CN107937573A - Quick detection bacillus rhusiopathiae suis and Streptococcus suis duplex PCR primer pair and kit and method - Google Patents
Quick detection bacillus rhusiopathiae suis and Streptococcus suis duplex PCR primer pair and kit and method Download PDFInfo
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Abstract
The invention discloses a kind of quick detection bacillus rhusiopathiae suis and Streptococcus suis duplex PCR primer pair and kit and method, which can quickly detect the bacillus rhusiopathiae suis and Streptococcus suis in sample by the PCR amplification to being detected sample DNA.Duplex PCR is on the basis of regular-PCR, two pairs of primers are added in the reaction system, the single infection and mixed infection of two kinds of bacteriums in clinical sample can be detected, therefore the duplex PCR detection kit and detection method of both pathogenic bacteria are established, the Molecule Epidemiology Investigation to fast and accurately identifying pathogenic bacteria in clinical sample and pathogenic bacteria is respectively provided with important meaning.
Description
Technical field
It is double in particular to a kind of quick detection bacillus rhusiopathiae suis and Streptococcus suis the present invention relates to PCR detection kit
PCR primer pair and kit and method.
Background technology
Bacillus rhusiopathiae suis can cause septicemia, polyarthirtis and endocarditis of pig etc., and people is easy to bacillus rhusiopathiae suis
Sense, shows as skin-type or septicemia, is known as " erysipeloid ".The national live pig report of infectious disease of the issue of the Ministry of Agriculture 2013 shows,
The morbidity quantity of brickpox case is far above swine fever, highly pathogenic PRRS, pork measles, anthrax, swine plague and cloth Lu Shi
Bacterium disease.Streptococcus suis is the main cause of disease of Chinese Pigs source streptococcosis.
Bacillus rhusiopathiae suis has 25 serotypes, and the predominant serotypes of China's prevalence are 1a and 2 types.Wang Lei etc. utilizes SpaA bases
Because designing PCR primer, can Rapid identification bacillus rhusiopathiae suis, and brickpox attenuated vaccine strain and other wild prevalences can be distinguished
Strain.Okwumabua O in 2003 detect pig hammer using gdh gene (gdh) the design primer of Streptococcus suis
Bacterium, in recent years, substantial amounts of clinical detection show that gdh genes are the specific genes of Streptococcus suis, exist only in Streptococcus suis
And it is widely present in all serotypes of Streptococcus suis.
Bacillus rhusiopathiae suis and Streptococcus suis and presence in the common PCR method detection sample of clinic, but repeatedly single PCR is anti-
Unnecessary waste should can be caused, while clinically there are a large amount of mixed infection cases, for bacillus rhusiopathiae suis and Streptococcus suis
Mixed infection sample, single PCR reactions are difficult to carry out comprehensive, quickly detection.Therefore, there is an urgent need to design bacillus rhusiopathiae suis
With the primer pair and kit and method of the detection of Streptococcus suis duplex PCR.
The content of the invention
The object of the present invention is to provide a kind of quick detection bacillus rhusiopathiae suis and Streptococcus suis duplex PCR primer pair and examination
Agent box and method.Its double PCR method produced on the basis of PCR, replaces single primer with two pairs of primers, can more accelerate
The purpose fragment of prompt amplification different bacterium, substantially reduces qualification time；It can be provided for the clinical detection of two kinds of pathogenic bacteria
More simple and efficient technological means, has great importance prevention and control bacillus rhusiopathiae suis and Streptococcus suis.
To achieve the above object, a kind of quick detection bacillus rhusiopathiae suis provided by the invention and Streptococcus suis duplex PCR draw
Thing pair, the duplex PCR primer pair are：
Present invention also offers a kind of bacillus rhusiopathiae suis and Streptococcus suis duplex PCR detection kit, the duplex PCR examination
Agent box includes primer mixed liquor, contains following primer pair in the primer mixed liquor：
Further, primer spaA-1F, spaA-1R, gdh-1F and gdh-1R concentration is in the primer mixed liquor
45~50 μM.
Yet further, the duplex PCR kit further includes 2 × PCR reaction buffers and ddH2O, wherein 2 × PCR
Mg containing archaeal dna polymerase EasyTaq, 2.5mM in reaction buffer2+Concentration magnesium chloride and dNTPs.
Present invention also offers a kind of detection method using above-mentioned duplex PCR detection kit, comprise the following steps：
1) measuring samples genomic DNA is extracted using boiling method,
2) PCR is carried out using mentioned reagent box, obtains PCR product, electrophoresis is completed after observation knot under gel imaging system
Fruit：
If fragment length is 770bp, it is bacillus rhusiopathiae suis to illustrate measuring samples；
If fragment length is 525bp, it is Streptococcus suis to illustrate measuring samples；
If observe there are two purpose fragments under gel imaging system, and two silver segment length are followed successively by from top to bottom
770bp and 525bp, then illustrate that measuring samples have bacillus rhusiopathiae suis and Streptococcus suis.
Preferably, in the step 1), boiling method extracting method：
1) measuring samples tissue is shredded into homogenate first and adds ddH2O is mixed and through Proteinase K digestion process,
2) ice bath immediately after boiling water bath again, it is measuring samples genomic DNA that supernatant, which is collected by centrifugation,.
Preferably, in the step 2),：
A.PCR reaction systems are：
B.PCR reaction conditions are：
(1) pre-degeneration：94 DEG C 2 minutes；
(2) circulate 35 times
Denaturation：94 DEG C 30 seconds,
Annealing：55 DEG C 30 seconds,
Extension：72 DEG C 1 minute；
(3) extension eventually：72 DEG C 2 minutes.
The beneficial effects of the present invention are：
The duplex PCR detection kit of the present invention can detect two kinds of bacteriums at the same time in same PCR reactions, can significantly save
Testing cost and save the time.Complicated test apparatus is not required in this method, it is not necessary to aseptic processing environment, boiling method extraction base
It is easy to operate because organizing.
Brief description of the drawings
Fig. 1 is the positive control and negative control figure of duplex PCR,
In figure, M is DNA Marker；Hole 1 is duplex PCR positive control；Hole 2,3 is respectively bacillus rhusiopathiae suis and pig chain
The respective positive control of coccus, hole 4 are negative control；
Fig. 2 is the specific test of duplex PCR detection kit,
In figure, M is DNA Marker, and hole 1 is positive control, and hole 2 is negative control, hole 3-11：Bacillus rhusiopathiae suis, pig chain
Coccus, Malian drainage, Escherichia coli, salmonella, haemophilus parasuis, pasteurella multocida, bronchus lose
Blood bordetella bacilli and Actinobacillus pleuropneumoniae；
Fig. 3 is the sensitivity tests of duplex PCR detection kit,
M is DNA Marker, and hole 1PCR products template is positive control；Hole 2PCR products template is negative control；3-
8PCR products template is bacillus rhusiopathiae suis, and amount of bacteria is respectively 2 × 105、2×104、2×103、2×102、2×10、2cfu；
Hole 9-14PCR products template is Streptococcus suis, and amount of bacteria is respectively 2 × 105、2×104、2×103、2×102、2×10、
2cfu。
Embodiment
In order to preferably explain the present invention, below in conjunction with the specific embodiment main contents that the present invention is furture elucidated, but
Present disclosure is not limited solely to following embodiments.
The preparation of 1 duplex PCR primer pair of embodiment and kit
Using 6 softwares of Primer Premier, to SpaA genes and gdh genes, (its sequence such as sequence is respectively SEQ respectively
ID No.1 and SEQ ID No.2) design PCR amplification primer, and entrust Wuhan Qing Ke bioengineering Co., Ltd synthetic primer.
DdH is used after primer synthesis2O is diluted to 50 μM, and primer sequence is as follows：
Each kit detects 100 samples, by following content assembling kit (whole operation requirement gnotobasis)：
1st, 2 × PCR reaction buffers (the Mg2+ concentration magnesium chloride containing archaeal dna polymerase EasyTaq, 2.5mM,
dNTPs)：1.25mL
2nd, primer mixed liquor (being 50 μM containing spaA-1F, spaA-1R, gdh-1F and gdh-1R concentration)：400μL
3rd, positive control solution：100μL
4、ddH2O：1mL
A.PCR reaction systems are：
B.PCR reaction conditions are：
(1) pre-degeneration：94 DEG C 2 minutes；
(2) circulate 35 times
Denaturation：94 DEG C 30 seconds,
Annealing：55 DEG C 30 seconds,
Extension：72 DEG C 1 minute；
(3) extension eventually：72 DEG C 2 minutes.
Embodiment 2：Bacillus rhusiopathiae suis and Streptococcus suis duplex PCR detection kit prepare and its detection of positive control
The bacillus rhusiopathiae suis SE10 and Streptococcus suis SC19 for taking separation to identify respectively, use boiling method two bacteriums of extraction
Genome；By following sequent synthesis 2 to primer, be diluted to 50 μM it is spare.
spaA-1F：5’-CAGCAATGCCACTACAAACAG-3’,
spaA-1R：5’-TACAACTTGAATTTGGCGATT-3’,
gdh-1F：5’-AAGCAGTCAAAGCCCGC-3’,
gdh-1R：5’-GGAGGCGTTTGTATTGACC-3’；
It is using the archaeal dna polymerase EasyTaq and its 10 supporting × Buffer, reaction system of Dalian treasured biotech firm：
4 μ l of primer mixed liquor (each 1 μ l containing spaA-1F, spaA-1R, gdh-1F, gdh-1R), 19.5 μ l of PCR buffer solutions (contain dNTPs
2 μ l, 10 × Buffer 2.5 μ l, ddH215 μ l of O), EasyTaq (Dalian treasured biotech firm) 0.5 μ l, 1 μ l of specimen dna.Make
Mixed with micropipettor pressure-vaccum, high speed brief centrifugation.
Reaction condition is：(1) pre-degeneration：94 DEG C 2 minutes, (2) circulate 35 times：Denaturation：94 DEG C 30 seconds；Annealing：56℃30
Second；Extension：72 DEG C 1 minute, (3) eventually extend：72 DEG C 2 minutes.PCR product recycles purpose bar through 10g/L agarose gel electrophoresis
Band.
Recovery product is inserted on carrier pMD18-T, and conversion enters Escherichia coli, uses the culture medium containing ampicillin
Cultivated, and send positive colony to Shanghai Sangon Biotech Company's sequencing identification.Sequencing result shows that purpose fragment has been correctly inserted into load
In body.Escherichia coli are the bacterium of the carrier containing purpose fragment.Use Shanghai Sangon Biotech Company's plasmid extraction kit extraction large intestine
The plasmid of bacillus, two kinds of recombinant plasmids press 1:1 mixing, uses ddH2It is in this detection kit that O, which is adjusted to 100ng/ μ l,
Positive control.
Embodiment 3：The detection of bacillus rhusiopathiae suis and Streptococcus suis duplex PCR detection kit to clinical sample
511 parts of hearts, lung, kidney,liver,spleen, brain and the joint sample gathered with this detection detection kit from slaughterhouse, detection
Step is as follows：
The extraction of specimen dna：The tissue of 10g lesions is homogenized, 5h is cultivated in 37 DEG C, through Proteinase K (25g/L) in 64-
65 DEG C of water-bath 2h.1ml digestion products are taken, 5min is heated in boiling water and is quickly placed into 5min in ice bath, 12000rpm centrifugation 2min,
It is sample DNA to take supernatant.
PCR amplification：PCR reactions are carried out by template of the pathological material of disease genome of extraction, while set positive control and feminine gender right
According to.PCR reaction systems and reaction condition are shown in embodiment 1.
Pcr amplification product is analyzed：After the completion of PCR reaction amplifications, appropriate bromophenol blue sample-loading buffer is added into product.Make
The GoldView dyestuffs that 0.005% is added with the Ago-Gel of electrophoresis buffer 10g/L, in gel are dyed.Make
Add 5 μ l amplified productions into the glue hole of each solidification with micropipettor, DNA Marker holes are set.It is electric under constant voltage
Swimming.Observe and take pictures using gel imaging system after the completion of electrophoresis, by measuring samples hole and positive control and negative control hole into
Row compares, so as to judge the positive and negative of two kinds of bacterium samples.
In 511 parts of clinical samples with the detection of this detection kit, it is the bacillus rhusiopathiae suis positive to have 37 parts；63 parts are pig chains
Coccus is positive；12 parts are that bacillus rhusiopathiae suis and Streptococcus suis are positive.It is all that 112 parts of positives are carried out with bacteria distribution identification
The positive, coincidence rate 100%, illustrates that this method has good sensitiveness.50 parts of samples are randomly selected from 399 parts of negative samples
Product carry out bacteria distribution identification, are not separated to bacillus rhusiopathiae suis and Streptococcus suis.
Embodiment 4：The specific test and sensitiveness of bacillus rhusiopathiae suis and Streptococcus suis duplex PCR detection kit try
Test.
Bacterial genomes extraction, PCR reaction systems and reaction condition are the same as embodiment 1 in the present embodiment.
Specific test：PCR testing inspections are carried out to bacillus rhusiopathiae suis and Streptococcus suis respectively using this detection kit,
Clearly purpose band is obtained, to Malian drainage, Escherichia coli, salmonella, haemophilus parasuis, killing property more
Pasteurella, bordetella branchiseptica and Actinobacillus pleuropneumoniae are detected, and are as a result showed no positive band.Explanation
This detection kit has good specificity (Fig. 2).
Sensitivity tests：The bacterium solution of known bacillus rhusiopathiae suis and Streptococcus suis bacterial population is used into bacterial genomes kit
Genome is extracted, the amount of bacteria using two kinds of genomic templates is respectively 2 × 105To 2 × 10cfu, PCR is carried out；
The result shows that：Using this detection kit detect bacillus rhusiopathiae suis and Streptococcus suis limit of identification be 2 ×
102cfu.Show that this detection kit has good sensitiveness (Fig. 3).
Other unspecified parts are the prior art.Although above-described embodiment is made that the present invention and retouches in detail
State, but it is only part of the embodiment of the present invention, rather than whole embodiments, people can also according to the present embodiment without
Other embodiment is obtained under the premise of creativeness, these embodiments belong to the scope of the present invention.
Sequence table
<110>Hua Zhong Agriculture University
<120>Quick detection bacillus rhusiopathiae suis and Streptococcus suis duplex PCR primer pair and kit and method
<160> 6
<170> SIPOSequenceListing 1.0
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<213>Bacillus rhusiopathiae suis (Erysipelothrix rhusiopathiae)
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atgaaaaaga aaaaacacct atttccgaaa gtaagtctta tgtcgtgctt acttttaaca 60
gcaatgccac tacaaacagc ttttgctgat tcgacagata tttctgtgat tccactaatc 120
ggtgaacaag ttggattgct cccagtttta cctgggacag gggtacatgc tcaggaatac 180
aacaaaatga ctgatgctta tattgaaaaa ttggtatctc taattaatca aaaagtgaag 240
ccgtttctta taaatgaacc aaaggggtac caaagtttcg aagcagtgaa tgaagagatt 300
aactcgattg taagtgaact taaaaatgaa ggaatgagtc ttcaaaacat tcaccatatg 360
tttaaacaaa gcatccaaaa cctagcaact agaatcggct acagaagttt tatgcaggat 420
gctatgtatc ttgaaaattt tgaaagatta acgattcctg aacttgatga agcatacgtt 480
gatttactcg tgaattacga ggtgaaacac cgtattttag taaaatatga aggtaaagtt 540
aaaggtagag ctcccttaga agcatttata gttcctctaa gagatagaat tcgtagtatg 600
aatgaaatgg ctgcagaagt aaattattta cctgaagcgc atgaggattt cttagtttca 660
gattcaagcg agtataatga caaactaaat aatatcaact ttgctttggg tctaggggtc 720
agcgagttta ttgactataa ccggctcgaa aatatgatgg aaaaagaaat tcatccactg 780
tatcttgaac tttatgctat gcggagaaat cgccaaattc aagttgtaag agatgtatat 840
ccaaacttgg aacgtgcgaa cgcggttgtt gaatccttaa agacaattaa agatataaaa 900
caaagaggga agaaactaca ggaacttctt gaaatttata tccaaagaag tggagatgtt 960
cgaaaaccag atgtactcca acgatttatt ggaaaatatc aatcagtagt tgatgaagaa 1020
aaaaataaac ttcaagatta tttagaatca gatatttttg attcatatag tgtggatggc 1080
gagaaaataa gaaataaaga aattacactc atcaatagag atgcatactt atctatgatt 1140
tacagagctc aatcgatttc ggaaattaag acgattcgtg cagatttaga atcacttgtc 1200
aaatcattcc aaaatgaaga aagtgactct aaagtagagc ctgaaagtcc cgttaaagta 1260
gaaaaaccag ttgatgaaga aaaacctaaa gatcaaaaga agctagttga tcaatcaaaa 1320
cccgaatcga attcaaaaga agggtggatt aagaaagata ataagtggtt ctatattgag 1380
aaatcaggtg gaatggcaac aggttggaag aaggtagcag acaaatggta ctacctcgat 1440
aatacgggtg ctatagttac gggttggaag aaggtagcaa acaaatggta ctatcttgaa 1500
aaatcaggtg cgatggcaac aggatggaag aaagtatcaa acaagtggta ctaccttgaa 1560
aactcaggtg caatggcaac aggatggaag aaagtatcaa acaagtggta ctaccttgaa 1620
aattcaggcg caatggctac aggatggaaa aaggtagcaa acaaatggta ctaccttgaa 1680
aactcaggtg cgatggcaac aggatggaag aaagtatcga acaagtggta ctaccttgaa 1740
aactcaggcg caatggctac aggatggaaa aaggtagcaa acaaatggta ctaccttgat 1800
aaatcaggaa tgatggttac aggttcaaaa tctattgatg gtaaaaagta tgcatttaag 1860
aacgatggaa gtttaaaata g 1881
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gaagcacacc cagaatacat cgaagaaaac atcttggctc gtatcgttga gcctgagcgt 180
atcatcagct tccgtgttcc atggacagat aaagatggaa atgttcaagt caaccgtggc 240
taccgtgttc agttcaactc agctgtaggt ccttataaag gcggtcttcg cttccaccca 300
actgtaaacc aatccatctt gaagttcctc ggttttgagc aaatcttcaa aaacgtcttg 360
actggtcttc caatcggcgg tggtaaaggt ggttcagact ttgatcctaa aggaaaaact 420
gatgctgaaa tcatgcgctt ctgccaaagc ttcatgactg aattgcaaaa acacatcgga 480
ccttcacttg acgtccctgc tggtgacatc ggtgtcggtg gtcgtgagat cggttacatg 540
tacggtcaat acaaacgcct ccgccagttt gatgcaggtg tcttgactgg taaacctctt 600
ggcttcggtg gttcattgat ccgcccagaa gcaactggtt acggtttggt ttacttcact 660
gataacatgt tggcagcaaa cggtaaatcc ttcaaagacc aaactgtcct tatctcaggt 720
tctggtaacg ttgcccaata tgctgttcaa aaagcgactg aacttggtgc aaaagttatt 780
tctgtttcag actcaaatgg ttacatcatt gacgaaactg gtatcgactt cgacctcttg 840
gtggacatca aagaaaaacg ccgcgctcgt ttgacagaat acgctgcaga aaaatcaact 900
gctaagtact tcaaaggttc tgtatggaac tacgatggca aggctgatat tgcccttcca 960
tgtgcgactc aaaatgagat caacggcaaa caagctgctg cccttgtaaa aaatggcgtg 1020
tactgtgtgg ctgaaggtgc caacatgcca tctgaccttg atgccatcaa agtctacaag 1080
gaaaatggcg ttctctacgg actcgcaaaa gctgccaacg ctggtggtgt agctgtatct 1140
gcccttgaaa tgagtcaaaa cagccttcgc ttgtcatgga ctcgtgaaga agtagacggc 1200
cgtcttaaag acatcatggc caacatcttc aacacagcca aagaaactgc tgaaaaatac 1260
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<213>Bacillus rhusiopathiae suis (Erysipelothrix rhusiopathiae)
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aagcagtcaa agcccgc 17
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ggaggcgttt gtattgacc 19
Claims (7)
1. a kind of quick detection bacillus rhusiopathiae suis and Streptococcus suis duplex PCR primer pair, it is characterised in that：The duplex PCR draws
Thing to for：
Bacillus rhusiopathiae suis primer pair：
spaA-1F：CAGCAATGCCACTACAAACAG,
spaA-1R：TACAACTTGAATTTGGCGATT；
Streptococcus suis primer pair：
gdh-1F：AAGCAGTCAAAGCCCGC,
gdh-1R：GGAGGCGTTTGTATTGACC.
2. a kind of bacillus rhusiopathiae suis and Streptococcus suis duplex PCR detection kit, it is characterised in that：The duplex PCR kit
Including primer mixed liquor, following primer pair is contained in the primer mixed liquor：
Bacillus rhusiopathiae suis primer pair：
spaA-1F：CAGCAATGCCACTACAAACAG,
spaA-1R：TACAACTTGAATTTGGCGATT；
Streptococcus suis primer pair：
gdh-1F：AAGCAGTCAAAGCCCGC,
gdh-1R：GGAGGCGTTTGTATTGACC.
3. bacillus rhusiopathiae suis and Streptococcus suis duplex PCR detection kit according to claim 2, it is characterised in that：It is described
Primer spaA-1F, spaA-1R, gdh-1F and gdh-1R concentration is 45~50 μM in primer mixed liquor.
4. bacillus rhusiopathiae suis and Streptococcus suis duplex PCR detection kit according to Claims 2 or 3, it is characterised in that：Institute
State duplex PCR kit and further include 2 × PCR reaction buffers and ddH2O, wherein gathering in 2 × PCR reaction buffers containing DNA
The Mg of synthase EasyTaq, 2.5mM2+Concentration magnesium chloride and dNTPs.
A kind of 5. detection method using duplex PCR detection kit described in claim 2, it is characterised in that：Including following step
Suddenly：
1) using boiling method extraction measuring samples genomic DNA；
2) PCR is carried out using mentioned reagent box, obtains PCR product, electrophoresis is completed after observing result under gel imaging system：
If fragment length is 770bp, it is bacillus rhusiopathiae suis to illustrate measuring samples；
If fragment length is 525bp, it is Streptococcus suis to illustrate measuring samples；
If observe there are two purpose fragments under gel imaging system, and from top to bottom two silver segment length be followed successively by 770bp and
525bp, then illustrate that measuring samples have bacillus rhusiopathiae suis and Streptococcus suis.
6. according to the method described in claim 5, it is characterized in that：In the step 1), boiling method extracting method：
1) measuring samples tissue is shredded into homogenate first and adds ddH2O is mixed and handled through protease K digesting,
2) ice bath immediately after boiling water bath again, it is measuring samples genomic DNA that supernatant, which is collected by centrifugation,.
7. according to the method described in claim 5, it is characterized in that：In the step 2)：
A.PCR reaction systems are：
B.PCR reaction conditions are：
(1) pre-degeneration：94 DEG C 2 minutes；
(2) circulate 35 times
Denaturation：94 DEG C 30 seconds,
Annealing：55 DEG C 30 seconds,
Extension：72 DEG C 1 minute；
(3) extension eventually：72 DEG C 2 minutes.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711071457.1A CN107937573A (en) | 2017-11-03 | 2017-11-03 | Quick detection bacillus rhusiopathiae suis and Streptococcus suis duplex PCR primer pair and kit and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN108913792A (en) * | 2018-08-03 | 2018-11-30 | 暨南大学 | Primer and probe and its kit and method based on digital pcr technology detection Streptococcus suis |
| CN110878337A (en) * | 2019-11-01 | 2020-03-13 | 拱北海关技术中心 | Detection method of erysipelothrix rhusiopathiae, primers and probe thereof |
| CN111763718A (en) * | 2020-07-09 | 2020-10-13 | 广东省农业科学院动物卫生研究所 | Nano PCR method for detecting streptococcus suis infection |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913792A (en) * | 2018-08-03 | 2018-11-30 | 暨南大学 | Primer and probe and its kit and method based on digital pcr technology detection Streptococcus suis |
| CN110878337A (en) * | 2019-11-01 | 2020-03-13 | 拱北海关技术中心 | Detection method of erysipelothrix rhusiopathiae, primers and probe thereof |
| CN111763718A (en) * | 2020-07-09 | 2020-10-13 | 广东省农业科学院动物卫生研究所 | Nano PCR method for detecting streptococcus suis infection |
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