CN106435007A - Primer, probe, kit and method for detecting bacillus erysipelatos-suis by fluorescent quantitative PCR (Polymerase Chain Reaction) - Google Patents
Primer, probe, kit and method for detecting bacillus erysipelatos-suis by fluorescent quantitative PCR (Polymerase Chain Reaction) Download PDFInfo
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- 238000003752 polymerase chain reaction Methods 0.000 title abstract 2
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Abstract
The invention discloses a primer, a probe, a kit and a method for detecting bacillus erysipelatos-suis by a fluorescent quantitative PCR (Polymerase Chain Reaction). The primer is shown as SEQ ID NO: 1 and SEQ ID NO: 2; the probe is shown as SEQ ID NO: 3. The method disclosed by the invention takes DNA (Deoxyribonucleic Acid) of a sample to be detected as a template for carrying out a fluorescent quantitative PCR amplification reaction; fluorescent signals are collected to draw a curve. The primer and the probe for detecting the bacillus erysipelatos-suis have very high specificity and sensitivity, can be used for detecting the bacillus erysipelatos-suis and can also be used for detecting various types of clinical samples, so that a clinical bacillus erysipelatos-suis infection condition can be rapidly monitored; the operation is simple and practical.
Description
Technical field
A kind of the invention belongs to animal virology and technical field of molecular biology, more particularly it relates to fluorescence
The primer of quantitative PCR detection bacillus rhusiopathiae suises, probe, test kit and method.
Background technology
Pig erysipelas are acute, the hot infectious disease of the one kind being caused by bacillus rhusiopathiae suises, and it is mainly characterized by hyperpyrexia, acute loses
Mass formed by blood stasis, skin rash block (subacute), chronic libman-Sack endocarditis and cutaneous necrosis and multiple non suppurative arthritis.Primary disease was once
Through being referred to as one of China's " three big infectious disease ", in recent years, gradually fade out the regarding of people with intensivization development pig erysipelas of raising pigs
Open country, but this disease is not cleaned completely, and area has fragmentary appearance always throughout the country, causes serious warp to pig farmer
Ji loss.
In Swine Production, conventional pig erysipelas attenuated vaccine strain carries out immune prevention and control, effect is significant to pig erysipelas.In recent years, in
Herd, release one after another swine fever, pig erysipelas, pig lung plague three of the vaccine producer such as Guangdong Yongshun is lived Seedling, a shot can prevent 3 kinds of biographies
Catch an illness, effect is close with 3 kinds of single Seedlings, simple and effective.However, because pig erysipelas are similar with pig lung plague pasteurellosis bacilluss nutritional need,
Two kinds of antibacterials are cultivated in same culture dish and interfere, and it is inaccurate that this will result in trigeminy vaccine finished product vaccine antigen content detection,
And attenuated vaccine is higher to antigen content requirement, antigenic content is low to cause immune efficacy low, and antigenic content height then can affect
The safety of vaccine.
Generally using viable bacteria culture counting method, the bacterium number containing in vaccine is measured in prior art, concrete grammar
For：With normal saline, vaccine is diluted to after 1 part/milliliter, takes 1 milliliter to do 10 times and be serially diluted, each dilution factor changes one
Vaccine is rule of thumb diluted to corresponding multiple by dilution tube, selects suitable dilution bacterium solution inoculation blood agar culture
Base flat board 3,0.1 milliliter of each plate, cultivate 24～48 hours, perusal bacterium colony, and in flat board ground points, calculate 3
The average colony number of flat board, is multiplied by extension rate and is multiplied by 10 again, the total bacteria count contained by as every part vaccine.
The method major defect that viable bacteria cultivates counting method is that the detection used time is longer, and same vaccine has two kinds of nutritional needs similar
Antigen, then can interfere.Detection duration is because needing to do antibacterial culturing, needs 24～48 hours；After vaccine dilution
Inoculation plating medium is it is impossible to determine inoculation position, two bacterium colonies overlap or cover, then be unable to accurate counting.In addition, it is thin
There is potentially danger in bacterium culture.
In sum, detection bacillus rhusiopathiae suises content that is simply accurate in the urgent need to one kind, not disturbed by other antibacterials
Detection method.Quantitative fluorescent PCR is so that high specificity, sensitivity is high, reproducible, fireballing plurality of advantages becomes molecular biosciences
Learn the important tool in research, because it detects specific nucleic acid, therefore testing result will not be affected by other cause of diseases, can be applicable to
The content detection of bacillus rhusiopathiae suises.
Content of the invention
Based on this, in order to overcome the defect of above-mentioned prior art, the present invention is by bacillus rhusiopathiae suises 16S ribosomal RNA
The analysis of gene, devises primer and the probe of reporter group labelling, and establishes fluorescence quantifying PCR method, can be red to pig
Bacillus venenosus is efficiently detected.
In order to realize foregoing invention purpose, this invention takes technical scheme below：
A kind of primer of fluorescence quantitative PCR detection bacillus rhusiopathiae suises, described primer such as SEQ ID NO:1 and SEQ ID NO:
Shown in 2.
The invention also discloses a kind of probe of fluorescence quantitative PCR detection bacillus rhusiopathiae suises, described probe such as SEQ ID
NO:Shown in 3.
5 ' ends of described probe and 3 ' ends flag F AM and BHQ1 respectively.
The application in the test kit of preparation fluorescence quantitative PCR detection bacillus rhusiopathiae suises of above-mentioned primer and probe.
A kind of test kit of fluorescence quantitative PCR detection bacillus rhusiopathiae suises, described test kit includes above-mentioned primer and probe.
Described test kit also includes PCR premixed liquid, plasmid control sample and sterile distilled water.
A kind of method of fluorescence quantitative PCR detection bacillus rhusiopathiae suises, using above-mentioned primer and probe, comprises the following steps：
With the DNA of detected sample as template, carry out fluorescent quantitative PCR reaction, collect fluorescence signal, draw curve.
The reaction system of described fluorescent quantitative PCR reaction is：PCR premixed liquid 12.5 μ L (2 times of quantitative PCR buffer,
Taq archaeal dna polymerase 6U/ μ L, primer SEQ ID NO:The NO of 1pm, SEQ ID shown in 1:1pm shown in 2), fluorescent probe 0.5 μ L
(10pm), sterile distilled water 10 μ L, adds the DNA profiling 2 μ L having extracted.
The response procedures of described fluorescent quantitative PCR reaction are：95 DEG C, 1 minute；95 DEG C 5 seconds, 60 DEG C 30 seconds, 40
Circulation, wherein 60 DEG C collection fluorescence.
Compared with prior art, the present invention has following remarkable result：
The present invention is directed to bacillus rhusiopathiae suises 16S ribosomal gene sequential design pair of primers, a label probe, and makes
Standby plasmid control sample, as internal reference, establishes fluorescence quantifying PCR method, and primer and probe have very high special
Property and sensitivity, detection by quantitative can go out bacillus rhusiopathiae suises, various vaccines and clinical sample can be carried out detecting, differentiate, inspection
The survey time is short, can obtain result within general 2～3 hours, such that it is able to efficiently make a distinction to bacillus rhusiopathiae suises, simple to operate,
Practical.
Brief description
Fig. 1 is the experimental result of the quantitative fluorescent PCR joining standard substance outside the present invention, wherein, curve 1,2,3,4,5 generation respectively
Every milliliter of plasmid copy number of table is：1.56×108、1.56×107、1.56×106、1.56×105、1.56×104；
Epidemic disease that Fig. 2 is the method 3 batch pig erysipelas each to producer A and B of the embodiment of the present invention 1, pig lung plague, swine fever three are lived
Seedling testing result, wherein, every milliliter of plasmid copy number that curve 1,2,3,4,5 represents respectively is 1.56 × 108、1.56×107、
1.56×106、1.56×105、1.56×104(standard curve)；Curve 6,7,8 is 3 batch vaccine amplifications of producer A respectively
Curve, calculates vaccine antigen content copy number and is respectively 2.52 × 1010、7.04×109、6.87×109, antigenic content all closes
Lattice；Curve 9,10,11 is 3 batch vaccine amplification curves of producer B respectively, calculates vaccine antigen content copy number and is respectively
5.27×109、4.95×109、1.36×106, wherein front two batches antigenic content is qualified, and later batch is unqualified.
Fig. 3 is the specific assay result of test example 1 of the present invention, and wherein curve 1 is pig erysipelas, and curve 2-5 is respectively chain
Coccus, escherichia coli, pasteurellosis bacilluss, staphylococcuses.
Fig. 4 is the sensitivity test result of test example 2 of the present invention, the plasmid that wherein curve 1,2,3,4,5,6,7,8 represents
Copy number is respectively 4.26 × 108、4.26×107、4.26×106、4.26×105、4.26×104、4.26×103、4.26×
102、4.26；Curve 9 is negative control；
Fig. 5 is the repeated experiment result of test example 3 of the present invention, wherein curve 1, and curve 2 and 3 is 3 weights of same sample
Renaturation is tested.
Specific embodiment
To describe the present invention below in conjunction with the drawings and specific embodiments in detail.
The experiment material being related in following examples if no special instructions, derives from commercially available, is adopted in following examples
The routine operation that operation is well known to the skilled person.
The primer of embodiment 1 fluorescence quantitative PCR detection bacillus rhusiopathiae suises, probe and method
1、Primer, probe design
According to 7 pig erysipelas bacterial strain 16S ribosomal gene sequences that NCBI logs in, (accession number is respectively：AB055908.1、
AB055907.1, AB055905.1, NR_040837.1, KP063151.1, KP063150.1 and KJ660062.1) letter that provides
Breath, through comparing analysis, devises primers F, R and the probe P of quantitative PCR, nucleotide sequence is as follows：
Forward primer F：AGGGAATTTTCGGCAATGG(SEQ ID NO:1)；
Downstream primer R：CCCGAAGGCCKTCTTCA(SEQ ID NO:2)；Its K represents T or G；
Probe P：AAGACTACCGACAAGCCCACTCACAAC(SEQ ID NO:3)
Wherein, the 5 ' ends of probe P and 3 ' ends flag F AM and BHQ1 respectively.
Carry out fluorescent quantitative PCR using above-mentioned primer and probe, bacillus rhusiopathiae suises can be detected by amplification curve.
2nd, plasmid control sample preparation
The pig erysipelas bacterial strain 16S ribosomal gene sequential design that logged according to NCBI primers F 1 and R1, nucleotide sequence
As follows：
F1：GAATTCCACTAACCTCTCCTG(SEQ ID NO:4)；
R1：CACAAYGAGATGGGCTTA(SEQ ID NO:5)；Wherein Y represents C or T.
With the extracting examination in a small amount of AxyGen body fluid viral DNA/RNA pillar method after the swine erysipelas vaccine dilution of Zhongmu Stocks Trading Co. company
Agent box (also can use other method for extracting nucleic acid) extracts nucleic acid, saves backup；The nucleic acid being extracted with primers F 1 and R1 amplification, reaction
System is：PCR premixed liquid 12.5 μ L (2 times of PCR buffer, Taq archaeal dna polymerase 6U/ μ L, primer SEQ ID NO:Shown in 4
1pm、SEQ ID NO:1pm shown in 5), sterile distilled water 10 μ L, the nucleic acid 3 μ L of extraction, response procedures are：98 DEG C, 15 seconds；53
DEG C, 30 seconds；72,45 seconds；72 DEG C, 45 seconds；30 circulations；72 DEG C, 2 minutes；PCR primer is cloned into after purification PMD-18T matter
Grain carrier, then convert to DH5 α escherichia coli propagation, carry out 10 times after purification and be serially diluted, choose wherein 5 dilution factors and make
For standard sample, measured plasmid concentration, the copy number of plasmid can be conversed, every milliliter of copy number is respectively：1.56×
108、1.56×107、1.56×106、1.56×105、1.56×104.
3rd, detection method
Comprise the following steps：
(1), measuring samples DNA extraction
Choose swine fever, pig erysipelas and the pig lung plague trigeminal live vaccine of producer A and producer B, respectively take 3 bottles, use sterile physiological salt
Water is diluted to 1 milliliter/part, respectively takes 200 μ L, extracts nucleic acid with AxyGen body fluid DNA/RNA pillar method a small amount of extraction agent box
(finally with 20 μ L water elutions), saves backup.
(2), quantitative fluorescent PCR
Each nucleic acid drawing 2 μ L extractions and plasmid control sample are added in quantitative PCR reaction system, and reaction system is prepared
For (every part)：PCR premixed liquid 12.5 μ L (2 times of quantitative PCR buffer, Taq archaeal dna polymerase 6U/ μ L, primer SEQ ID NO:1
Shown 1pm, SEQ ID NO:1pm shown in 2), fluorescent probe 0.5 μ L (10pm), sterile distilled water 10 μ L.
After reaction system mix homogeneously, quantitative PCR apparatus are expanded, response procedures are：95 DEG C, 1 minute；95 DEG C, 5
Second；60 DEG C, 30 seconds；40 circulations, wherein 60 DEG C collection fluorescence.
(3), result judgement
After amplification terminates, according to the CT value of plasmid control sample and vaccine sample fluorescent PCR, calculate sample size, then
It is multiplied by 50, bacillus rhusiopathiae suises content in as 1 part vaccine.Calculate every part vaccine pig erysipelas content >=2.5 × 109, epidemic disease
Seedling antigenic content is qualified；Calculate every part vaccine pig erysipelas content ＜ 2.5 × 109, unqualified (the vaccine matter of vaccine antigen content
Amount standard is that every part vaccine pig erysipelas content is more than 5 × 108, said method detection calculate 2.5 × 109With count of bacteria
The 5 × 10 of method detection8Corresponding).
The testing result of the sample of the present embodiment is shown in Fig. 1 and Biao 1.
Table 1 measuring samples testing result
Test example 1 specific assay
Swine erysipelas vaccine strain, pasteurellosis bacilluss vaccine strain is contained, the sample of escherichia coli and Streptococcus suis, after process known to taking
Take each 200 μ L AxyGen body fluid viral DNAs/RNA pillar method a small amount of extraction agent box to extract nucleic acid, then carry out fluorescent quantitation
PCR operates, and obtains a result as shown in figure 3, the sample amplification containing swine erysipelas vaccine strain goes out A curve in Fig. 1, Ct value is 9.76,
Result judgement is the positive；Sample containing pasteurellosis bacilluss vaccine strain, escherichia coli and Streptococcus suis does not amplify specific curve,
It is judged to feminine gender.Thus result understands that the present invention program has good specificity.
Test example 2 sensitivity test
Concentration known is 1.75 × 1011The PMD-18T plasmid of copy/mL (has been cloned into sequence 5-AGGGAATTTTCGG
CAATGGGGGAAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGCCTTCGGG-3) carry out 10 times to be serially diluted, dilute
Release to 1.75 × 103Copy/mL, each dilution factor takes 1 μ L, together with negative control, adds in quantitative PCR reaction system, mixes
Quantitative PCR reaction is carried out on quantitative real time PCR Instrument, wherein reaction system is afterwards：Premixed liquid 12.5 μ L, distilled water 11 μ L, glimmering
Light probe 0.5 μ L；Response procedures are：95 DEG C, 1 minute；95 DEG C, 5 seconds；60 DEG C, 30 seconds；40 circulations, wherein 60 DEG C collections are glimmering
Light.Result as shown in Figure 4, the Ct value of curve 1,2,3,4,5,6,7,8 and 9 is 9.54 respectively, 12.17,14.68,19.21,
22.08th, 25.01,28.32,31.36 and 35.19, corresponding plasmid copy number is respectively 1.75 × 108、1.75×107、1.75
×106、1.75×105、1.75×104、1.75×103、1.75×102, 1.75 × 10,1.75, curve 10 be feminine gender, thus tie
Fruit understands, the solution of the present invention has very great number sensitivity.
Test example 3 Repeatability checking
Take the known sample containing swine erysipelas vaccine strain to be divided into 3 parts, after process, take 200 μ L AxyGen body fluid DNA/RNA
Pillar method a small amount of extraction agent box extracts nucleic acid, then carries out fluorescence quantitative PCR detection, obtains a result as shown in Figure 5, Fig. 5
Middle curve 1,2,3, corresponding Ct value is for 14.35,14.19 and 14.27 respectively, and result judgement is the positive；Thus result understands
The present invention has good repeatability.
Embodiment described above only have expressed the several embodiments of the present invention, and its description is more concrete and detailed, but simultaneously
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, some deformation can also be made and improve, these broadly fall into the guarantor of the present invention
Shield scope.Therefore, the protection domain of patent of the present invention should be defined by claims.
<110>Guangdong Wen'S Foodstuffs Group Co., Ltd.
<120>A kind of primer of fluorescence quantitative PCR detection bacillus rhusiopathiae suises, probe, test kit and method
<160> 5
<170> PatentIn version 3.3
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Claims (7)
1. a kind of primer of fluorescence quantitative PCR detection bacillus rhusiopathiae suises is it is characterised in that described primer such as SEQ ID NO:1 He
SEQ ID NO:Shown in 2.
2. a kind of probe of fluorescence quantitative PCR detection bacillus rhusiopathiae suises is it is characterised in that described probe such as SEQ ID NO:3 institutes
Show.
3. the probe of fluorescence quantitative PCR detection bacillus rhusiopathiae suises according to claim 2 is it is characterised in that described probe
5 ' end and 3 ' end be marked with FAM and BHQ1 respectively.
4. the primer of fluorescence quantitative PCR detection bacillus rhusiopathiae suises described in claim 1 or the detection described in Claims 2 or 3
Application in the test kit of preparation detection bacillus rhusiopathiae suises for the probe of bacillus rhusiopathiae suises.
5. a kind of test kit of fluorescence quantitative PCR detection bacillus rhusiopathiae suises is it is characterised in that described test kit includes claim
The primer of detection bacillus rhusiopathiae suises described in 1 and the probe of the detection bacillus rhusiopathiae suises described in Claims 2 or 3.
6. the test kit of fluorescence quantitative PCR detection bacillus rhusiopathiae suises according to claim 5 is it is characterised in that described examination
Agent box also includes PCR premixed liquid, plasmid standard and sterile distilled water.
7. a kind of method of fluorescence quantitative PCR detection bacillus rhusiopathiae suises is it is characterised in that adopt the detection described in claim 1
The probe of the detection bacillus rhusiopathiae suises described in the primer of bacillus rhusiopathiae suises and Claims 2 or 3, comprises the following steps：To treat
The DNA of detection sample is template, carries out fluorescent quantitative PCR reaction, collects fluorescence signal, draws curve；
Wherein, the reaction system of described fluorescent quantitative PCR reaction is：PCR premixed liquid 12.5 μ L (2 times of quantitative PCR bufferings
Liquid, Taq archaeal dna polymerase 6U/ μ L, primer SEQ ID NO:The NO of 1pm, SEQ ID shown in 1:1pm shown in 2), fluorescent probe 0.5 μ
L (10pm), sterile distilled water 10 μ L, add the DNA 2 μ L having extracted.
The response procedures of described fluorescent quantitative PCR reaction are：95 DEG C, 1 minute；95 DEG C 5 seconds, 60 DEG C 30 seconds, 40 circulation,
Wherein 60 DEG C collection fluorescence.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106916910A (en) * | 2017-05-10 | 2017-07-04 | 广东温氏食品集团股份有限公司 | Sai Nika paddy virus (PRRSV) TaqMan MGB fluorescence quantitative PCR detections primer, probe and detection method |
| CN107119121A (en) * | 2017-05-05 | 2017-09-01 | 广西壮族自治区兽医研究所 | A kind of real-time quantitative LAMP primer, kit and method for detecting bacillus rhusiopathiae suis |
| CN107937573A (en) * | 2017-11-03 | 2018-04-20 | 华中农业大学 | Quick detection bacillus rhusiopathiae suis and Streptococcus suis duplex PCR primer pair and kit and method |
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| CN105925729A (en) * | 2016-06-29 | 2016-09-07 | 广东温氏食品集团股份有限公司 | Primer, probe, kit and method for fluorogenic quantitative PCR detection on pig delta coronavirus |
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| JP2000279179A (en) * | 1999-03-31 | 2000-10-10 | National Institute Of Animal Health | Swine erysipelas recombinant subunit vaccine |
| CN105925729A (en) * | 2016-06-29 | 2016-09-07 | 广东温氏食品集团股份有限公司 | Primer, probe, kit and method for fluorogenic quantitative PCR detection on pig delta coronavirus |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107119121A (en) * | 2017-05-05 | 2017-09-01 | 广西壮族自治区兽医研究所 | A kind of real-time quantitative LAMP primer, kit and method for detecting bacillus rhusiopathiae suis |
| CN106916910A (en) * | 2017-05-10 | 2017-07-04 | 广东温氏食品集团股份有限公司 | Sai Nika paddy virus (PRRSV) TaqMan MGB fluorescence quantitative PCR detections primer, probe and detection method |
| CN107937573A (en) * | 2017-11-03 | 2018-04-20 | 华中农业大学 | Quick detection bacillus rhusiopathiae suis and Streptococcus suis duplex PCR primer pair and kit and method |
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Application publication date: 20170222 |