CN106967839A - Primer, probe, kit and the method for B races streptococcus fluorescence quantitative PCR detection - Google Patents
Primer, probe, kit and the method for B races streptococcus fluorescence quantitative PCR detection Download PDFInfo
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Abstract
The invention discloses a kind of primer, probe, kit and the method for B races streptococcus fluorescence quantitative PCR detection, belong to Bacteria Detection field.The primer and probe includes amplification B streptococcic primer pair GBS F and the GBS R of the race and detection B streptococcic probe GBS P of race, and expands people β actin primer pair β actin F and β actin R and detection people β actin probe β actin P；The kit contains above-mentioned primer pair and probe.Primer pair and probe that the present invention is used, can be specific while composite amplification B races streptococcus gene and mankind's β actin genes, as a result more reliable, accurate due to adding mark system in the autosome that sample collection is monitored.
Description
Technical field
The present invention relates to the apparatus and method field of Bacteria Detection, and in particular to a kind of B races streptococcus quantitative fluorescent PCR inspection
Primer, probe, kit and the method for survey.
Background technology
B races streptococcus (group B streptococcus, GBS) be one kind parasitize human urogenital and under disappear
Change the conditioned pathogen in road, healthy population bacterial bearing rate is up to 35%.Early in 1970s GBS just it is verified that to enclose production
One of important pathogenic bacteria of phase mother and baby infection.The B races streptococcus of puerpera's genital tract is colonized in, vertical transmission can give new in childbirth
Raw youngster, and the disease fatal rate such as neonate GBS infection caused sepsis of the newborn, meningitis and pneumonia is high, and can cause
Nervous system sequelae.Center for Disease Control of the U.S. formulates within 2010《Perinatal period GBS prevention guideline》, it is proposed that pregnant woman is in gestation
Carry out GBS examinations within 35-37 weeks.
The domestic examination to GBS at present has three kinds of methods：
(1) bacterial cultivation：The streptococcic presence of B races is detected using normal bacterial cultural method, is about needed after sampling
Want obtain testing result within 18-48 hours, it is relatively time-consuming, it is impossible to effectively and quickly to detect the streptococcic presence of B races, and sun
Property rate is influenced by factors, and drawback is obvious.
(2) amynologic diagnostic method：Serological method is unable to high flux processing sample.Due to the presence of window phase, serum
Whether there is hysteresis quality in diagnosis, it is impossible to accurately reflect and currently infect.This method is time-consuming, laborious, it is impossible to meet quick, early stage
The requirement of diagnosis.
(3) fluorescent PCR method detection method：The detection of fluorescent PCR method is not only time-consuming short, also superior to training in sensitivity and specificity
The method of supporting, therefore develop and a kind of there is important clinical meaning using simple, the accurate B races streptococcus PCR detection reagents of result.
But this method has a common issue with cultivation, i.e. testing result is influenceed larger by sampling error, vagina and rectum sample
This sampling operation difficulty is big, and sampling result has influence on follow-up testing result.Sampling failure will cause the diagnosis of false negative.
The content of the invention
It is an object of the invention to provide a kind of primer of B races streptococcus fluorescence quantitative PCR detection, probe, kit and
Method, causes the problem of detecting false negative result to solve existing sampling error or sampling failure.
To achieve the above object, the present invention adds people's conservative gene while based on fluorescence quantitative PCR detection GBS
As internal reference target gene, β-actin internal standards are devised, the internal standard can be from sample collection, to nucleic acid extraction and GBS bases
Gene-amplification whole process supervision experimental implementation links so that testing result is relatively reliable, stably.Specifically, B races streptococcus is glimmering
Fluorescent Quantitative PCR detection primer and correspondence probe, including：
Expand the B streptococcic primer pair GBS-F and GBS-R of race and detection the B streptococcic probe GBS-P of race, the GBS-
F, GBS-R and GBS-P nucleotide sequence are respectively successively such as SEQ ID NO:1~SEQ ID NO:Shown in 3；
And amplification people β-actin primer pair β-actin-F and β-actin-R and detection people β-actin probe β-
Actin-P, the β-actin-F, β-actin-R and β-actin-P nucleotide sequence are respectively successively such as SEQ ID NO:4~
SEQ ID NO:Shown in 6.Above-mentioned SEQ ID NO:1~SEQ ID NO:6 nucleotide sequences are specific referring to table 1.
Table 1 detects B race streptococcus and people β-actin primer and probe
| Title | Nucleotide sequence | SEQ ID NO： |
| GBS-F | 5’-GAGGCTATTACTAGCGTTGAA-3’ | 1 |
| GBS-R | 5’-GGCTTCTACACGACTACCAAT-3’ | 2 |
| GBS-P | 5’-CTTCATTGCGTGCCAACCCTGAGACA-3’ | 3 |
| β-actin-F | 5’-TGGCAAGAAAGTGCTCGGTG-3’ | 4 |
| β-actin-R | 5’-CAGCTTGTCACAGTGCAGCTCA-3’ | 5 |
| β-actin-P | 5’-ATGGCCTGGCTCACCTGGACAAC-3’ | 6 |
The end of nucleotide sequence 5 ' of the GBS-P and β-actin-P marks different fluorescence report groups respectively, described glimmering
Light report group is in FAM, HEX, CY3, CY5, JOE, TET or LC RED640；The nucleosides of the GBS-P and β-actin-P
Group is quenched in mark fluorescent respectively at 3 ' ends of acid sequence, and the fluorescent quenching group is selected from BHQ1, BHQ2, BHQ3 or Dabcy1.
It is preferred that, the end of GBS-P nucleotide sequences 5 ' the flag F AM fluorescence reports group, 3 ' end mark BHQ1 fluorescence are quenched
Go out group；The end of probe β-actin-P nucleotide sequences 5 ' the mark HEX fluorescence reports group, 3 ' mark BHQ1 fluorescent quenchings
Group.
Present invention also offers the kit for above-mentioned primer and probe, including pcr amplification reaction liquid, enzyme mixation,
In positive control and negative control, the pcr amplification reaction liquid containing the streptococcic primer pair GBS-F of above-mentioned amplification B races and
The GBS-R and detection B streptococcic probe GBS-P of race, and expand people β-actin primer pair β-actin-F and β-actin-R
And detection people β-actin probe β-actin-P.Also contain other routines expanded into performing PCR in the pcr amplification reaction liquid
Reagent, such as PCR reaction buffers, MgCl2, dNTPs etc.；Mentioned reagent is conventional constituents, voluntarily can configure or buy, can also
All it is fitted into kit.
The enzyme mixation includes hot start Taq polymerase and uracil-N-glycosylase.
The positive control contains B races streptococcus conserved sequence, can for the plasmid containing B races streptococcus conserved sequence or
For the B races streptococcus culture fluid of inactivation.The negative control is without the streptococcic purified water of B races or physiological saline.
Mentioned reagent box is used present invention also offers one kind, the detection method of quantitative fluorescent PCR, including following step is carried out
Suddenly：
A, extraction sample DNA, prepare DNA profiling；
B, sample-adding：Sample DNA, positive control or negative control are corresponded to add respectively pcr amplification reaction liquid and enzyme are housed
In the PCR pipe of mixed liquor, corresponding sample PCR reaction systems, positive PCR reaction systems or feminine gender PCR reaction systems are obtained；
C, PCR are expanded：By sample PCR reaction systems, positive PCR reaction systems or feminine gender PCR reaction systems obtained by step B
Reaction tube is placed on quantitative real time PCR Instrument respectively, sets loop parameter, enters performing PCR amplification；
After D, PCR reaction terminate, infection B races streptococcus is judged whether according to fluorescence curve.
Due to adding people conservative gene β-actin in pcr amplification reaction liquid, (i.e. flesh moves egg in above-mentioned technical proposal
In vain) as internal reference target gene, with B races streptococcus gene while composite amplification.If there is no depositing for any DNA in testing result
It then can't detect any fluorescence, it may be possible to which sampling failure or other operation links go wrong；If only someone β-actin bases
Gene-amplification, then it is negative for B races streptococcus；If there are two kinds of fluorescence to occur simultaneously, B races streptococcus is only positive.It therefore, it can exclude
The situation of B races streptococcus false negative.
The kit and method of the present invention has the following advantages that：(1) present invention uses fluorescent quantitative PCR detection method, with
Traditional bacterial cultivation is compared, and detection time has been greatly shortened；Compared with serological method, eliminate previous infection IgG
The false positive results that positive patient is brought；(2) present invention is used primer pair and probe, can specific composite amplification B races simultaneously
Mark system in streptococcus gene and mankind's β-actin genes, the autosome due to adding sample collection monitoring, as a result more may be used
Lean on, accurately.
Brief description of the drawings
Fig. 1 is the sample amplification curve of the embodiment of the present invention 2, and wherein abscissa represents period, and ordinate represents fluorescence
Intensity.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
The B races streptococcus fluorescent quantificationally PCR detecting kit of embodiment 1.
The B races streptococcus fluorescence PCR detection reagent kit of the present embodiment, including pcr amplification reaction liquid, enzyme mixation, the positive
Control and negative control.
Wherein described pcr amplification reaction liquid mainly includes：
1. PCR cushioning liquid (PCR Buffer), the pH provided needed for reaction is made up of tris-HCl, KCl and purified water
Value and salt ion environment；
②MgCl2There is provided the catalyst of reaction enzymes；
3. dNTPs forms required raw material there is provided DNA in course of reaction；
4. B race streptococcus and people β-actin primer pair and corresponding probe are detected.
Primer pair GBS-F, GBS-R and probe GBS-P as described in Table 1 is derived from drawing for the streptococcic conservative region of B races
Thing and probe；Primer pair β-actin-F, β-actin-R and β-actin-P are derived from the primer of people β-actin conservative region
And probe.In GBS-P 5 ' end flag F AM fluorescence reports groups, 3 ' end mark BHQ1 fluorescent quenchings groups, in β-actin-P
5 ' end mark HEX fluorescence reports groups, 3 ' mark BHQ1 fluorescent quenchings groups.Above-mentioned primer pair and probe can be specific simultaneously multiple
Close amplification B race's streptococcus genes and people's β-actin genes.
Wherein described enzyme mixation is Taq enzyme and UNG enzymes, and the Taq enzyme is hot start Taq polymerase, and the UNG enzymes are phonetic to urinate
Pyridine-N- glycosylases.
The positive control is the plasmid containing B races streptococcus conserved sequence or the B races streptococcus culture fluid of inactivation, and its is dense
Spend for 1000~10000CFU/mL, the negative control is does not contain the streptococcic purified water of B races or physiological saline.
The streptococcic detection of the B races of embodiment 2.
Step A, extraction sample DNA, prepare DNA profiling.
12 cultures from hospital are detected as feminine gender, the anus swab and vaginal swab sample of 10 culture test positive
This, nucleic acid is extracted according to kit specification, prepares DNA profiling, and the present embodiment is used by all biotechnologies (Beijing) of helping
FinePure viral DNAs/RNA pillar extracts kits that Co., Ltd provides, are operated according to the specification of kit, also
Can be using other DNA extraction methods in the prior art.
Step B, sample-adding：By the positive control and negative control in 22 sample DNAs and kit that are prepared in step A
Correspond to and added in the PCR pipe equipped with pcr amplification reaction liquid respectively, obtain corresponding sample PCR reaction systems, positive PCR reactants
System and feminine gender PCR reaction systems.
The composition of PCR reaction systems is referring to table 2 in the present embodiment.
The composition of the PCR reaction systems of table 2
Step C, PCR is expanded：The PCR reaction tubes of the PCR reaction systems of gained in step B are placed in quantitative real time PCR Instrument
On, loop parameter is set, enters performing PCR amplification, amplified reaction program is as described in Table 3.
The amplified reaction program of table 3
After the reaction of step D, PCR terminates, infection B races streptococcus is judged whether according to fluorescence curve, referring to Fig. 1.
Result judgement：It is negative for B races streptococcus if only someone β-actin gene magnifications；If having two kinds of fluorescence while going out
Now or only FAM fluorescence is expanded, then it is that B races streptococcus is positive；If two kinds of fluorescence are not all expanded, testing result without
Effect.Final testing result is as shown in table 4.
The comparison of the testing result of the sample 1~22 of table 4 and culture testing result
Interpretation of result：Comparison by bacterial cultivation and fluorescence quantifying PCR method is visible, fluorescence quantifying PCR method sun
Property rate is higher.
, can be in the unknown samples such as genital secretion and rectal secretions using the kit and method of the present invention
B races streptococcus-DNA is used for quickly detecting, and reliable experimental basis is provided for the streptococcal infection of diagnosis B races.
Although above with general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
SEQUENCE LISTING
<110>Help all biotechnologies（Beijing）Co., Ltd
<120>Primer, probe, kit and the method for B races streptococcus fluorescence quantitative PCR detection
<130> 2010
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> Artifical sequence
<400> 1
gaggctatta ctagcgttga a 21
<210> 2
<211> 21
<212> DNA
<213> Artifical sequence
<400> 2
ggcttctaca cgactaccaa t 21
<210> 3
<211> 26
<212> DNA
<213> Artifical sequence
<400> 3
cttcattgcg tgccaaccct gagaca 26
<210> 4
<211> 20
<212> DNA
<213> Artifical sequence
<400> 4
tggcaagaaa gtgctcggtg 20
<210> 5
<211> 22
<212> DNA
<213> Artifical sequence
<400> 5
cagcttgtca cagtgcagct ca 22
<210> 6
<211> 23
<212> DNA
<213> Artifical sequence
<400> 6
atggcctggc tcacctggac aac 23
Claims (9)
1. a kind of primer and probe of B races streptococcus fluorescence quantitative PCR detection, it is characterised in that the primer and correspondence probe
Including：
Expand the B streptococcic primer pair GBS-F and GBS-R of the race and detection B streptococcic probe GBS-P of race, the GBS-F,
GBS-R and GBS-P nucleotide sequence is respectively successively such as SEQ ID NO:1~SEQ ID NO:Shown in 3；
And amplification people β-actin primer pair β-actin-F and β-actin-R and detection people β-actin probe β-
Actin-P, the β-actin-F, β-actin-R and β-actin-P nucleotide sequence are respectively successively such as SEQ ID NO:4~
SEQ ID NO:Shown in 6.
2. the primer and probe of B races streptococcus fluorescence quantitative PCR detection according to claim 1, it is characterised in that described
GBS-P the and β-actin-P end of nucleotide sequence 5 ' marks different fluorescence report groups, the fluorescence report group choosing respectively
From in FAM, HEX, CY3, CY5, JOE, TET or LC RED640；3 ' ends of the nucleotide sequence of the GBS-P and β-actin-P
Group is quenched in mark fluorescent respectively, and the fluorescent quenching group is selected from BHQ1, BHQ2, BHQ3 or Dabcy1.
3. the primer and probe of B races streptococcus fluorescence quantitative PCR detection according to claim 2, it is characterised in that described
The end of GBS-P nucleotide sequences 5 ' flag F AM fluorescence reports group, 3 ' end mark BHQ1 fluorescent quenchings groups；The probe β-
The end of actin-P nucleotide sequences 5 ' mark HEX fluorescence reports group, 3 ' mark BHQ1 fluorescent quenchings groups.
4. a kind of kit of B races streptococcus fluorescence quantitative PCR detection, including pcr amplification reaction liquid, enzyme mixation, the positive are right
According to and negative control, it is characterised in that contain primer as described in any one of claims 1 to 3 in the pcr amplification reaction liquid
And correspondence probe.
5. the kit of B races streptococcus fluorescence quantitative PCR detection according to claim 4, it is characterised in that detection B races
The proportioning of streptococcic primer and probe is GBS-F：GBS-R：GBS-P=2:2:1；Detect people β-actin primer and probe
Proportioning be β-actin-F：β-actin-R:β-actin-P=2:2:1.
6. the kit of B races streptococcus fluorescence quantitative PCR detection according to claim 4, it is characterised in that the enzyme is mixed
Closing liquid includes hot start Taq polymerase and uracil-N-glycosylase.
7. the kit of B races streptococcus fluorescence quantitative PCR detection according to claim 4, it is characterised in that the positive
The B races streptococcus culture fluid for the plasmid containing B races streptococcus conserved sequence or inactivation is compareed, the negative control is without B
The streptococcic purified water of race or physiological saline.
8. a kind of method of B races streptococcus fluorescence quantitative PCR detection, using the kit described in claim 4, its feature exists
In comprising the following steps：
A, extraction sample DNA, prepare DNA profiling；
B, sample-adding：Sample DNA, positive control or negative control are corresponded to respectively to add and mixed equipped with pcr amplification reaction liquid and enzyme
In the PCR pipe of liquid, corresponding sample PCR reaction systems, positive PCR reaction systems or feminine gender PCR reaction systems are obtained；
C, PCR are expanded：By sample PCR reaction systems, positive PCR reaction systems or feminine gender PCR reaction systems difference obtained by step B
It is placed on quantitative real time PCR Instrument, loop parameter is set, enters performing PCR amplification；
After D, pcr amplification reaction terminate, infection B races streptococcus is judged whether according to fluorescence curve.
9. the method for B races streptococcus fluorescence quantitative PCR detection according to claim 8, it is characterised in that PCR in step C
The program of amplification is：
Step 1: pre-degeneration 95 DEG C 5 minutes；
Step 2: deform 95 DEG C 15 seconds,
Step 3: annealing, extension and fluorescent collecting 60 DEG C 15 seconds,
Repeat step two and step 3, set 45 circulations altogether.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557443A (en) * | 2017-10-27 | 2018-01-09 | 广州赛哲生物科技股份有限公司 | Group B streptococcus fluorescence quantitative PCR detection kit |
| CN109136395A (en) * | 2018-08-27 | 2019-01-04 | 郑州安图生物工程股份有限公司 | A kind of B race streptococcus kit for detecting nucleic acid |
| CN111020042A (en) * | 2020-01-02 | 2020-04-17 | 圣湘生物科技股份有限公司 | Compositions and methods for detecting group A streptococci |
| CN112226522A (en) * | 2019-07-15 | 2021-01-15 | 中生方政生物技术股份有限公司 | Primer, probe, kit and detection method for detecting group B streptococcus |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557443A (en) * | 2017-10-27 | 2018-01-09 | 广州赛哲生物科技股份有限公司 | Group B streptococcus fluorescence quantitative PCR detection kit |
| CN109136395A (en) * | 2018-08-27 | 2019-01-04 | 郑州安图生物工程股份有限公司 | A kind of B race streptococcus kit for detecting nucleic acid |
| CN112226522A (en) * | 2019-07-15 | 2021-01-15 | 中生方政生物技术股份有限公司 | Primer, probe, kit and detection method for detecting group B streptococcus |
| CN111020042A (en) * | 2020-01-02 | 2020-04-17 | 圣湘生物科技股份有限公司 | Compositions and methods for detecting group A streptococci |
| CN111020042B (en) * | 2020-01-02 | 2023-06-16 | 圣湘生物科技股份有限公司 | Compositions and methods for detecting group A streptococci |
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Application publication date: 20170721 |