CN102242195B - Multiple polymerase chain reaction (PCR) method for detecting extracellular protein factor (EF) and hemolysin (SLY) gene of streptococcus suis type 2 - Google Patents
Multiple polymerase chain reaction (PCR) method for detecting extracellular protein factor (EF) and hemolysin (SLY) gene of streptococcus suis type 2 Download PDFInfo
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Abstract
The invention relates to a multiple polymerase chain reaction (PCR) method for detecting an extracellular protein factor (EF) and a hemolysin (SLY) gene of streptococcus suis type 2. A pair of specific primers is respectively designed and synthesized according to the conserved sequences of the EF and the SLY gene of the streptococcus suis type 2, which are disclosed by GenBank, a multiple PCR system and reaction conditions are optimized, and the multiple PCR method for detecting the EF and the SLY gene of the streptococcus suis type 2 simultaneously is established. By the established multiple PCR method, the EF and the SLY gene of the streptococcus suis type 2 can be quickly authenticated and detected.
Description
Technical field
The present invention relates to a kind of multiple PCR method that detects simultaneously streptococcus suis 2-type extracellular protein factor and hemolysin gene, belong to the detection of nucleic acids field.
Technical background
Swine streptococcus (Streptococcus suis, SS) be worldwide distribution, perplex for many years the sound development of pig industry, pig acute infection swine streptococcus often shows as hueppe's disease and meningitis always, and chronic infection then shows as sacroiliitis, endocarditis, lymphoglandula suppuration.Swine streptococcus or important Zoonosis disease pathogen threaten human life security, and the people infects suis and suffers from toxic shock and meningitis.
Swine streptococcus serotype is numerous.According to Lan Shi (Lancefield) serological classification, suis can be divided into 19 serotypes such as A, B, C, D, E, F, G, H, K, L, M, N, O, P, Q, R, S, T, U, wherein the group streptococcus such as C, D, E, L, R, S can cause Streptococcus suis, as C group's streptococcus zooepidemicus (S.zooepidemicus) and streptococcus equisimilis (S.epuisimilis), D group's swine streptococcus (S.suis), R group's pig 2 type suis (S.suis subtype 2, SS2).The popular Streptococcus suis of China's eighties in 20th century shows as sacroiliitis and meningitis clinically, and the nineties is because C group streptococcus vaccine immunity is alleviated the state of an illness to some extent.Nineteen ninety finds to have in the swinery similar 2 type streptococcicosises first in China Guangdong Province, but has no people's infection morbidity.1998-999, Jiangsu Province and Zhejiang Province is twice outburst pig 2 type streptococcicosis successively, up to ten thousand pig morbidities, tens practitioners occur that meningitis, sacroiliitis and toxic hemorrhage shock are levied, the multi-organ function infringement, isolate the streptococcus suis 2-type bacterial strain from patient and sick pig, people source strain isolated and pig source strain isolated are the homology strain.In June, 2005, the ground such as Ziyang, Sichuan Province, inland river nearly 80 routine patients acuity SS2 infect, and wherein 12 routine patients with severe symptoms are dead.At present the popular Streptococcus suis of China is take SS2 as main.
There is complicated mechanism of causing a disease in SS2.The pathogenic power of SS2 bacterial strain is with whether to have virulence factor closely related.Have been found that SS2 produces multiple virulence factor, such as muramidase-released protein (MRP), extracellular protein factor (EF), hemolysin (SLY), capsular polysaccharide (CPS), fibronectin binding protein (FBPS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), IgG in conjunction with albumen (IBP), arginine deiminase (AD), dipeptidyl peptidase IV (DPP IV) etc.Wherein MRP, EF, SLY are three most important virulence factors.The SS2 of twice outburst of China is MRP
+EF
+SLY
+Phenotype.
The method of the detection SS2 virulence factor of report has PCR and multiple PCR method.Smith etc. utilize the gene order of pod membrane biosynthesis gene CPS to set up type specificity PCR authentication method for 1,2,9 type swine streptococcus.Ma Qingxia, what Kong Wang, Lu Cheng equality design and synthesize primer, set up and to have detected simultaneously CPS, the MRP of SS2, the multiple PCR method of EF, this method high specificity, susceptibility are high, can directly detect SS2 and can identify its virulence factor phenotype from clinical pathological material of disease.He Kongwang etc. detect MRP and the EF of SS2 with PCR method, susceptibility can reach 100 bacteriums.
EF, two kinds of virulence factors of SLY are secreted protein, be secreted into outside the born of the same parents, can be diffused into host's blood host's whole body, may be to the diffusion of SS2 self, to the damage of host's general, play a role with interaction aspect between other pathogenic micro-organism, be necessary to strengthen monitoring for molecular epidemiology and the heritable variation situation of these two virulence factors.
The present invention sets up multiple PCR method according to EF and the SLY gene order design primer of disclosed SS2, can detect simultaneously EF and the SLY gene of SS2 by a PCR reaction.
Summary of the invention
The invention provides a kind of multiple PCR method that detects simultaneously streptococcus suis 2-type extracellular protein factor and hemolysin gene, quick, the discriminating that are used for streptococcus suis 2-type extracellular protein factor and hemolysin gene detect.
By EF and the comparison of SLY gene order to disclosed SS2 among the Genbank, choose the EF of SS2 and the conserved sequence in the SLY gene order, with PremierPrimers 5.0 softwares and Oligo 6.0 software design primers.The EF upstream primer is: 5 '-GAAGAAGAACCCAAGGAACC-3 '; The EF downstream primer is: 5 '-ACATTCTGACCACTCGCATC-3 '; The EF clip size of the SS2 of amplification is 158bp; The SLY upstream primer is: 5 '-TTCCGATTTCGTATTCAACC-3 '; The SLY downstream primer is: 5 '-AACTGTTCTCCACCACTCCC-3 '; The SLY clip size of amplification is 338bp.Primer is given birth to worker's biotechnology company limited by Shanghai and is synthesized.
PCR reaction system of the present invention is the 25ul system, is sequentially added into following reagent:
Above operation is all carried out on ice, after all reagent add, and lid lid, low-speed centrifugal (1500g * 2min), put the enterprising performing PCR reaction of PCR instrument.
PCR reaction conditions of the present invention is:
95 ℃ of denaturation 5min
94 ℃ of sex change 1min, 55 ℃ of annealing 1min; 72 ℃ are extended 40s; 35 circulations of increasing
72 ℃ are extended 10min
4 ℃ of cooling forever
The electrophoresis detection condition is:
With 0.5 * tbe buffer liquid compound concentration be 2% sepharose, gel is put into electrophoresis chamber, add electrophoresis liquid, with the electrophoresis sample with sample is added in the well successively applied sample amount 6ul after the tetrabromophenol sulfonphthalein sample-loading buffer mixes in 5: 1 ratios.Open electrophoresis apparatus, 180V constant voltage electrophoresis makes nucleic acid samples to anodal swimming.After electrophoresis finishes gel put in the ultraviolet-visible light gel imaging instrument and observe electrophoresis result.
Step of the present invention comprises:
(1) operation on ice is sequentially added into a certain amount of H
2O, Buffer, Mg
2+, dNTP, primer, template, enzyme etc., lid lid, low-speed centrifugal.
(2) reaction system with above-mentioned 25ul is put on the PCR instrument, sets the PCR response procedures, carries out pcr amplification.
(3) agarose electrophoresis detects: with 0.5 * tbe buffer liquid compound concentration be 2% sepharose, gel is put into electrophoresis chamber, add electrophoresis liquid, with the electrophoresis sample with sample is added in the well successively applied sample amount 6-10ul after the tetrabromophenol sulfonphthalein sample-loading buffer mixes in 5: 1 ratios.Open electrophoresis apparatus, 180V constant voltage electrophoresis makes nucleic acid samples to anodal swimming.Observe electrophoresis result in the rearmounted ultraviolet-visible light gel imaging of the electrophoresis instrument.
(4) criterion: if electrophoresis detection occurs 158, the purpose band of 338bp, then be judged to be EF and the SLY gene test is all positive; If there is not the purpose band, then be judged to be feminine gender.
That the present invention has is highly sensitive, detection efficiency is high, can rapid detection go out EF, the SLY gene of streptococcus suis 2-type by a PCR reaction.Detection sensitivity of the present invention can reach 0.1ng/ul.Because the penetration and promotion of PCR instrument, that the present invention has advantages of is practical, testing cost is low.
Description of drawings
Accompanying drawing 1 multiplex PCR amplified production electrophoresis detection result.Purpose band molecular weight is respectively 338bp and 158bp, respectively corresponding SLY and EF gene fragment.Swimming lane 1 is DNA Marker, and swimming lane 2 is the multiplex PCR amplification, and swimming lane 3 is SLY gene PCR amplification, and swimming lane 4 is EF gene PCR amplification, swimming lane 5 negative contrasts.
The sensitivity of accompanying drawing 2 multiplex PCRs amplification.The template concentrations of swimming lane 1-7 is followed successively by 1,0.1,0.01,0.001,0.0001,0.00001,0.000001ng/ul, swimming lane 8 negative contrasts.Purpose band molecular weight in the swimming lane 1,2 is respectively 338bp and 158bp, respectively corresponding SLY and EF gene fragment.Amplified production electrophoresis detection result shows that the sensitivity of multiplex PCR detection SLY and EF gene is 0.1ng/ul.
Embodiment:
The design of embodiment 1 primer is with synthetic
According to EF, the SLY gene order of disclosed streptococcus suis 2-type among the GenBank, by the gene order comparison, choose conserved sequence, the GeneBank accession number of the EF that chooses, the open sequence of SLY is respectively DQ417121.1, DQ443530.1.Use Primer PremierS 5.0 and Oligo 6 software designed, designed EF, SLY gene PCR primer, primer is given birth to worker biotech company by Shanghai and is synthesized.
The EF upstream primer is: 5 '-GAAGAAGAACCCAAGGAACC-3 '; The EF downstream primer is: 5 '-ACATTCTGACCACTCGCATC-3 '.The EF clip size of amplification is 158bp.
The SLY upstream primer is: 5 '-TTCCGATTTCGTATTCAACC-3 '; The SLY downstream primer is: 5 '-AACTGTTCTCCACCACTCCC-3 '.The SLY clip size of amplification is 338bp.
The extraction of embodiment 2 DNA of bacteria
With streptococcus suis 2-type HA9801 strain inoculation sterilization Todd-Hewitt broth culture, 37 ℃ of incubated overnight, get bacterium liquid 1mL, the centrifugal 2min of 12000rpm, precipitate resuspendedly with the TE damping fluid (pH8.0) of 567 μ L, add the Proteinase K of the 20mg/mL of the 10%SDS of 30 μ L and 3 μ L, fully mixing, 37 ℃ of water-bath 1h, the NaCl that adds 100 μ L 5mol/L, fully mixing adds 80 μ LCTAB/NaCl solution, mixing again, 65 ℃ of water-bath 10min, add isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1) mixing, the centrifugal 10min of 12000rpm draws supernatant and adds isopyknic chloroform: primary isoamyl alcohol (24: 1) mixing, the centrifugal 10min of 12000rpm, draw supernatant, add the Virahol of 0.6 volume, put into-20 ℃ of 20min, DNA is fully precipitated, the centrifugal 10min of 12000rpm, precipitation is with 70% washing with alcohol 2 times, then drying at room temperature, make the ethanol volatilization, precipitation is dissolved with the TE (the Pancreatic RNase 20 μ g/mL that do not contain the DNA enzyme) of 25 μ LpH8.0.
Measure concentration and the purity of extracting dna solution.Adjust the concentration of DNA during Fluorescence PCR, draw an amount of DNA, make that template content is 50-100ng in the PCR reaction system of 25ul.
The foundation of embodiment 3PCR method
PCR reaction system of the present invention is the 25ul system, is sequentially added into following reagent:
Above operation is all carried out on ice, after all reagent add, and lid lid, low-speed centrifugal (1500g * 2min), put the enterprising performing PCR reaction of PCR instrument.
PCR reaction conditions of the present invention is:
95 ℃ of denaturation 5min
94 ℃ of sex change 1min, 55 ℃ of annealing 1min; 72 ℃ are extended 40s; 35 circulations of increasing
72 ℃ are extended 10min
4 ℃ of cooling forever
The electrophoresis detection condition is:
With 0.5 * tbe buffer liquid compound concentration be 2% sepharose, gel is put into electrophoresis chamber, add electrophoresis liquid, with the electrophoresis sample with sample is added in the well successively after the tetrabromophenol sulfonphthalein sample-loading buffer mixes in 5: 1 ratios.Applied sample amount 6ul.Open electrophoresis apparatus, 180V constant voltage electrophoresis makes nucleic acid samples to anodal swimming.Electrophoresis finishes to observe electrophoresis result in the rearmounted gel ultraviolet-visible light gel imaging instrument.
Criterion: if the purpose band of electrophoresis detection appearance 158,338bp then is judged to be EF and SLY gene PCR and detects all positive; If there is not the purpose band to occur, then is judged to be EF and SLY gene PCR and detects negative.Experiment need be established positive control and negative control.The results are shown in accompanying drawing 1.
Get the bacterium liquid 1mL of streptococcus suis 2-type HA9801 bacterial strain, centrifugal, get precipitation, extract DNA of bacteria.Ultraviolet spectrophotometer is measured purity and the concentration of DNA.With the sterilization distilled water DNA of bacteria is become 10 with 10 times of doubling dilutions
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, 10
-9, 10
-10The different concns gradient.Get each dilution DNA and make template, carry out pcr amplification.Behind the pcr amplification, the agarose gel electrophoresis with 2% detects the pcr amplification result, with the sensitivity of the detected minimum template concentrations of PCR method energy as the inventive method.
Detected result shows, the concentration of the DNA of bacteria of extraction is 2000ng/ul, and purity is fit to carry out PCR and detects.When template concentrations during greater than 0.1ng/ul, PCR detects the band that two clauses and subclauses appear in the product electrophoresis detection; When template concentrations during less than 0.1ng/ul, PCR detects the band that two clauses and subclauses do not appear in the product electrophoresis detection.The detection sensitivity of indicating the inventive method is 0.1ng/ul.The results are shown in accompanying drawing 2.
The bacterial strain list bacterium colonies such as difference picking intestinal bacteria, Salmonellas, streptococcus aureus, streptococcus equi epizootic disease subspecies, riemerella anatipestifer, shaking culture 24h in the LB liquid nutrient medium gets respectively 1ml bacterium liquid, and the centrifuging and taking thalline extracts DNA of bacteria.With the positive contrast of DNA of bacteria of streptococcus suis 2-type HA9801 strain, with the negative contrast of sterilization distilled water.The DNA of bacteria of above-mentioned each bacterial strain of PCR method amplification of setting up with the present invention, the specificity of checking the inventive method.
Experimental result shows the high specificity of the inventive method.When the DNA of the bacteriums such as pcr amplification intestinal bacteria, Salmonellas, streptococcus aureus, streptococcus equi epizootic disease subspecies, riemerella anatipestifer and distilled water, the electrophoresis detection of pcr amplification product does not all occur 158, the purpose band of 338bp; And the positive control pcr amplification rear electrophoresis of streptococcus suis 2-type detects 158, the purpose band of 338bp, and is consistent with the purpose stripe size of EF, SLY gene, indicate the present invention specifically PCR detect EF, the SLY gene of streptococcus suis 2-type.
Get 37 ℃ of three different streptococcus suis 2-type HA9801 strains of cultivating batch and cultivated bacterium liquid 1ml in 20 hours, extract DNA of bacteria.With the inventive method to the sample duplicate detection of each batch three times, the repeatability of checking present method.With the positive contrast of DNA of bacteria of streptococcus suis 2-type HA9801 strain, with the negative contrast of sterilization distilled water.
The repeatability detected result shows, the result of three batches sample duplicate detection is consistent, and the result that each sample duplicate detection is 3 times is also consistent, and the pcr amplification product electrophoresis detection all goes out to reveal the size amplified fragments consistent with the purpose band.The variation coefficient (CV%) that detects between batch is 1.50%, and the variation coefficient (CV%) that each sample duplicate detection is 3 times is 1.14%.The result shows that the inventive method has good repeatability, thereby has guaranteed to detect the comparability of data.
The EF of embodiment 7SS2 strain isolated, SLY gene test
Get the 19 strain SS2 isolates that our unit separates voluntarily between 2008-2010, preserve, single bacterium colony of each bacterial strain that the picking solid medium is preserved, 37 ℃ leave standstill cultivation 24 hours in the T-H meat soup, extract DNA of bacteria.Detect EF, the SLY gene of each strain isolated with the inventive method.With the positive contrast of the DNA of HA9801 strain, with the negative contrast of sterile purified water.
The multiplex PCR detected result shows, the multiplex PCR amplified production electrophoresis detection of the DNA of 19 strain SS2 strain isolateds and positive control all is positive, and negative control does not detect the purpose band, and the EF of 19 strain SS2 strain isolateds, SLY gene masculine rate are 100%.
Claims (3)
1. multiplex PCR detection reagent that is used for detecting simultaneously streptococcus suis 2-type extracellular protein factor EF and hemolysin gene SLY, it is characterized in that this reagent comprises streptococcus suis 2-type extracellular protein factor PCR primer and hemolysin gene PCR primer, Buffer, template, enzyme, wherein the upstream primer of extracellular protein factor gene EF be 5 '-GAAGAAGAACCCAAGGAACC-3 ', downstream primer is 5 '-ACATTCTGACCACTCGCATC-3 ', the EF clip size of amplification is 158bp; The upstream primer of hemolysin gene SLY is 5 '-TTCCGATTTCGTATTCAACC-3 ', downstream primer is 5 '-AACTGTTCTCCACCACTCCC-3 ', the SLY clip size of amplification is 338bp.
2. multiplex PCR detection reagent according to claim 1 is characterized in that the PCR reaction system is the 25ul system, is sequentially added into following reagent: Buffer 2.5ul, Mg
2+1.5ul, dNTP 1ul, each 0.5ul of EF upstream and downstream primer (10 *), each 0.5ul of SLY upstream and downstream primer (10 *), template 1ul, enzyme 0.25ul, aqua sterilisa complements to 25ul, aforesaid operations carries out on ice, after all reagent add, lid lid, low-speed centrifugal, centrifugal condition is 1500g * 2min, puts the enterprising performing PCR reaction of PCR instrument.
3. multiplex PCR detection reagent claimed in claim 1 is characterized in that the PCR reaction conditions is 95 ℃ of denaturation 5min, 94 ℃ of sex change 1min, 55 ℃ of annealing 1min; 72 ℃ are extended 40s; 35 circulations of increasing, 72 ℃ are extended 10min, 4 ℃ of cooling forever.
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