CN106834463A - Fluorescence PCR primer, kit and detection method for detecting Si Shi arch bacterium - Google Patents
Fluorescence PCR primer, kit and detection method for detecting Si Shi arch bacterium Download PDFInfo
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- CN106834463A CN106834463A CN201710060204.8A CN201710060204A CN106834463A CN 106834463 A CN106834463 A CN 106834463A CN 201710060204 A CN201710060204 A CN 201710060204A CN 106834463 A CN106834463 A CN 106834463A
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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 fluorescence PCR primer for detecting Si Shi arch bacterium, kit and detection method.The nucleotides sequence of the primer and probe is classified as：Sense primer：5’‑ATGCTCGTCATCGTAGGGTT‑3’（SEQ ID NO.1）；Anti-sense primer：5’‑AGCGGATTTGCCTACTTAACAAC‑3’ （SEQ ID NO.2）；Probe：HEX ‑ATCGAGGTCACGGATGGAAGTGT‑ BHQ1（SEQ ID NO.3）.The real-time fluorescence PCR reaction method that the present invention is provided has the characteristics that with reference to above-mentioned primer and probe groups：High specificity, sensitivity is high, detection is fast and reliable, it is easy to operate the characteristics of.
Description
Technical field
The invention belongs to field of food safety, and in particular to Si Shi arch bacterium detection primer and probe groups, kit and
Detection method.
Background technology
Arch bacterium (Arcobacter) is a kind of new food-borne pathogens, the international food microbial standard committee
(International Commission on Microbiological Specifications for Foods, ICMSF)
Advised within 2002 is have serious harm quasi-microorganism to human health.Bu Shi arch bacterium (A.butzleri) in arch bacterium,
Thermophilic low temperature arch bacterium (A.cryaerophilus) and Si Shi arch bacterium (A.skirrowii) are considered as related to human diseases.
Arch bacterium is near to Campylobacter spp affiliation, form is similar, in bending or spirality it is shaft-like, do not produce brood cell, but both
Growth characteristics have different, and arch bacterium is stronger compared with Campylobacter spp growth temperature range wider, oxygen tolerance, thus compared with Campylobacter spp
It is more easy to survival.Growth temperature, the difference of oxygen tolerance and other biochemical characteristics be usually used in differentiating arch Pseudomonas and Campylobacter spp this 2
Individual category.Since Ellis etc. establishes the separation method of arch bacterium for 1977 first, different researchers establish various different
Increase the conventional method of bacterium and flat board clastotype, wherein being considered as most in the method that Johnson in 1999 and Murano set up
Good method.But, because the physio-biochemical characteristics phenotypic difference of arch bacterium is larger, cause it to be difficult to rely on traditional physiology
Biochemical characteristic carries out Classification Identification between arch strain.The artificial addition for having scholar is tested and shown, with the addition of certain of Si Shi arch bacterium
A little samples cannot isolate this bacterium using traditional detection method.This is quicker to antibiotic than other arch bacterium with Si Shi arch bacterium
Sense, its growth and breeding speed are also relatively slow relevant, thus are more difficult to be grown on the culture medium containing antibiotic than other arch bacterium.Arch
The tradition of bacterium detection increases bacterium flat board separation method as other microorganism traditional detection methods, and the detection for arch bacterium is same
There is many deficiencies such as step is complicated, detection cycle is long, be highly desirable to set up a kind of fast and accurately detection Si Shi arch
The method of bacterium.
The content of the invention
It is an object of the invention to provide a kind of fluorescence PCR primer for detecting Si Shi arch bacterium, kit and detection
Method.
The technical solution used in the present invention is：
Fluorescence PCR primer and probe for detecting Si Shi arch bacterium, it is the 23S rRNA bases according to Si Shi arch bacterium
Because designed by specific sequence.
The nucleotide sequence of the primer and probe is as follows：
Sense primer：5’-ATGCTCGTCATCGTAGGGTT-3’(SEQ ID NO.1)；
Anti-sense primer：5’-AGCGGATTTGCCTACTTAACAAC-3’(SEQ ID NO.2)；
Probe：HEX-ATCGAGGTCACGGATGGAAGTGT-BHQ1(SEQ ID NO.3).
A kind of kit for detecting Si Shi arch bacterium, it includes above-described fluorescence PCR primer and probe.
The fluorescence PCR detecting method of Si Shi arch bacterium, comprises the following steps：
(1) sample DNA is extracted；
(2) with sample DNA as template, fluorescent PCR amplification is carried out using above-mentioned fluorescence PCR primer, probe；
(3) whether Si Shi arch bacterium are contained in judgement sample according to amplification curve.
Contain following components in fluorescent PCR amplification system：
Fluorescent PCR amplification program be：95 DEG C of predegeneration 5min；95 DEG C of denaturation 20s, 60 DEG C of annealing 1min, collect fluorescence,
40 circulations.
The beneficial effects of the invention are as follows：
The real-time fluorescence PCR reaction method that the present invention is provided has the characteristics that with reference to above-mentioned primer and probe groups：Specifically
Strong, sensitivity is high, detection is fast and reliable for property, it is easy to operate the characteristics of.
Brief description of the drawings
Fig. 1 is to carry out the result schematic diagram of real-time fluorescence PCR in embodiment 1；
Fig. 2, Fig. 3 be embodiment 1 in carry out 2 plants of result schematic diagrams of the real-time fluorescent PCR amplification curve of Si Shi arch bacterium；
Fig. 4, Fig. 5 are to carry out the result schematic diagram of sensitivity experiment in embodiment 3.
Specific embodiment
The foundation of the fluorescence PCR detecting method of the detection Si Shi arch bacterium of embodiment 1
1st, design of primers
23S rRNA gene specific sequences according to Si Shi arch bacterium devise a pair of specific primers, the following institute of sequence
Show：
Sense primer：5’-ATGCTCGTCATCGTAGGGTT-3’(SEQ ID NO.1)；
Anti-sense primer：5’-AGCGGATTTGCCTACTTAACAAC-3’(SEQ ID NO.2)；
Probe：HEX-ATCGAGGTCACGGATGGAAGTGT-BHQ1(SEQ ID NO.3).
2nd, fluorescent PCR detection
Configuration cumulative volume is the reaction system of the real-time fluorescence PCR of 25 μ L, specially：
| Component | Working solution concentration | Sample-adding amount (μ L) |
| Sense primer | 10μmol/L | 1 |
| Anti-sense primer | 10μmol/L | 1 |
| Probe | 10μmol/L | 1 |
| ROX Reference Dye II | 50× | 0.5 |
| dNTPs | 10mmol/L | 1 |
| PCR buffer solutions | 10× | 2.5 |
| Taq polymerase | 5U/μL | 0.5 |
| Template DNA to be checked | / | 2 |
| Deionized water | / | 16.5 |
Wherein, sense primer, anti-sense primer, probe correspond to the real-time fluorescent PCR testing primer of the Si Shi arch bacterium
With the primer and probe in probe groups.Real-time fluorescence PCR reaction system is placed in real-time fluorescence PCR instrument, by following reaction bars
Part carries out Fluorescence PCR：95 DEG C of predegeneration 5min；95 DEG C of denaturation 20s, 60 DEG C of annealing 1min, collect fluorescence, 40 circulations.
Result judges：According to Ct value judged results, Ct value≤35.0 can determine that the sample is the positive, and Ct value >=40 can be sentenced
It is feminine gender to determine sample result；Ct values ＞ 35.0 and ＜ 40, sample of reforming, result of reforming Ct value >=40 are feminine gender, are otherwise sun
Property.
Selecting above-mentioned primer carries out real-time PCR detection, and template to be checked is replaced with the standard DNA solution of Si Shi arch bacterium
DNA, replaces template DNA to be checked as negative control using deionized water, is additionally carried out above-mentioned steps；Both real-time fluorescence PCRs
Testing result is shown in Fig. 1 and Fig. 2, corresponds to standard DNA solution, the deionized water of Si Shi arch bacterium respectively in Fig. 1 and Fig. 2.
The specificity experiments of embodiment 2
The present embodiment is mainly carries out specificity experiments to the primer and probe groups of the offer of embodiment 1, specifically：
Replace the step described in template DNA additional embodiment one to be checked respectively with the DNA of the coli strain in following table
Rapid 2；Using deionized water as negative control.
Table 2
Experimental result is：2 plants of Si Shi arch bacteria strains amplifications are the positive in upper table, and other bacterial strain amplifications are
Feminine gender, the real-time fluorescent PCR amplification curve of 2 plants of Si Shi arch bacterium is shown in Fig. 2 and Fig. 3.
As seen from the figure, above-mentioned primer and probe groups have specificity to Si Shi arch bacterium, therefore, above-mentioned primer and probe groups
High specificity, therefore, herein under the premise of, the present invention provide detection method it is very reliable.
The sensitivity experiment of embodiment 3
The present embodiment is mainly carries out sensitivity experiment to the primer and probe groups of the offer of embodiment 1, specifically：
By the μ L of standard DNA solution 2 of Si Shi arch bacterium ATTCC 51132, measured using nucleic acid-protein analyzer and obtain DNA
Solution concentration is 18ng/ μ L.10 times of solution deionized water is incremental to be diluted to concentration 1.8ng/ μ L, 180pg/ μ L, 18pg/ respectively
μ L, 1.8pg/ μ L, 0.18pg/ μ L, 2 the step of replace template DNA to be checked to operate in addition described in embodiment one.Experimental result
As shown in Figure 4, in figure amplification curve from left to right successively distinguish corresponding concentration be 18ng/ μ L, 1.8ng/ μ L, 180pg/ μ L,
The amplification curve of 18pg/ μ L, the standard DNA solution of the Si Shi arch bacterium ATTCC 51132 of 1.8pg/ μ L, therefore, can determine whether this
Real-time PCR detection is high to the detection sensitivity of this standard DNA, and its detection sensitivity is reacted up to 3.6pg/.
By the μ L of standard DNA solution 2 of Si Shi arch bacterium ATTCC 51400, measured using nucleic acid-protein analyzer and obtain DNA
Solution concentration is 24ng/ μ L.10 times of solution deionized water is incremental to be diluted to concentration 2.4ng/ μ L, 240pg/ μ L, 24pg/ respectively
μ L, 2.4pg/ μ L, 0.24pg/ μ L, 2 the step of replace template DNA to be checked to operate in addition described in embodiment one.Experimental result
As shown in Figure 4, in figure amplification curve from left to right successively distinguish corresponding concentration be 24ng/ μ L, 2.4ng/ μ L, 240pg/ μ L,
The amplification curve of 24pg/ μ L, the standard DNA solution of the Si Shi arch bacterium ATTCC 51132 of 2.4pg/ μ L, therefore, can determine whether this
Real-time PCR detection is high to the detection sensitivity of this standard DNA, and its detection sensitivity is reacted up to 4.8pg/.
Standard DNA sensitivity test according to Si Shi arch bacterium ATTCC 51132 and Si Shi arch bacterium ATTCC 51400 can
Judge the characteristics of detection method of the invention has sensitivity high, its detection sensitivity is up to pg grades/reaction.
Embodiment 4
The present embodiment is mainly carries out sample contamination investigation to the primer and probe groups of the offer of embodiment 1, specifically：
All samples are aseptic to weigh the 25g homogenizing bags for having filter screen extremely, adds 225mL arch bacterium to increase bacterial context soup, slap
Formula homogenizer homogeneous 2min, above-mentioned filtered solution is carried out to put 36 DEG C of micro- aerobic (5%O2,79.8%N2,7.6%CO2,7.6%
H2) increase bacterium 24h, extract measuring samples template DNA by embodiment one and detected using real-time fluorescence PCR method.
Totally 52 parts of fresh fowl (chicken, duck) product is purchased and collected from the market, has detected Si Shi arch bacterium positive 2
Individual, positive rate 3.8% shows that the detection method can be efficiently applied to the detection of Si Shi arch bacterium of food samples.
Above example shows：The real-time fluorescence PCR reaction method that the present invention is provided combines above-mentioned primer and probe set
There are following characteristics：High specificity, sensitivity is high, detection is fast and reliable, it is easy to operate the characteristics of.
SEQUENCE LISTING
<110>Inspection ＆ Quarantine Technology Center of Zhuhai Entry-Exit Inspection ＆ Quarantine Bureau
<120>Fluorescence PCR primer, kit and detection method for detecting Si Shi arch bacterium
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
atgctcgtca tcgtagggtt 20
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence
<400> 2
agcggatttg cctacttaac aac 23
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<400> 3
atcgaggtca cggatggaag tgt 23
Claims (6)
1. it is used to detect the fluorescence PCR primer and probe of Si Shi arch bacterium, it is characterised in that the primer and probe are according to this
Designed by the specific sequence of the 23S rRNA genes of family name arch bacterium.
2. fluorescence PCR primer according to claim 1 and probe, it is characterised in that the nucleotides of the primer and probe
Sequence is as follows：
Sense primer：5’-ATGCTCGTCATCGTAGGGTT-3’；
Anti-sense primer：5’-AGCGGATTTGCCTACTTAACAAC-3’；
Probe：5’-ATCGAGGTCACGGATGGAAGTGT-3’.
3. a kind of kit for detecting Si Shi arch bacterium, it includes the fluorescence PCR primer and spy described in claim 1 or 2
Pin.
4. the fluorescence PCR detecting method of Si Shi arch bacterium, comprises the following steps：
(1) sample DNA is extracted；
(2) with sample DNA as template, fluorescent PCR amplification is carried out using the fluorescence PCR primer described in claim 1 or 2, probe；
(3) whether Si Shi arch bacterium are contained in judgement sample according to amplification curve.
5. detection method according to claim 4, it is characterised in that contain following components in fluorescent PCR amplification system：
6. detection method according to claim 4, it is characterised in that the program of fluorescent PCR amplification is：95 DEG C of predegenerations
5min；95 DEG C of denaturation 20s, 60 DEG C of annealing 1min, collect fluorescence, 40 circulations.
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| CN201710060204.8A CN106834463A (en) | 2017-01-24 | 2017-01-24 | Fluorescence PCR primer, kit and detection method for detecting Si Shi arch bacterium |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114438235A (en) * | 2020-11-04 | 2022-05-06 | 深圳市南山区疾病预防控制中心 | Primer probe assembly and kit for simultaneously detecting low-temperature toxoplasma gondii and toxoplasma stewardii and application of primer probe assembly and kit |
Citations (2)
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| CN102268478A (en) * | 2011-07-13 | 2011-12-07 | 中国疾病预防控制中心传染病预防控制所 | Primers, probe, method and kit for detecting campylobacter jejuni |
| KR101478921B1 (en) * | 2013-08-22 | 2015-01-05 | 중앙대학교 산학협력단 | Primer for loop-mediated isothermal amplification reaction for detecting Arcobacter spp., and method for detecting Arcobacter spp. using the same |
-
2017
- 2017-01-24 CN CN201710060204.8A patent/CN106834463A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268478A (en) * | 2011-07-13 | 2011-12-07 | 中国疾病预防控制中心传染病预防控制所 | Primers, probe, method and kit for detecting campylobacter jejuni |
| KR101478921B1 (en) * | 2013-08-22 | 2015-01-05 | 중앙대학교 산학협력단 | Primer for loop-mediated isothermal amplification reaction for detecting Arcobacter spp., and method for detecting Arcobacter spp. using the same |
Non-Patent Citations (5)
| Title |
|---|
| GALE BRIGHTWELL ET AL.: "Development of a multiplex and real time PCR assay for the specific detection of Arcobacter butzleri and Arcobacter cryaerophilus", 《JOURNAL OF MICROBIOLOGICAL METHODS》 * |
| KABEYA H ET AL.: "One-step polymerase chain reaction-based typing of Arcobacter species", 《INT J FOOD MICROBIOL.》 * |
| 刘敦华等: "《食品安全与清真食品检测》", 30 September 2015 * |
| 毕水莲: "应用gyrA基因聚合酶链反应检测食品中弓形菌", 《食品与发酵工业》 * |
| 游淑珠等: "PCR法检测动物源性食品中布氏弓形菌", 《现代食品科技》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114438235A (en) * | 2020-11-04 | 2022-05-06 | 深圳市南山区疾病预防控制中心 | Primer probe assembly and kit for simultaneously detecting low-temperature toxoplasma gondii and toxoplasma stewardii and application of primer probe assembly and kit |
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Application publication date: 20170613 |