CN109136395A - A kind of B race streptococcus kit for detecting nucleic acid - Google Patents
A kind of B race streptococcus kit for detecting nucleic acid Download PDFInfo
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- CN109136395A CN109136395A CN201810979694.6A CN201810979694A CN109136395A CN 109136395 A CN109136395 A CN 109136395A CN 201810979694 A CN201810979694 A CN 201810979694A CN 109136395 A CN109136395 A CN 109136395A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a kind of B race streptococcus kit for detecting nucleic acid, including PCR reaction solution, the PCR reaction solution includes PCR reaction solution 1 and PCR reaction solution 2, wherein PCR reaction solution 1 includes for the upstream primer and downstream primer of target polynucleotide amplification and for the probe of target polynucleotide detection, dual-function dna polymerase, uracil dna glycosylase, dUTP, PCR amplification reinforcing agent；The PCR reaction solution 2 includes manganese ion.The advantage of the invention is that easy to operate, detection sensitivity is high, and specificity is good, and repeatability is strong, and testing result is quick, and reagent facilitates storage and transport without -20 DEG C of preservations.B race streptococcus-DNA in the unknown samples such as pregnant women of perinatal period genital secretion and rectal secretions can be used for quickly detecting using kit of the present invention, provide reliable experimental basis for diagnosis B race streptococcal infection.
Description
Technical field
The present invention relates to Measurements for Biotechnique, more particularly, to a kind of B race streptococcus kit for detecting nucleic acid.
Background technique
B race streptococcus (group B streptococcus, GBS) be one kind parasitize human urogenital and under exhale
The conditioned pathogen in road is inhaled, healthy population bacterial bearing rate is up to 35%.Early in the 1970s, B race streptococcus just it is verified that
For one of the important pathogenic bacteria of perinatal period mother and baby infection.The B race streptococcus for being colonized in puerpera's genital tract can vertically pass in childbirth
It broadcasts to newborn, and the diseases lethality such as sepsis of the newborn, meningitis and pneumonia caused by the streptococcal infection of neonatal B race pole
Height, and can lead to nervous system sequelae.Center for Disease Control of the U.S. in 2010 has formulated " perinatal period GBS prevention guideline ", builds
Pregnant woman is discussed in progress GBS screening in pregnant 35-37 weeks.
Currently, domestic mainly have cultivation, immunology diagnosis method and diagnosis of molecular biology method to the diagnostic method of GBS.
Bacterial cultivation is the laboratory diagnosis conventional method of GBS, is the goldstandard for detecting GBS, it may be assumed that takes within pregnant 35-37 weeks vagina and straight
Intestines sample is positive in broth bouillon.Culture-based method specificity and sensibility are high, but clinical detection time length (at least needs
24-48h), process is cumbersome, and positive rate is influenced by factors, and is not suitable for detecting on a large scale.Serological method is due to window
Whether the presence of mouth phase, there are hysteresis qualitys for diagnosis, not can accurately reflect and currently infect, although specificity up to 95%, quick
Perceptual inadequate, verification and measurement ratio is lower.Molecular diagnostics method includes gene order sequencing and nucleic acid detection method, wherein gene order
Sequencing has the disadvantages of expensive, step complexity is cumbersome, and time-consuming, is not suitable for clinical detection use.And bacterial nucleic acid fluorescent PCR
Detection method has many advantages, such as rapidity, accuracy and high sensitivity, and early diagnosis can be made to bacterium infection, is suitble to clinical sieve
It looks into and diagnoses, while providing strong technical support to the quick analysis and control of epidemic situation.Studies have shown that real-time fluorescence PCR
The method for cultivation of bacteria of detection method and standard can reach 90% or more in terms of the sensibility and specificity that GBS is detected, and meet
Screening criteria has obtained the approval of United States Food and Drag Administration (FDA) and has been applied to clinic.Therefore, it uses at home
It is most quick, reliable method that real-time fluorescent PCR technology, which carries out GBS screening,.
But Standard PCR fluorogenic quantitative detection DNA cloning uses hot start Taq polymerase amplification system, RNA amplification uses Taq
Enzyme and reverse transcription Mlv enzymatic amplification system, the Buffer that the two is selected is also different, and two kinds of amplification systems can not achieve unification.?
In terms of detection time, DNA cloning needs about 1.5h, and RNA amplification needs about 2h, time-consuming also longer.And hot start Taq polymerase expands
Reagent condition of storage is necessary for -20 DEG C of preservations, dry ice or ice bag transport, and validity period is shorter, is using and inconvenience in transportational process
It is prompt.
Summary of the invention
It is an object of the invention to for above-mentioned detection of the existing technology, time-consuming, amplification system disunity and reagent
Inconvenient defect is stored, a kind of B race streptococcus kit for detecting nucleic acid is provided.
To achieve the above object, the present invention can take following technical proposals:
B race streptococcus kit for detecting nucleic acid of the present invention, including PCR reaction solution, the PCR reaction solution include PCR anti-
Answer liquid 1 and PCR reaction solution 2, wherein PCR reaction solution 1 include for target polynucleotide amplification upstream primer and downstream primer with
And the probe for target polynucleotide detection, dual-function dna polymerase, uracil dna glycosylase, dUTP, PCR amplification enhancing
Agent；The PCR reaction solution 2 includes manganese ion.
It is streptococcic that upstream primer, downstream primer and the detection probe for target polynucleotide amplification is derived from B race
The primer and probe of conservative region, sequence are respectively as follows:
Upstream primer (SEQ ID No.1): 5 '-AGCAATCACACATGCTGTTGGA-3 '；
Downstream primer (SEQ ID No.2): 5 '-TAATGCTGTTTGAAGTGCTGCT-3 '；
Probe (SEQ ID No.3): 5 '-CAGTTGAATCCAAATGTTACGGTACAAC-3 '.
The kit further includes internal standard, and the internal standard uses human beta-globin gene magnification, can monitor clinical sampling
Process again can monitor sample nucleic acid extraction process.
The kit further includes for the upstream and downstream primer of internal standard fragment amplification and for detecting interior target probe, institute
Target upstream and downstream primer and probe sequence in detection is stated to be respectively as follows:
Upstream primer (SEQ ID No.4): 5 '-GAAGGCTCATGGCAAGAAAG-3 '；
Downstream primer (SEQ ID No.5): 5 '-CTCACTCAGTGTGGCAAAGG-3 '；
Probe (SEQ ID No.6): 5 '-CTTGAGGTTGTCCAGGTGAGCCAG-3 '.
The kit further includes positive control and negative control.
The concentration of the upstream primer expanded for target polynucleotide and downstream primer is 0.2 μm of ol/L-1.0 μm of ol/
L；The concentration of the detection probe is 0.2 μm of ol/L-1.0 μm of ol/L.
The concentration of the upstream and downstream primer for internal standard fragment amplification is 0.1 μm of ol/L-0.5 μm of ol/L；Detect internal standard
The concentration of probe is 0.1 μm of ol/L-0.5 μm of ol/L.
The advantage of the invention is that easy to operate, detection sensitivity is high, and specificity is good, and repeatability is strong, and testing result is fast
Speed, reagent facilitate storage and transport without -20 DEG C of preservations.It can be to pregnant women of perinatal period genital tract point using kit of the present invention
B race streptococcus-DNA in the unknown samples such as secretion and rectal secretions is used for quickly detecting, and is mentioned for diagnosis B race streptococcal infection
For reliable experimental basis.
Detailed description of the invention
Fig. 1 is the amplification curve diagram of kit of the present invention detection detection limit reference material.
Fig. 2 is the amplification curve diagram that kit of the present invention detects yin-yang reference material.
Fig. 3 is kit specificity experiments amplification curve diagram of the present invention.
Fig. 4 a, 4b, 4c are kit specificity experiments amplification curve diagram of the present invention.
Fig. 5 is that the 20170919th batch reagent withinrun precision of kit of the present invention detects amplification curve.
Fig. 6 is that the 20171010th batch reagent withinrun precision of kit of the present invention detects amplification curve.
Fig. 7 is that the 20171128th batch reagent withinrun precision of kit of the present invention detects amplification curve.
Specific embodiment
Further more detailed description is done to the present invention combined with specific embodiments below.Those skilled in the art should
Understand, examples are merely exemplary, and it is not intended to limit the scope of the present invention in any way, without departing from of the invention
Can be with the details and forms of the technical scheme of the invention are modified or replaced under spirit and scope, but these modifications and replacement are equal
It falls within the scope of protection of the present invention.
Embodiment 1 prepares B race streptococcus kit for detecting nucleic acid
1, PCR reaction solution 1
Its component includes:
The probe for target polynucleotide detection of 0.2 μm of ol/L-1.0 μm of ol/L, 0.2 μm of ol/L-1.0 μm of ol/L's is used for target
The upstream primer and downstream primer of polynucleotides detection；
The probe for the detection of human beta-globin internal standard nucleotide of 0.1 μm of ol/L-0.5 μm of ol/L, 0.1 μm of ol/L-0.5 μ
The upstream primer and downstream primer for the detection of human beta-globin internal standard of mol/L；
0.01U/ μ l-0.1U/ μ l dual-function dna polymerase, 0.001U/ μ l-0.01U/ μ l uracil dna glycosylase and
0.1mmol/L-0.5mmol/LdUTP；Using UNG enzyme can degrade the DNA chain containing dU the characteristics of, be added in PCR system
UNG enzyme and dUTP can prevent the pollution of previous PCR product, prevent pattern detection false positive.
(methylol) methylglycine of pH7.0-pH9.0 1mol/L tri- solution, 6.25%-12.5%PCR expand reinforcing agent.
2, PCR reaction solution 2: acetic acid manganese salt solution.
3, positive control: synthesis B race streptococcus target sequence plasmid, concentration 1.00-5.00E+05copies/ml.
4, negative control: the physiological saline after sterilizing.
Embodiment 2 is unknown using kit detection genital secretion and rectum road secretion etc. prepared by embodiment 1
The method of B race streptococcus-DNA in sample
One, reagent prepares
According to sample to be tested, negative control and positive control quantity, (1 20 μ l/ person-portion+PCR of PCR reaction solution reaction in proportion
2 10 μ l/ person-portion of liquid) the PCR reaction solution 1, the PCR reaction solution 2 that take corresponding amount, PCR-MIX mixed liquor is mixed well into, instantaneously
It is spare after centrifugation.
Two, sample process
1, swab to be measured is put into 1ml physiological saline, is rinsed 5-10 times, it is spare to rinse rear liquid；
2,600 μ l is taken to rinse rear liquid sample, cooperation Zhengzhou Autobio Engineering Co., Ltd.'s paramagnetic particle method nucleic acid extraction examination
B race streptococcus DNA nucleic acid in sample to be tested is extracted in agent, spare；
3,600 μ l Yin/Yang quality-control products are taken simultaneously, cooperate Zhengzhou Autobio Engineering Co., Ltd.'s paramagnetic particle method nucleic acid extraction
Reagent extracts positive and negative quality-control product DNA nucleic acid, spare；
4, it takes PCR reaction tube several, 30 μ l of PCR-MIX mixed liquor is added, then be separately added into the sample to be tested of extraction, Yin/Yang
Each 20 μ l of quality-control product nucleic acid product, lid upper tube cap (after removing bubble removing), brief centrifugation 10 seconds.
Three, Fluorescence PCR
1, PCR reaction tube is put into amplification instrument sample cell, sample to be tested title is set by corresponding sequence,
2, fluorescence detection channel selects: selection FAM Air conduct measurement GBS target；Select CY5 Air conduct measurement internal standard；Reference fluorescent is set
None is set to,
3, Fluorescence PCR condition is shown in Table 1:
Table 1: Fluorescence PCR condition:
Four, interpretation of result
After reaction, instrument is automatically saved and (can also be adjusted manually as a result, can use the included software of instrument and automatically analyzed
Initial value, end value and the threshold line of section baseline are analyzed), then record sample Ct value result.Amplification curve and threshold value
(i.e. cycle threshold refers to that the fluorescence signal in PCR reaction tube reaches the threshold value when institute of setting for the focus of line, referred to as Ct value
The cycling numerical value of experience)；Instrument software is according to each sample Ct value size, it can be determined that testing result.For measuring value≤38 Ct
Sample, be reported as GBS-DNA the positive；For measuring the sample of Ct value > 38, while internal standard test positive (value≤40 Ct),
It is reported as GBS-DNA and is less than detection limit；For measuring the sample without Ct value, while internal standard test positive (value≤40 Ct), report
Accusing is GBS-DNA negative；If internal standard Ct value > 40 or without display, the testing result of the sample is invalid, should search and exclude original
Cause, and this sample is carried out to repeat test.
The kit performance measurement of the present invention of embodiment 3
1, sensitivity experiment
Cooperate Zhengzhou Autobio Engineering Co., Ltd.'s paramagnetic particle method nucleic acid extracting reagent, the reagent prepared using embodiment 1
Box detects enterprise work reference material, detection limit reference material recall rate > 95%.The detection detection limit enterprise work reference of this kit
Product each 10, Fig. 1 show the amplification curve of kit detection detection limit reference material.
Conclusion: showing according to testing result, and kit of the present invention cooperates Zhengzhou Autobio Engineering Co., Ltd.'s magnetic
Pearl method nucleic acid extracting reagent, detection enterprise detection limit operating reference product 10, amplification curve is standard " S " type curve, wherein 10
Example full inspection goes out, and recall rate is greater than 95%, this kit high sensitivity.
2, accuracy is tested
Cooperate Zhengzhou Autobio Engineering Co., Ltd.'s paramagnetic particle method nucleic acid extracting reagent, the reagent prepared using embodiment 1
Box, detects enterprise work reference material, and yin and yang attribute reference material coincidence rate is 100%.This kit detects the reference of yin and yang attribute enterprise work
Product each 10, testing result is as shown in table 2 below:
Table 2: accuracy testing result
Fig. 2 show the amplification curve of kit detection positive reference product；Fig. 3 show the expansion of kit detection negative reference product
Increase curve.
Conclusion: from Fig. 2 and Fig. 3 it is found that Zhengzhou Autobio Engineering Co., Ltd.'s magnetic bead with kit of the present invention
Method nucleic acid extracting reagent detects enterprise's positive and negative operating reference product each 10, and amplification curve is standard " S " type curve, wherein yin
Property coincidence rate 100%, positive coincidence rate 100%, it was demonstrated that this kit accuracy is high.
3, specificity experiments
Zhengzhou Autobio Engineering Co., Ltd.'s paramagnetic particle method nucleic acid extracting reagent is cooperated to carry out specificity experiments, it is normal with clinic
See pathogen and microorganism: chlamydia trachomatis (CT), gonococcus (NG), ureaplasma urealyticum (UU), hepatitis type B virus (HBV),
Hepatitis C Virus (HCV), human cytomegalovirus (HCMV), Bacterium enteritidis, micrococcus scarlatinae, Klebsiella Pneumoniae,
Streptococcus pneumonia, solution gallic acid streptococcus, pseudomonas aeruginosa, staphylococcus aureus, enterococcus faecalis, epidermis grape ball
Bacterium, Escherichia coli, Candida glabrata, Candida tropicalis, Candida albicans, haemophilus influenzae, citrobacter freundii are without friendship
Fork reaction, testing result are feminine gender.
Fig. 4 a, b4,4c are that (wherein, Fig. 4 a is chlamydia trachomatis to kit specificity experiments amplification curve diagram of the present invention
(CT), gonococcus (NG), ureaplasma urealyticum (UU) cross reaction detect amplification curve diagram；Fig. 4 b be hepatitis type B virus (HBV),
Hepatitis C Virus (HCV), human cytomegalovirus (HCMV) cross reaction detect amplification curve diagram；Fig. 4 c is Salmonella
Bacterium, micrococcus scarlatinae, Klebsiella Pneumoniae, streptococcus pneumonia, solution gallic acid streptococcus, pseudomonas aeruginosa, golden yellow
Staphylococcus, enterococcus faecalis, staphylococcus epidermis, Escherichia coli, Candida glabrata, Candida tropicalis, Candida albicans, influenza
Haemophilus, citrobacter freundii cross reaction detect amplification curve diagram).
Conclusion: testing result shows that common clinical substance and microorganism: chlamydia trachomatis (CT), solves gonococcus (NG)
Urea urea substance (UU), hepatitis type B virus (HBV), Hepatitis C Virus (HCV), human cytomegalovirus (HCMV), enteritis sramana
It is Salmonella, micrococcus scarlatinae, Klebsiella Pneumoniae, streptococcus pneumonia, solution gallic acid streptococcus, pseudomonas aeruginosa, golden yellow
Color staphylococcus, enterococcus faecalis, staphylococcus epidermis, Escherichia coli, Candida glabrata, Candida tropicalis, Candida albicans, stream
Haemophilus influenza, citrobacter freundii detect no cross reaction to GBS, it was demonstrated that this kit specificity is higher.
4, Precision Experiment
Respectively with three batch kits (lot number 1:20170919, lot number 2:20171010, lot number 3:20171128) detection it is high,
In, low three concentration samples, each sample respectively detects 8 repetitions.Specific testing result is as shown in table 3-6 and Fig. 5-7, Fig. 5-
7 are respectively shown in and detect amplification curve for 1 withinrun precision of lot number, and 2 withinrun precision of lot number detects amplification curve, in lot number 3 batches
Precision detects amplification curve.
Table 3: 1 reagent withinrun precision testing result of lot number statistics
Table 4: 2 reagent withinrun precision testing result of lot number statistics
Table 5: 3 reagent withinrun precision testing result of lot number statistics
Table 6: betweenrun precision testing result statistics
Experiment shows: in kit of the present invention batch and batch between reproducible, the coefficient of variation < 10% of testing result Ct value.
5,2-8 DEG C of stability experiment
Cooperate Zhengzhou Autobio Engineering Co., Ltd.'s paramagnetic particle method nucleic acid extracting reagent, the reagent prepared using embodiment 1
PCR reaction solution 1 and PCR reaction solution 2 are placed 37 DEG C of acceleration by box simultaneously, respectively in 0 day, 3 days, 7 days, 4 time point inspections in 14 days
High, medium and low three concentration samples are surveyed, each sample each time point respectively detects 2 repetitions, N1-N10 negative reference product, P1-
P10 positive reference product, yin and yang attribute compare each 1 repetition.Testing result is as shown in table 7:
Table 7
It according to the above experimental result, is calculated according to Arrhenius formula, 37 DEG C of kit acceleration prepared by the embodiment of the present invention 1
It 14 days, can be at least 18-24 months in 2-8 DEG C of storage.
Sequence table
<110>Zhengzhou Autobio Engineering Co., Ltd.
<120>B group of streptococcus kit for detecting nucleic acid
<141> 2018-08-16
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213>artificial synthesized
<400> 1
AGCAATCACACATGCTGTTGGA 22
<210> 2
<211> 22
<212> DNA
<213>artificial synthesized
<400> 2
TAATGCTGTTTGAAGTGCTGCT 22
<210> 3
<211> 28
<212> DNA
<213>artificial synthesized
<400> 3
CAGTTGAATCCAAATGTTACGGTACAAC 28
<210> 4
<211> 20
<212> DNA
<213>artificial synthesized
<400> 4
GAAGGCTCATGGCAAGAAAG 20
<210> 5
<211> 20
<212> DNA
<213>artificial synthesized
<400> 5
CTCACTCAGTGTGGCAAAGG 20
<210> 6
<211> 24
<212> DNA
<213>artificial synthesized
<400> 6
CTTGAGGTTGTCCAGGTGAGCCAG 24
Claims (7)
1. a kind of B race streptococcus kit for detecting nucleic acid, including PCR reaction solution, it is characterised in that: the PCR reaction solution includes
PCR reaction solution 1 and PCR reaction solution 2, wherein PCR reaction solution 1 includes to draw for the upstream primer of target polynucleotide amplification and downstream
Object and the probe detected for target polynucleotide, dual-function dna polymerase, uracil dna glycosylase, dUTP, PCR amplification
Reinforcing agent；The PCR reaction solution 2 includes manganese ion.
2. B race streptococcus kit for detecting nucleic acid according to claim 1, it is characterised in that: described to be used for target multicore glycosides
The sequence of upstream primer, downstream primer and detection probe that acid expands is respectively as follows:
Upstream primer: 5 '-AGCAATCACACATGCTGTTGGA-3 '；
Downstream primer: 5 '-TAATGCTGTTTGAAGTGCTGCT-3 '；
Probe: 5 '-CAGTTGAATCCAAATGTTACGGTACAAC-3 '.
3. B race streptococcus kit for detecting nucleic acid according to claim 1, it is characterised in that: the kit further includes
Internal standard, the internal standard use human beta-globin gene magnification.
4. B race streptococcus kit for detecting nucleic acid according to claim 3, it is characterised in that: the kit further includes
For the upstream and downstream primer of internal standard fragment amplification and for detecting interior target probe, in the detection target upstream and downstream primer and
Probe sequence is respectively as follows:
Upstream primer: 5 '-GAAGGCTCATGGCAAGAAAG-3 '；
Downstream primer: 5 '-CTCACTCAGTGTGGCAAAGG-3 '；
Probe: 5 '-CTTGAGGTTGTCCAGGTGAGCCAG-3 '.
5. B race streptococcus kit for detecting nucleic acid according to claim 1, it is characterised in that: the kit further includes
Positive control and negative control.
6. B race streptococcus kit for detecting nucleic acid according to claim 2, it is characterised in that: described to be used for target multicore glycosides
The upstream primer of acid amplification and the concentration of downstream primer are 0.2 μm of ol/L-1.0 μm of ol/L；The concentration of the detection probe is 0.2
μmol/L-1.0μmol/L。
7. B race streptococcus kit for detecting nucleic acid according to claim 4, it is characterised in that: described to be used for internal standard segment
The concentration of the upstream and downstream primer of amplification is 0.1 μm of ol/L-0.5 μm of ol/L；The concentration for detecting internal standard probe is 0.1 μm of ol/L-
0.5μmol/L。
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Cited By (1)
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| CN112695109A (en) * | 2020-12-25 | 2021-04-23 | 中生北控生物科技股份有限公司 | Group B streptococcus nucleic acid detection kit and detection method thereof |
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