receive control
System, but shortly there are the S. aureus L-forms to Penicillin-resistant since bacterium produces the resistance mechanism of penicillase.20 generation
It records the beginning of the sixties, the development and application of the penicillinase-fast penicillins such as methicillin, benzene XiLin once overcome staphylococcus penicillin
Resistance problems, but then shortly have found methicillin-resistant staphylococcus aureus (MRSA), subsequent MRSA and methoxy
XiLin drug resistance coagulase-negative staphylococci (MRCNS) is rapidly in world's scope spreading.Now, methicillin-resistant Staphylococci
(MRS) most important nosocomial infection pathogen is had become.Due to caused by drug-fast bacteria infection with sensitive strain compared with severe symptoms,
The features such as case fatality rate is high, treatment is difficult and somewhat expensive, it is bigger to the harm of human health.The recall rate of MRS had been just in recent years
It is rising year by year, to endanger caused by the infection for effectively controlling MRS, and reduction disease, is establishing accurately and rapidly detection method
Be clinical diagnosis there is an urgent need to.
Traditional staphylococcus identifies the method for being mainly based upon culture, including conventional Gram's staining, is separately cultured
And catalase test, coagulase test of blood plasma etc., the detection of these methods usually require 3~5 days, and often because of the use of antibiotic
And it is suppressed, the failure detected is caused to nutritional requirement height and slow growth.
Summary of the invention
One of the technical problems solved by the invention is to provide one kind for detecting coagulase-negative staphylococci, golden yellow Portugal
The primer and probe composition of grape coccus and methicillin resistance gene.
Present invention solves the technical problem that two be to provide it is a kind of for detecting coagulase-negative staphylococci, golden yellow Portugal
The kit of grape coccus and methicillin resistance gene.
Present invention solves the technical problem that three be to provide it is a kind of for detecting coagulase-negative staphylococci, golden yellow Portugal
The application method of the kit of grape coccus and methicillin resistance gene.
Present invention application real-time fluorescence quantitative PCR detects coagulase-negative staphylococci, staphylococcus aureus and methoxy
XiLin drug resistant gene, detection cycle greatly shortens compared with current routine clinical detection method, improves efficiency and detection flux,
Detection demand, and specificity, sensitivity and accuracy with higher are catered to, simultaneously because in experiment after no amplification
Detection process, can effectively solve the problem that experiment in pollution problem.
It is as follows that the present invention solves technical solution used by above-mentioned technical problem:
A kind of primer and probe composition, the primer and probe composition for detect coagulase-negative staphylococci,
Staphylococcus aureus and methicillin resistance gene；The coagulase-negative staphylococci detection target is sodA gene, institute
Stating staphylococcus aureus detection target is femA gene, and the methicillin resistance gene is mecA gene；The primer and
Probe compositions include be respectively used to detect the sodA gene, the femA gene and the mecA gene forward primer,
Reverse primer and probe；For detect the forward primer of the sodA gene of coagulase-negative staphylococci, reverse primer and
The sequence of probe is respectively as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3；For detecting golden yellow grape
The sequence of the forward primer of the femA gene of coccus, reverse primer and probe is respectively such as SEQ ID NO.4, SEQ ID
Shown in NO.5 and SEQ ID NO.6；For detecting the forward primer, anti-of the mecA gene of pathogen methicillin resistance
To primer and probe sequence respectively as shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9.
Preferably, for detecting forward primer, reverse primer and the probe groups of the sodA gene and the femA gene
At the first primer probe compositions；Forward primer, reverse primer and probe for detecting the mecA gene form second and draw
Physical prospecting injection composition.
Preferably, the probe for detecting the sodA gene is MGB probe, and 5 ' end label reporter fluorescence groups are FAM；
5 ' end label reporter fluorescence groups of the probe for detecting the femA gene are ROX, 3 ' end label quenching fluorescence groups
BHQ2；5 ' end label reporter fluorescence groups of the probe for detecting the mecA gene are FAM, 3 ' end label quenching fluorescences
Group BHQ1.
Preferably, the first primer probe compositions and the second primer combination of probe object are respectively included for detecting
Forward primer, reverse primer and the probe of internal standard gene.
It is highly preferred that the internal standard gene is internal standard int gene.
It is further preferred that the sequence point of forward primer, reverse primer and probe for detecting the internal standard int gene
Not as shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.
Most preferably, 5 ' end mark fluorescent groups of the probe for detecting the internal standard int gene are JOE, 3 ' end marks
Remember quenching group TAMRA.
It is specific as follows:
1. designing pair of primers and a probe according to sodA gene order (AJ343947.1) by sequence alignment:
SodA forward primer: 5 '-TTCAGCAGTTGAAGGGACAGATT-3 ' (SEQ ID NO.1)；
SodA reverse primer: 5 '-GATGGCACACTATCTAAATTAGCAACA-3 ' (SEQ ID NO.2)；
SodA probe: 5 '-FAM-AGAAGCTAAATCAATCGAAG-MGB-3 ' (SEQ ID NO.3).
2. designing pair of primers and a probe according to femA gene order (CPO20618.1) by sequence alignment:
FemA forward primer: 5 '-ATGAAGTTTACAAATTTAACAGCTAC-3 ' (SEQ ID NO.4)；
FemA reverse primer: 5 '-GTGTCTTATACACTGAATACAAAGG-3 ' (SEQ ID NO.5)；
FemA probe:
5’-ROX-AATGACTCCAACATCTTCAGCGTCTT-BHQ2-3’(SEQ ID NO.6)。
3. designing pair of primers and a probe according to mecA gene order (NG_047945.1) by sequence alignment:
MecA forward primer: 5 '-GGATCTGTACTGGGTTAATC-3 ' (SEQ ID NO.7)；
MecA reverse primer: 5 '-AGGTGTTGGTGAAGATATAC-3 ' (SEQ ID NO.8)；
MecA probe: 5 '-FAM-CACCTTGTCCGTAACCTGAATCA-BHQ2-3 ' (SEQ ID NO.9).
4. internal standard gene (internal amplification control, int): for identifying instrument failure, reagent
The reason undesirable there are result caused by mortifier etc. in factor, polymerase activity factor or sample.The present invention chooses rice
NrDNA ITS ITS1 genetic fragment is as internal standard.Int gene and aforementioned testing goal gene order are carried out
It compares, chooses specific primer and fluorescence labeling probe that specific fragment design is directed to the int gene:
Int forward primer: 5 '-GCGATACCACGAGCTAAATC-3 ' (SEQ ID NO.10)；
Int reverse primer: 5 '-GCATTTCGCTACGTTCTTCAT-3 ' (SEQ ID NO.11)；
Int probe:
5’-JOE-ACTCTCGGCAACGGATATCTCGGCTC-TAMRA-3’(SEQ ID NO.12)。
A kind of kit, the kit is for detecting coagulase-negative staphylococci, staphylococcus aureus and methoxy
XiLin drug resistant gene；The kit include nucleic acid extraction liquid, PCR reaction enzyme system, internal standard, negative controls, positive reference substance,
The first primer probe mixed liquor and the second primed probe mixed liquor.
Preferably, the nucleic acid extraction liquid is by 2% (M/V) Chelex-100,1% (V/V) Tris-HCl, 1M, pH9.0
Composition.
Preferably, the PCR reaction enzyme system is by 5U/ μ L Taq archaeal dna polymerase and 2U/ μ L uracil-N-glycosylase
(UNG enzyme) 3: 1 ratios mixing by volume composition.
Preferably, the plasmid containing internal standard int genetic fragment is designated as in described.
Preferably, the negative controls are the distilled water purified by Millipore pure water meter.
Preferably, the positive reference substance is the T load for carrying detection coagulase-negative staphylococci target gene (sodA)
Constitution grain, the carrier T plasmid for carrying detection staphylococcus aureus target gene (femA) and carrying methicillin resistance gene
(mecA) carrier T plasmid or the colibacillus engineering containing above-mentioned plasmid, the bacterium solution bacteria concentration 10 as reference substance5
CFU/mL。
Preferably, the first primer probe mixed liquor is drawn by deoxyribonucleoside triphosphate dN (U) TP and described first
Physical prospecting injection composition composition.
It is highly preferred that the first primer probe mixed liquor is by 2 μ L 10 × PCR Buffer, 2 μ L 25mmol/L
MgCl2, 1.6 μ L 2.5mmol/L dN (U) TP, 0.6 μ L, 10 μm of ol/L be respectively used to detection sodA gene and femA gene
Forward primer and reverse primer, 0.2 μ L, 10 μm of ol/L be respectively used to detection sodA gene and femA gene probe, 0.6
Point of 10 μm of ol/L of forward primer and reverse primer and 0.2 μ L for being respectively used to detection internal standard int gene of 10 μm of ol/L of μ L
The probe and 7 μ L sterilizing purified water composition of internal standard int gene Yong Yu not detected
Preferably, the second primed probe mixed liquor is drawn by deoxyribonucleoside triphosphate dN (U) TP and described second
Physical prospecting injection composition composition.
It is highly preferred that the second primed probe mixed liquor is by 2 μ L 10 × PCR Buffer, 2 μ L 25mmol/L
MgCl2, 1.6 μ L 2.5mmol/L dN (U) TP, the forward primer for detecting mecA gene of 0.6 μ L, 10 μm of ol/L and anti-
To primer, 0.2 μ L, 10 μm of ol/L for detect the probe of mecA gene, 0.6 μ L, 10 μm of ol/L for detecting internal standard int
The probe for being used to detect internal standard int gene and 7 μ L sterilizing of the forward primer and reverse primer of gene and 0.2 μ L, 10 μm of ol/L
Purified water composition.
Preferably, the kit includes separating and concentrating to pack the reagent bottle of the reagent or the packing box of pipe.
A kind of application method of kit, the kit is for detecting coagulase-negative staphylococci, golden yellow grape
Coccus and methicillin resistance gene；The application method of the kit includes the following steps:
(1) prepared by sample nucleic acid, nucleic acid-templated to obtain；
(2) it takes negative controls, positive reference substance to be placed in centrifuge tube, is separately added into nucleic acid extraction liquid and mixes well, and
Heating and centrifugal treating are carried out, supernatant is taken to detect for fluorescent PCR；
(3) enzymatic reagent is prepared, wherein the enzyme is by thermus aquaticus archaeal dna polymerase (Taq enzyme) and uracil-N- glycosyl
Change enzyme (UNG enzyme) mixing composition；
(4) PCR detection mixed liquor and enzymatic reagent concussion are mixed, carries out centrifugal treating；The PCR detects mixed liquor
The first primer probe mixed liquor and the second primed probe mixed liquor；
(5) the PCR detection mixed liquor after taking enzyme mixing respectively is placed in fluorescent PCR pipe, samples this nucleic acid extraction supernatant
Existing PCR detection mixing is added in liquid, negative controls nucleic acid extraction supernatant, positive reference substance nucleic acid extraction supernatant and internal standard
In the fluorescent PCR pipe of liquid, fluorescent PCR amplification, reaction condition are carried out are as follows: 37 DEG C × 2min, 94 DEG C × 2min, recycle 1 time；93
DEG C × 15s, it 60 DEG C × 60s, recycles 40 times；Single-point fluorescence detection acquires fluorescence signal at 60 DEG C herein, it is described in be designated as containing
There is the plasmid of internal standard int genetic fragment；
(6) it for sodA gene and mecA gene, is detected with the channel FAM on fluorescent PCR instrument, for femA base
Cause is detected with the channel ROX on fluorescent PCR instrument, for int gene, is detected with the channel JOE on fluorescent PCR instrument,
The genotype of detection site is determined by data collected by fluorescence quantitative PCR instrument.
Basic principle of the invention:
The present invention to coagulase-negative staphylococci target gene sodA, staphylococcus aureus target gene femA and
On the basis of methicillin resistance gene mecA sequence is furtherd investigate, the conservative and special section of each gene is selected, point
The primer and probe of specificity is not devised, and interfering with each other between primer, finally establishes when effectively preventing tube amplification
Specificity and the good multiple fluorescence quantitative PCR reaction system of sensitivity.
Compared with prior art, the beneficial effects of the present invention are:
(1) present invention establishes specificity and the good multiple fluorescence quantitative PCR reaction system of sensitivity, in two PCR
Detected in pipe 3 target genes (i.e. a single tube detects two different target genes and an identical internal standard gene respectively,
Another single tube detects a target gene and an identical internal standard gene respectively), it can be determined in sample by one-time detection
Whether coagulase-negative staphylococci or staphylococcus aureus are had, and whether containing because carrying mecA gene in judgement sample
And lead to the pathogen of corresponding drug resistance, foundation is provided for the diagnosis state of an illness；
(2) present invention is using real-time fluorescence quantitative PCR detection coagulase-negative staphylococci, staphylococcus aureus and first
Oxygen XiLin drug resistant gene, detection cycle greatly shortens compared with current routine clinical detection method, improves efficiency and detection is logical
Amount has catered to detection demand, and specificity, sensitivity and accuracy with higher, simultaneously because not expanding in experiment
Detection process later can effectively solve the problem that the pollution problem in experiment.
Detailed description of the invention
Fig. 1 is the quantitative fluorescent PCR of sodA gene and femA gene masculine reference substance amplification in the embodiment of the present invention 2
Curve graph；
Fig. 2 is the quantitative fluorescent PCR curve graph of mecA gene masculine reference substance amplification in the embodiment of the present invention 2.
Specific embodiment
In order to better understand the content of the present invention, it is described further combined with specific embodiments below with attached drawing.Ying Li
Solution, these embodiments are only used for that the present invention is further described, rather than limit the scope of the invention.In addition, it should also be understood that,
After having read content of the present invention, person skilled in art makes some nonessential changes or adjustment to the present invention,
Still fall within protection scope of the present invention.
Experimental method in the following example is unless otherwise specified routine experiment method.
Embodiment 1
The composition of kit:
1, nucleic acid extraction liquid: 2% (M/V) Chelex-100,1% (V/V) Tris-HCl, 1M, pH9.0 composition；
2, PCR reacts enzyme system: 5U/ μ L Taq archaeal dna polymerase and 2U/ μ L uracil-N-glycosylase (UNG enzyme) press body
Product is than 3: 1 ratios mixing composition；
3, internal standard: the plasmid containing internal standard int genetic fragment；
4, negative controls: the distilled water purified by Millipore pure water meter；
5, positive reference substance: carrying the carrier T plasmid of coagulase-negative staphylococci target gene (sodA), carries gold
The carrier T plasmid of staphylococcus aureus target gene (femA) and the carrier T plasmid for carrying methicillin resistance gene (mecA)
Or the colibacillus engineering containing above-mentioned plasmid, the bacterium solution bacteria concentration 10 as reference substance5CFU/mL；
6, the first primer probe mixed liquor: by deoxyribonucleoside triphosphate dN (U) TP, coagulase-negative staphylococci target
Mark forward and reverse the drawing of forward and reverse primer and fluorescence labeling probe, staphylococcus aureus target gene (femA) of gene (sodA)
Object and fluorescence labeling probe, forward and reverse primer of internal standard int gene and fluorescence labeling probe composition；
7, the second primed probe mixed liquor: by deoxyribonucleoside triphosphate dN (U) TP, methicillin resistance gene
(mecA) the forward and reverse primer and fluorescence labeling probe of forward and reverse primer and fluorescence labeling probe, internal standard int gene form.
The first primer probe mixed liquor is by 2 μ L 10 × PCR Buffer, 2 μ L, 25 mmol/LMgCl2、1.6μL
2.5mmol/L dN (U) TP, be respectively 0.6 μ L, 10 μm of ol/L coagulase-negative staphylococci target gene (sodA) just
The solidification of forward and reverse primer of reverse primer and staphylococcus aureus target gene (femA), respectively 0.2 10 μm of ol/L of μ L
The fluorescence labeling probe of enzyme negative Staphylococcus target gene (sodA) and staphylococcus aureus target gene (femA) it is glimmering
Signal probe, forward and reverse primer of 0.6 μ L, 10 μm of ol/L internal standard int genes and 0.2 μ L 10 μm of ol/L internal standard int genes
Fluorescence labeling probe and 7 μ L sterilizing purified water composition.
The second primed probe mixed liquor is by 2 μ L 10 × PCR Buffer, 2 μ L, 25 mmol/LMgCl2、1.6μL
Forward and reverse primer, the 0.2 μ L 10 of the methicillin resistance gene (mecA) of 2.5mmol/L dN (U) TP, 0.6 μ L, 10 μm of ol/L
10 μm of ol/L internal standard int genes of fluorescence labeling probe, 0.6 μ L of the methicillin resistance gene (mecA) of μm ol/L it is positive and negative
To the fluorescence labeling probe of 10 μm of ol/L internal standard int genes of primer and 0.2 μ L and 7 μ L sterilizing purified water composition.
Wherein, the primer nucleotide sequences such as SEQ ID of coagulase-negative staphylococci target gene (sodA) is detected
Shown in NO.1 and SEQ ID NO.2, the nucleotide sequence of the probe of coagulase-negative staphylococci target gene (sodA) is detected
As shown in SEQ ID NO.3, the probe for detecting the sodA gene is MGB probe, and 5 ends label reporter fluorescence group is
FAM；
Detect primer nucleotide sequences such as SEQ ID NO.4 and the SEQ ID of staphylococcus aureus target gene (femA)
Shown in NO.5, the nucleotide sequence such as SEQ ID NO.6 institute of the probe of staphylococcus aureus target gene (femA) is detected
Show, it is ROX that 5 ends, which mark reporter fluorescence group, and 3 ends mark quenching fluorescence group BHQ2；
Detect the primer nucleotide sequences such as SEQ ID NO.7 and SEQ ID NO.8 of methicillin resistance gene (mecA)
It is shown, the nucleotide sequence of the probe of methicillin resistance gene (mecA) is detected as shown in SEQ ID NO.9,5 ends label report
Announcement fluorophor is FAM, and 3 ends mark quenching fluorescence group BHQ1；
The primer nucleotide sequences of internal standard int gene are detected as shown in SEQ ID NO.10 and SEQ ID NO.11, detection
For the probe nucleotide sequence of internal standard int gene as shown in SEQ ID NO.12,5 ' end mark fluorescent groups are JOE, 3 ' end labels
Quenching group TAMRA.
8, separate and concentrate the reagent bottle of packaging mentioned reagent or the packing box of pipe.
Embodiment 2
The foundation of kit test method
1, reference substance prepares
It takes each 50 μ L of positive reference substance, negative controls to be respectively placed in 1.5mL (or 0.5mL) centrifuge tube and (freezes reagent
Oscillation mixes 10s after thawing), it is separately added into 50 μ L of nucleic acid extraction liquid and mixes well, 98 DEG C of 10min, then 12,000rpm
5min takes 2 μ L of supernatant to do PCR reaction.
2, reaction system is prepared
16 μ L the first primer probe mixed liquors are taken to react enzyme system mixing with 0.2 μ L PCR, oscillation mixes several seconds, 3000rpm
Centrifugation mixes the several seconds；16 μ L the second primed probe mixed liquors are taken to react enzyme system mixing with 0.2 μ L PCR, oscillation mixes the several seconds,
3000rpm centrifugation mixes the several seconds.
3, PCR amplification
Each 2 μ L of the processing supernatant of negative controls, positive reference substance is separately added into reaction system described in step 2
In the PCR pipe of mixed liquor, and 2 μ L internal standards are added into negative controls, positive reference substance PCR pipe respectively, cover pipe lid, immediately
Carry out fluorescent PCR amplified reaction.
Reaction condition is as follows:
It 37 DEG C × 2min, 94 DEG C × 2min, recycles 1 time；It 93 DEG C × 15s, 60 DEG C × 60s, recycles 40 times；The inspection of single-point fluorescence
It surveys at 60 DEG C, reaction system is 20 μ L.
Fluorescence channel detects and selects: sodA, mecA gene select the channel FAM, and femA gene selects the channel ROX, internal standard int
Gene selects the channel JOE.
The amplification of positive reference substance is shown in Fig. 1 and Fig. 2.
4, result judgement
After detection, baseline adjustment takes the fluorescence signal of 6~15 circulations, and threshold value setting principle is just super with threshold line
Cross the highest point of negative controls detection fluorescence curve.Testing result is determined according to the CT value of target gene amplification, if CT
Value >=35 indicates that test sample lower than detection limit, is reported as feminine gender；If value≤35 CT, test results report is the positive；CT is shown
It is gray area between 35~40.
Embodiment 3
The specificity experiments of kit
1, experimental material (being shown in Table 1)
The experimental material of the present invention of table 1
2, experimental method
It is caused a disease using the experimental method established in embodiment 2 to above-mentioned 17 kinds using kit described in embodiment 1
Bacterium is detected respectively, carries out specific assay to this kit.
3, experimental result
The kit described in embodiment 1 detects 15 kinds of common pathogens (enterococcus faecalis, enterococcus faecium, corynebacterium diphtheriae, streams
Haemophilus influenza, proteus, proteus mirabilis, enterobacter cloacae, Acinetobacter bauamnnii, Neisseria meningitidis, blood hammer
Bacterium, escherichia coli, Klebsiella oxytoca, Burkholderia cepacia, pseudomonas aeruginosa, Candida albicans), result is
It is negative, it was demonstrated that this kit detection specificity is good (being shown in Table 2).
The kit specificity verification result of the present invention of table 2
Embodiment 4
The sensitivity experiment of kit
1, experimental material
Establish 4 concentration gradients (5 × 105copies/mL、5×104copies/mL、5×103 copies/mL、5×
102Copies/mL sensitivity reference material of the plasmid reference material) as this kit.
2, experimental method
The plasmid positive reference material of 4 concentration gradients of sodA, femA and mecA gene is detected respectively, determines this
Kit detects the sensitivity of plasmid concentration (copies/mL).
Specific method is that plasmid reference material is measured OD value (260nm), is scaled copies/mL unit, then carry out gradient
It dilutes, 4 concentration gradients is selected in test, i.e., with 5 × 105 copies/mL、5×104copies/mL、5×103copies/
mL、5×102Copies/mL is as reference material.Sensitivity test is carried out to above-mentioned reference material using kit described in embodiment 1.
Each concentration reference material respectively repeats detection 20 times, counts each concentration positive detection number, determines product minimum detectability.
3, experimental result
Using kit described in embodiment 1, using the experimental method established in embodiment 2, to various concentration plasmid
Positive reference product are detected, and kit can stablize detection 5 × 104Copies/mL plasmid positive reference material, according to sensitivity
Test result, this kit are limited to 5 × 10 according to the lowest detection that plasmid copy Particle density determines4Copies/mL the results are shown in Table
3。
3 each concentration plasmid positive reference material of the present invention of table detects number
Embodiment 5
The repeated experiment of kit (in batch and between criticizing)
1, experimental material
5 × 10 described in embodiment 44Copies/mL plasmid positive reference material is that experimental subjects carries out this kit
Precision research.
2, experimental method
(1) repeat to test in criticizing: kit described in the embodiment 1 prepared using the same time detects plasmid positive reference
Product, each reference material repeat detection 3 times according to 2 the method for embodiment；
(2) repeat to test between criticizing: kit described in the embodiment 1 prepared using 3 batches of (P1, P2, P3) different times is detected
Plasmid positive reference material, each reference material repeat detection 3 times according to 2 the method for embodiment.
3, experimental result
Using kit described in 3 batches of embodiments 1, using the experimental method established in embodiment 2, respectively to sodA,
FemA, mecA gene plasmid positive reference product carry out precision research.As the result is shown: batch interior and betweenrun precision coefficient of variation
(CV%) it is respectively less than and is equal to 5% (being shown in Table 4 and table 5).
Repetitive research result in sodA, femA, mecA gene plasmid the positive reference product of the present invention of table 4 batch
Repetitive research result between sodA, femA, mecA gene plasmid the positive reference product of the present invention of table 5 batch
Embodiment 6
The detection of clinical sample
Using the experimental method established in kit described in embodiment 1 and embodiment 2,30 are detected by the state the Yin people
The collected clinical sample of hospital, wherein coagulase-negative staphylococci is 10 positive, and S. aureus-positive 11,
And in the coagulase-negative staphylococci of detection, methicillin resistance 7, in the staphylococcus aureus of detection, methoxy
XiLin drug resistance 5, specific sample situation is shown in Table 6.It is consistent with clinical bacteria culture identification and drug sensitive test result to will test result
As a result it is compared, counts coincidence rate.
Testing result shows that this kit test result is consistent with clinical bacteria culture identification and drug sensitive test result.
The kit coincidence rate experimental result of the present invention of table 6
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common
Within the essential scope of the present invention, the variations, modifications, additions or substitutions made also should belong to protection of the invention to technical staff
Range.
Sequence table
<110>Geneinn Biotechnology (Ningbo) Co., Ltd.
<120>a kind of primer and probe composition and kit
<130> 2019.05.08
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial synthesized (Unknown)
<400> 1
ttcagcagtt gaagggacag att 23
<210> 2
<211> 27
<212> DNA
<213>artificial synthesized (Unknown)
<400> 2
gatggcacac tatctaaatt agcaaca 27
<210> 3
<211> 20
<212> DNA
<213>artificial synthesized (Unknown)
<400> 3
agaagctaaa tcaatcgaag 20
<210> 4
<211> 26
<212> DNA
<213>artificial synthesized (Unknown)
<400> 4
atgaagttta caaatttaac agctac 26
<210> 5
<211> 25
<212> DNA
<213>artificial synthesized (Unknown)
<400> 5
gtgtcttata cactgaatac aaagg 25
<210> 6
<211> 26
<212> DNA
<213>artificial synthesized (Unknown)
<400> 6
aatgactcca acatcttcag cgtctt 26
<210> 7
<211> 20
<212> DNA
<213>artificial synthesized (Unknown)
<400> 7
ggatctgtac tgggttaatc 20
<210> 8
<211> 20
<212> DNA
<213>artificial synthesized (Unknown)
<400> 8
aggtgttggt gaagatatac 20
<210> 9
<211> 23
<212> DNA
<213>artificial synthesized (Unknown)
<400> 9
caccttgtcc gtaacctgaa tca 23
<210> 10
<211> 20
<212> DNA
<213>artificial synthesized (Unknown)
<400> 10
gcgataccac gagctaaatc 20
<210> 11
<211> 21
<212> DNA
<213>artificial synthesized (Unknown)
<400> 11
gcatttcgct acgttcttca t 21
<210> 12
<211> 26
<212> DNA
<213>artificial synthesized (Unknown)
<400> 12
actctcggca acggatatct cggctc 26
Claims (10)
1. a kind of primer and probe composition, which is characterized in that the primer and probe composition is for detecting coagulase-negative
Staphylococcus, staphylococcus aureus and methicillin resistance gene；The coagulase-negative staphylococci detects target
SodA gene, the staphylococcus aureus detection target are femA gene, and the methicillin resistance gene is mecA gene；
The primer and probe composition includes being respectively used to detect the sodA gene, the femA gene and the mecA gene
Forward primer, reverse primer and probe；For detecting the forward primer of the sodA gene, the sequence point of reverse primer and probe
Not as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3；For detect the femA gene forward primer,
The sequence of reverse primer and probe is respectively as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6；For detecting
The sequence of the forward primer of mecA gene, reverse primer and probe is stated respectively such as SEQ ID NO.7, SEQ ID NO.8 and SEQ
Shown in ID NO.9.
2. a kind of primer and probe composition as described in claim 1, which is characterized in that for detect the sodA gene and
Forward primer, reverse primer and the probe of the femA gene form the first primer probe compositions；For detecting the mecA
Forward primer, reverse primer and the probe of gene form the second primer combination of probe object.
3. a kind of primer and probe composition as claimed in claim 2, which is characterized in that for detecting the sodA gene
Probe is MGB probe, and 5 ' end label reporter fluorescence groups are FAM；For detecting 5 ' end labels of the probe of the femA gene
Reporter fluorescence group is ROX, 3 ' end label quenching fluorescence group BHQ2；For detecting 5 ' end marks of the probe of the mecA gene
Note reporter fluorescence group is FAM, 3 ' end label quenching fluorescence group BHQ1.
4. a kind of primer and probe composition as claimed in claim 3, which is characterized in that the first primer probe compositions
The forward primer, reverse primer and probe for detecting internal standard int gene are respectively included with the second primer combination of probe object；
For detecting the sequence of the forward primer of the internal standard int gene, reverse primer and probe respectively such as SEQ ID NO.10, SEQ
Shown in ID NO.11 and SEQ ID NO.12.
5. a kind of primer and probe composition as claimed in claim 4, which is characterized in that for detecting the internal standard int base
5 ' end mark fluorescent groups of the probe of cause are JOE, 3 ' end label quenching group TAMRA.
6. a kind of kit, which is characterized in that the kit is for detecting coagulase-negative staphylococci, Staphylococcus aureus
Bacterium and methicillin resistance gene；The coagulase-negative staphylococci detection target is sodA gene, the golden yellow grape
It is femA gene that coccus, which detects target, and the methicillin resistance gene is mecA gene；The kit includes nucleic acid extraction
Liquid, PCR reaction enzyme system, internal standard, negative controls, positive reference substance, the first primer probe mixed liquor and the second primed probe are mixed
Close liquid；The first primer probe mixed liquor includes the first primer probe compositions, the first primer probe compositions by with
In the forward primer of detection sodA gene and femA gene, reverse primer and probe composition；The second primed probe mixed liquor
Including the second primer combination of probe object, the second primer combination of probe object is sensed by the forward primer, anti-of mecA gene
It is formed to primer and probe；For detecting the sequence of the forward primer of the sodA gene, reverse primer and probe respectively such as SEQ
Shown in ID NO.1, SEQ ID NO.2 and SEQ ID NO.3；For detecting forward primer, the reverse primer of the femA gene
With the sequence of probe respectively as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6；For detecting the mecA base
The sequence of the forward primer of cause, reverse primer and probe is respectively such as SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 institute
Show.
7. a kind of kit as claimed in claim 6, which is characterized in that the first primer probe compositions and described second
Primer combination of probe object respectively includes the forward primer, reverse primer and probe for detecting internal standard int gene；For detecting
State the sequence of the forward primer of internal standard int gene, reverse primer and probe respectively such as SEQ ID NO.10, SEQ ID NO.11 and
Shown in SEQ ID NO.12.
8. a kind of kit as claimed in claim 7, which is characterized in that the probe for detecting the sodA gene is MGB
Probe, 5 ' end label reporter fluorescence groups are FAM；For detecting 5 ' end label reporter fluorescence bases of the probe of the femA gene
Group is ROX, 3 ' end label quenching fluorescence group BHQ2；For detecting 5 ' end label reporter fluorescences of the probe of the mecA gene
Group is FAM, 3 ' end label quenching fluorescence group BHQ1；5 ' the end labels for detecting the probe of the internal standard int gene are glimmering
Light group is JOE, 3 ' end label quenching group TAMRA.
9. a kind of kit as claimed in claim 8, which is characterized in that the nucleic acid extraction liquid is by 2% (M/V) Chelex-
100,1% (V/V) Tris-HCl, 1M, pH9.0 composition；The PCR reaction enzyme system is by 5U/ μ L Taq archaeal dna polymerase and 2U/ μ L
The uracil-N-glycosylase composition of 3: 1 ratios mixing by volume；The plasmid containing internal standard int genetic fragment is designated as in described；
The negative controls are the distilled water purified by Millipore pure water meter；The positive reference substance is to carry sodA gene
Carrier T plasmid, carry femA gene carrier T plasmid and carry mecA gene carrier T plasmid or contain above-mentioned plasmid
Colibacillus engineering, the bacterium solution bacteria concentration 10 as reference substance5CFU/mL；The first primer probe mixed liquor includes deoxidation
Ribonucleotide triphosphate dN (U) TP；The second primed probe mixed liquor includes deoxyribonucleoside triphosphate dN (U) TP.
10. a kind of kit as claimed in claim 9, the first primer probe mixed liquor is by 2 10 × PCR of μ L
Buffer、2μL 25mmol/L MgCl2, 1.6 μ L 2.5mmol/L dN (U) TP, 0.6 μ L, 10 μm of ol/L are respectively used to examine
Survey the forward primer and reverse primer of sodA gene and femA gene, 0.2 μ L, 10 μm of ol/L are respectively used to detection sodA gene
With the forward primer and reverse primer for being respectively used to detection internal standard int gene of the probe of femA gene, 0.6 μ L, 10 μm of ol/L
And the probe for being respectively used to detection internal standard int gene and 7 μ L sterilizing purified water composition of 0.2 μ L, 10 μm of ol/L；Described second draws
Physical prospecting needle mixed liquor is by 2 μ L 10 × PCR Buffer, 2 μ L 25mmol/L MgCl2、1.6μL 2.5mmol/L dN(U)TP、
The forward primer for detecting mecA gene and reverse primer of 0.6 10 μm of μ L ol/L, 0.2 μ L, 10 μm of ol/L for detecting
The probe of mecA gene, the forward primer for being used to detect internal standard int gene of 0.6 μ L, 10 μm of ol/L and reverse primer and 0.2 μ L
The probe and 7 μ L sterilizing purified water composition for detecting internal standard int gene of 10 μm of ol/L.
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| CN110669851A (en) * | 2019-10-30 | 2020-01-10 | 宁波基内生物技术有限公司 | Primer and/or probe composition for detecting cocci causing bloodstream infections and use thereof |
| CN112410343A (en) * | 2020-12-19 | 2021-02-26 | 郑州大学 | Kit based on CRISPR and application thereof |
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Application publication date: 20190806 |