CN109136396A - A kind of specific detection primer and detection kit of clostridium welchii disease - Google Patents
A kind of specific detection primer and detection kit of clostridium welchii disease Download PDFInfo
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- CN109136396A CN109136396A CN201811096473.0A CN201811096473A CN109136396A CN 109136396 A CN109136396 A CN 109136396A CN 201811096473 A CN201811096473 A CN 201811096473A CN 109136396 A CN109136396 A CN 109136396A
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- 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
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- 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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- 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
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses the specific detection primer groups and detection kit of a kind of clostridium welchii disease.As shown in SEQ ID NO:1 and SEQ ID NO:2, the specific detection agents box of clostridium welchii disease are prepared for using the primer respectively for specific detection primer group nucleotide sequence, establish a kind of new PCR detection method for clostridieum welchii.The present invention designs specific detection primer according to clostridieum welchii distinguished sequence, establishes quick, special, the sensitive PCR detection method for clostridieum welchii epidemiological survey and clinical infection situation, can accurately detect clostridieum welchii.Kit of the invention sequencing easy to operate, method high specificity, sensibility is high, result judgement is objective, the achievable epidemiological survey to clostridium welchii disease, the detection and clinical diagnosis of subclinical infection, application prospect are good, it is worth being widely applied, for the diagnosis of clostridium welchii disease and Prevention Technique etc., further research is laid a good foundation.
Description
Technical field
The present invention relates to disease detection reagent preparation technical fields, are related to a kind of specific detection primer of clostridium welchii disease
And detection kit.
Background technique
Clostridium welchii disease is a kind of zoonosis as caused by clostridieum welchii.Clostridium welchii disease can cause many animals
Enterotoxemia and necrotic enteritis, it is closely related with " Sudden Death Syndrome " of livestock and poultry, and also the food for infecting clostridieum welchii can lead to people
Appearance is poisoned by food and emphysematous gangrene, causes public health security problem.Clostridium welchii disease morbidity is anxious, the course of disease is short, lethal fast, hair
Sick animal usually has little time diagnosis will be dead, causes biggish loss to aquaculture.In addition, only by clinical diagnosis and
Pathological anatomy is difficult with multiple infectious disease relatively to distinguish it.Therefore, the epidemic prevention and quarantine work carried out usually is critically important.
For a long time, detection clostridieum welchii is most classical, most accurate method is toxin neutralization test, but exists by non-specific
Property death cause erroneous judgement the shortcomings that, and it is time-consuming and laborious, the test period is long.The identification of bacterium at present is mostly still using traditional thin
Mycology method and zoopery, but there are complex steps, the disadvantages such as the period is long, verification and measurement ratio is low, and cannot be guaranteed experimental result
Accuracy.Other diagnostic methods such as ELISA detection method, is mainly used for the parting of serotype, in laboratory diagnosis and clinic
In diagnosis using less；It is very numerous to prepare antitoxin process although sensibility and specificity is good for antitoxic detection
It is trivial, and Venom antigens need to be detected and purified, it is also easy to appear nonspecific reaction, so kind method also and is of little use；
Report that detection method is substantially detected with alpha-toxin gene, but other bacteriums, as clostridium septicum also has alpha-toxin
Gene, it is possible to can also amplify and, testing result is impacted, therefore there is limitation with alpha-toxin gene specific PCR.
To sum up, although these identification methods are still used for so far in the diagnosis of clostridium welchii disease, clinically it is badly in need of more
Accurately, quick and easy to operate detection method.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect of the prior art and deficiency, a kind of clostridium welchii disease is provided
Specific detection primer, detection kit and its detection method.Primer provided by the invention has high specificity, sensibility high
Advantage can accurately detect clostridieum welchii, and kit of the invention sequencing easy to operate, method high specificity, sensibility is high,
Result judgement is objective and accurate.
The object of the present invention is to provide the primers of a pair of of detection chicken clostridium welchii disease.
It is a further object to provide detected in chicken clostridium welchii disease kit using the primer in preparation
Using.
The purpose of the present invention is what is be achieved by the following technical programs:
The primer of a pair of detection chicken clostridium welchii disease, the nucleotide sequence of the primer is as shown in NO:1~2 SEQ ID.
SEQ ID NO:1:CATCATTCAACCAAA(TTF-2)；
SEQ ID NO:2:TTCCGCTCACGGAC(TTR-2).
Application of the primer in preparation detection chicken clostridium welchii disease kit, also belongs to protection scope of the present invention.
A kind of kit of specific detection chicken clostridium welchii disease, including the primer.
It is highly preferred that the concentration of the primer is respectively 50 pmol/ μ L.
Preferably, the kit further includes PCR reaction solution.
It is highly preferred that the PCR reaction solution is Premix Ex Taq Version.
Preferably, the PCR reaction system of the kit are as follows: 2 μ L, Premix Ex Taq Version of sample DNA
12.5 μ L, each 0.5 μ L of primer, distilled water supply surplus, and total volume is 25 μ L.
Preferably, the PCR amplification condition of the kit are as follows: 94 DEG C of 5 min；94 DEG C of 30 s, 52.3 DEG C of 30 s, 72
DEG C 80 s, 25 circulations；72℃ 10 min.
Preferably, the test object of the kit is chicken manure.
Preferably, the kit further includes sample DNA extracting solution and positive reference substance.
It is highly preferred that the positive reference substance is clostridieum welchii genomic DNA.
It is highly preferred that the sample DNA extracting solution is faeces DNA extracting solution.DNA of bacteria commonly used in the prior art extracts
The concrete component of liquid, extracting solution is unrestricted.
It is further preferred that the composition of the faeces DNA extracting solution includes: ASL Buffer, Proteinase K, Buffer
AL, Buffer AW1, Buffer AW2, Buffer AE and 96%~100% alcohol.
A kind of detection chicken clostridium welchii disease kit,
(1) it forms: the primer, sample DNA extracting solution, PCR reaction reagent and positive control solution,
Wherein, for the nucleotide sequence of the primer as shown in NO:1~2 SEQ ID, concentration is 50 pmol/ μ L；
The sample DNA extracting solution is the faeces DNA extracting solution of independent packaging；
The faeces DNA extracting solution include ASL Buffer, Proteinase K, Buffer AW1, Buffer AW2, Buffer AE and
96%~100% alcohol；
The composition of the PCR reaction solution are as follows: Premix Ex Taq Version 2.0(Loading dye Mix)；
The positive control is clostridieum welchii genomic DNA, and effect is to compare PCR product and monitor PCR operating process to be
It is no correct.
(2) operating method, specifically includes the following steps:
S1. chicken manure sample DNA is extracted；
S2. the primer is utilized, PCR amplification is carried out as template using the sample DNA that step S1 is obtained；
Wherein, the reaction system of PCR are as follows: 2 μ L, Premix Ex Taq Version of sample DNA, 12.5 μ L, primer each 0.5
μ L, surplus are distilled water, and the total volume of PCR reaction system is 25 μ L.
The reaction condition of PCR are as follows: 94 DEG C of 5 min of initial denaturation；94 DEG C of 30 s of denaturation, 52.3 DEG C of 30 s of annealing, 72 DEG C extend
80 s, totally 25 recycle；Extend 10 min after 72 DEG C.
S3. agarose gel electrophoresis.
S4. result judgement: if amplifying the band of 1225 bp, illustrate that the sample is the sample for having infected clostridium welchii disease
This, conversely, the sample does not infect clostridium welchii disease.
Compared with prior art, the invention has the following advantages:
Utilize the primer detection chicken clostridium welchii disease, high specificity, high sensitivity；PCR reaction condition is more suitable, the method for PCR
As a result it is easy to determine；The kit of detection clostridium welchii disease is made using the primer, easy to operate and sequencing can be used for clinic
Accurate, quick, the sensitive PCR of sample is detected, and result judgement is objective, it can be achieved that epidemiological survey to clostridium welchii disease,
The detection and clinical diagnosis of subclinical infection, for the diagnosis of clostridium welchii disease and Prevention Technique etc., further research is laid a good foundation.
Detailed description of the invention
Fig. 1 is the 5 pairs of primer pair clostridieum welchiis designed based on specific gene in clostridieum welchii 16s rRNA gene order
PCR specific amplification gel electrophoresis figure；Wherein, figure A is the electrophoretogram carried out after PCR amplification using primer 1；Scheming B is to utilize
Primer 2 carries out the electrophoretogram after PCR amplification；Scheming C is the electrophoretogram carried out after PCR amplification using primer 3；Figure D is to utilize primer 4
Electrophoretogram after carrying out PCR amplification；Scheming E is the electrophoretogram carried out after PCR amplification using primer 5.
Fig. 2 is to carry out temperature optimization PCR amplification gel electrophoresis figure to primer 2.
Fig. 3 is the PCR amplification gel electrophoresis figure that specific test is carried out using kit.
Fig. 4 is the PCR amplification gel electrophoresis figure that sensitivity tests is carried out using kit.
Fig. 5 is the PCR amplification gel electrophoresis figure that repetitive test is carried out using kit.
Fig. 6 is the PCR amplification gel electrophoresis figure that clinical sample detection is carried out using kit；Wherein, figure A is Guangdong Province four
6 parts of chicken manure samples of meeting city chicken house；Scheme 10 parts of chicken manure samples that B is Yunfu City Xinxing County town Le Zhu chicken house
This；Scheme 6 parts of chicken manure samples that C is Guangzhou, Guangdong chicken house；Scheme 45 that D is 8 chicken houses in Yunfu City Xinxing County
Part chicken manure sample.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
The extraction of faeces DNA: referring to the specification of QIAamp company.
1 design of primers of embodiment and screening
1, experimental procedure
(1) it according to the full gene sequence of coding clostridieum welchii 16s rRNA, is carried out with other bacterium 16s rRNA gene orders
It compares, the target gene for selecting specific gene to diagnose as clostridium welchii disease, special primer is designed according to its gene order, is shown in Table
1。
1 clostridieum welchii specific PCR primers of table
(2) extraction of faeces DNA: being extracted using commercial reagent box, specification of the specific steps referring to QIAamp company.
(3) PCR reaction system are as follows: Premix Ex Taq Version 2.0(Loading dye Mix) 12.5 μ L, just
To each 0.5 μ L of primer and reverse primer, 9.5 μ L of distilled water, 2 μ L of template DNA, amplified sample number n(n=sample number+3).It will
Above-mentioned reaction solution is mixed in centrifuge tube, is mixed, packing.It takes each sample DNA to be added in corresponding reaction tube, is mixed after each reaction tube label
Even centrifugation is placed in PCR instrument and reacts.
PCR amplification condition are as follows: 94 DEG C of 5 min of initial denaturation；94 DEG C of 30 s of denaturation, 52.3 DEG C of 30 s of annealing, 72 DEG C extend 80
S, totally 25 recycle；Extend 10 min after 72 DEG C.
(4) agarose gel electrophoresis.
(5) result judgement: if amplifying the band of 1225 bp, illustrate that the primer is suitble to detect clostridieum welchii, Ke Yizuo
For specific primer, conversely, then illustrating that the primer is not suitable for.
2, experimental result
The result shows that: primer 2, i.e. TTF(R) -2 specific best (Fig. 1), the nucleotide sequence such as institute of SEQ ID NO:1~2
Show.
SEQ ID NO:1::CATCATTCAACCAAA(TTF-2)；
SEQ ID NO:2::TTCCGCTCACGGAC(TTR-2).
Wherein, figure A is the electrophoretogram carried out after PCR amplification using primer 1；Scheming B is after carrying out PCR amplification using primer 2
Electrophoretogram；Scheming C is the electrophoretogram carried out after PCR amplification using primer 3；Scheming D is the electricity carried out after PCR amplification using primer 4
Swimming figure；Scheming E is the electrophoretogram carried out after PCR amplification using primer 5；Wherein, " sun " represents the DNA of clostridieum welchii, and " sky " represents
Blank control, " 1~8 " respectively represent staphylococcus, Escherichia coli, salmonella, clostridium botulinum, gangrenous toxin fusiform brood cell's bar
Bacterium, Pasteurella, streptococcus, clostridium septicum DNA.
The optimization of 2 PCR reaction condition of embodiment
1, experimental procedure
Its nucleotide series of primer TTF-2/TTR-2(obtained using the screening of embodiment 1 are as shown in NO:1~2 SEQ ID) into
Row PCR amplification, PCR reaction system with embodiment 1, annealing temperature between 44.3 DEG C to 55.3 DEG C, recurring number 25 to 45 it
Between be adjusted, concrete operations and determination method are referring to embodiment 1.
2, experimental result
The result shows that: to use its nucleotide series of TTF-2/TTR-2(as shown in NO:1~2 SEQ ID) as primer PCR most
Good annealing temperature is 52.3 DEG C (Fig. 2)；Recurring number is 25.Wherein, " 1~12 " respectively represent 44.3 DEG C of annealing temperature, 45.3 DEG C,
46.3 DEG C, 47.3 DEG C, 48.3 DEG C, 49.3 DEG C, 50.3 DEG C, 51.3 DEG C, 52.3 DEG C, 53.3 DEG C, 54.3 DEG C, 55.3 DEG C, " 13 " generation
Table blank control.
A kind of clostridium welchii disease detection kit of embodiment 3
1, kit forms
The kit forms of the present embodiment include: its nucleotide series of primer TTF-2/TTR-2(such as institute of SEQ ID NO:1~2
Show), sample DNA extracting solution, PCR reaction reagent and positive control.
Wherein, sample DNA extracting solution is the faeces DNA extracting solution of independent packaging, concrete composition are as follows: the 140 of 50 reactions
ASL Buffer of mL, the Proteinase K of 1.4 mL, Buffer AL of 33 mL, Buffer AW1 of 19 mL, 13 mL
96%~100% alcohol of Buffer AW2, Buffer AE of 12 mL, 10 mL.
PCR reaction reagent are as follows: 500 μ L Premix Ex Taq Version 2.0(Loading dye of 40 reactions
Mix).
Primer TTF-2/TTR-2: for its nucleotide sequence as shown in NO:1~2 SEQ ID, concentration is 50pmol/ μ L.
Positive control is clostridieum welchii genomic DNA, and effect is to compare PCR product and monitor PCR operating process to be
It is no correct.
2, application method
(1) DNA in sample is extracted using sample DNA extracting solution；
(2) PCR reaction is carried out
The reaction system of PCR are as follows: 13.5 μ L of PCR reaction solution, 2 μ L of template DNA, surplus are distilled water, 25 μ L of total volume；
PCR amplification: the reaction condition of PCR is carried out using the kit are as follows: 94 DEG C of 5 min of initial denaturation；94 DEG C of denaturation 30 s, 52.3
DEG C annealing 30 s, 72 DEG C of 80 s of extensions, totally 25 recycle；Extend 10 min after 72 DEG C；
(3) agarose gel electrophoresis；
(4) result judgement
If amplifying the band of 1225 bp, illustrate that the kit and detecting step are suitble to detect clostridium welchii disease, conversely, then
Illustrate that the kit and detecting step are not suitable for detection clostridium welchii disease.
A kind of specific test of the specific detection agents box of the clostridium welchii disease of embodiment 4
1, experimental procedure
With by the clostridieum welchii of DNA validation verification, staphylococcus, Escherichia coli, salmonella, clostridium botulinum, gangrene poison
9 check sample DNA such as plain clostruidium, Pasteurella, streptococcus, clostridium septicum are template, use the Wei of embodiment 3
The specific detection agents box of family name's clostridial disease detect, while setting distilled water as blank control.
2, experimental result
The results show that the DNA sample of only clostridieum welchii amplifies the band of about 1225 bp, and other above-mentioned 8 kinds of comparison bacteriums
There is (Fig. 3) without band with blank control.Wherein, " sun " represents the DNA of clostridieum welchii, and " sky " represents blank control, " 1~
8 " respectively represent staphylococcus, Escherichia coli, salmonella, clostridium botulinum, gangrenous toxin clostruidium, Pasteurella,
The DNA of streptococcus, clostridium septicum.
A kind of sensitivity of the specific detection agents box of the clostridium welchii disease of embodiment 5 is tested
1, experimental procedure
Respectively to contain 2000 Marker, 1.2 × 109 CFU/ml、1.2×108 CFU/ml、1.2×107 CFU/ml、1.2×
106 CFU/ml、1.2×105 CFU/ml、1.2×104 CFU/ml、1.2×103 CFU/ml、120 CFU/ml、12 CFU/ml、
The DNA of 1.2 CFU/ml clostridieum welchiis is template, is examined using the specific detection agents box of the clostridium welchii disease of embodiment 3
It surveys.
2, experimental result
The results show that specific PCR contains 1.2 × 10 in the sample3 There is band when the DNA of CFU/ml clostridieum welchii, can obtain spy
The minimal detectable concentration 1.2 × 10 of different PCR3 CFU/ml clostridieum welchii (Fig. 4).Wherein " 1~11 " respectively represents 1.2 × 109
CFU/ml、1.2×108 CFU/ml、1.2×107 CFU/ml、1.2×106 CFU/ml、1.2×105 CFU/ml、1.2×104
CFU/ml、1.2×103 CFU/ml, 120 CFU/ml, 12 CFU/ml, 1.2 CFU/ml clostridieum welchiis DNA, " 11 " represent sky
White control.
A kind of repetitive test of the specific detection agents box of the clostridium welchii disease of embodiment 6
1, experimental procedure
Carry out the DNA of 4 plants of different clostridieum welchiis using the specific detection agents box of the clostridium welchii disease of embodiment 3
3 specific PCR amplifications.
2, experimental result
As the result is shown the DNA of same strain clostridieum welchii 3 PCR carried out are amplified with the band of about 1225 bp, and band is all
Very clear, bright (Fig. 5).Wherein, " 1~3 ", " 4~6 ", " 7~9 ", " 10~12 " respectively represent 4 plants of different clostridieum welchiis
DNA, " 13 " represent blank control.
Embodiment 7 utilizes the detection clinical sample of the specific detection agents box of clostridium welchii disease
1, experimental procedure
It is carried out using the specific detection agents box of the clostridium welchii disease of embodiment 3 to the excrement sample from 11, Guangdong Province chicken house
This is detected, and detects 78 parts of fecal sample altogether.
2, experimental result
Wherein, figure A is 6 parts of chicken manure samples of Sihui, Guangdong chicken house, and " 1~6 " represents the DNA of chicken manure sample, " 7 " generation
The DNA of table clostridieum welchii, " 8 " represent blank control；Scheme 10 portions of chickens that B is Yunfu City Xinxing County town Le Zhu chicken house
Excrement sample, " 1 " represent the DNA of clostridieum welchii, and " 2~11 " represent the DNA of chicken manure sample, and " 12 " represent blank control；It is wide for scheming C
6 parts of chicken manure samples of the Guangzhou Dong Sheng chicken house, " 1~6 " represents the DNA of chicken manure sample, and " 7 " represent the DNA of clostridieum welchii,
" 8 " represent blank control；Scheme D be 8 chicken houses in Yunfu City Xinxing County 45 parts of chicken manure samples, " 1~3 ", " 4~9 ",
" 10~15 ", " 16~21 ", " 22~24 ", " 27~29 ", " 30~35 ", " 36~41 ", " 42~47 " respectively represent 8 chicken houses
Chicken manure sample DNA (wherein " 22~24 ", " 27~29 " be same chicken house chicken manure sample), " 25 ", " 48 " represent Wei Shi
The DNA of clostridium, " 26 ", " 49 " represent blank control.
Wherein test positive has 9 parts of samples (Fig. 6)；Positive sample is that clostridieum welchii is positive through sequencing, it was demonstrated that examination
The reliability of agent box detection.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>Agricultural University Of South China
<120>specific detection primer and detection kit of a kind of clostridium welchii disease
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
catcattcaa ccaaa 15
<210> 2
<211> 14
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ttccgctcac ggac 14
Claims (10)
1. the primer of a pair of detection chicken clostridium welchii disease, which is characterized in that the nucleotide sequence of the primer such as SEQ ID NO:1
Shown in~2.
2. application of the primer described in claim 1 in preparation detection chicken clostridium welchii disease kit.
3. a kind of kit of specific detection chicken clostridium welchii disease, which is characterized in that including primer described in claim 1.
4. kit according to claim 3, which is characterized in that the concentration of the primer is respectively 50 pmol/ μ L.
5. kit according to claim 3, which is characterized in that further include PCR reaction reagent.
6. kit according to claim 5, which is characterized in that the PCR reaction reagent is Premix Ex Taq
Version。
7. kit according to claim 3, which is characterized in that the PCR reaction system of the kit are as follows: sample DNA 2
12.5 μ L of μ L, Premix Ex Taq Version, each 0.5 μ L of primer described in claim 1, distilled water supply surplus, always
Volume is 25 μ L.
8. kit according to claim 3, which is characterized in that the PCR amplification condition of the kit are as follows: 94 DEG C 5
min；94 DEG C of 30 s, 52.3 DEG C of 30 s, 72 DEG C of 80 s, 25 circulations；72℃ 10 min.
9. kit according to claim 3, which is characterized in that the kit further includes sample DNA extracting solution and the positive
Reference substance.
10. kit according to claim 3, which is characterized in that the kit test object is chicken manure.
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| CN110760601A (en) * | 2019-12-11 | 2020-02-07 | 中国农业科学院兰州兽医研究所 | Primer group and kit for simultaneously detecting Brucella, Chlamydia abortus and Clostridium perfringens and application of primer group and kit |
| CN110760601B (en) * | 2019-12-11 | 2022-01-04 | 中国农业科学院兰州兽医研究所 | Primer group and kit for simultaneously detecting Brucella, Chlamydia abortus and Clostridium perfringens and application of primer group and kit |
| CN113151108A (en) * | 2021-05-18 | 2021-07-23 | 佛山市正典生物技术有限公司 | Separation method of clostridium perfringens |
| CN114517231A (en) * | 2022-03-03 | 2022-05-20 | 山东第一医科大学(山东省医学科学院) | Specific primer, detection method and kit for Eggerthella lenta |
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