CN102605046A - Kit and method for detecting streptococcus suis type 2 - Google Patents
Kit and method for detecting streptococcus suis type 2 Download PDFInfo
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Abstract
The invention provides a kit and a method for detecting streptococcus suis type 2. The kit comprises an upstream primer, a downstream primer and a probe; the 5' end of the upstream primer and/or 5' end of the downstream primer are/is modified by a biotin; the 5' end and/or the 3' end of the of the probe are/is modified by digoxin; the upstream primer and the downstream primer can amplify the DNA molecule D or the fragment of the DNA molecule D through polymerase chain reaction to obtain a double-stranded DNA amplification product; the base sequence of the DNA molecule D is shown as in the sequence table 1; and the probe can hybridize flexibly with a biotin modified single-stranded DNA in the double-stranded DNA amplification product. The kit and the method have the advantages of high sensitivity, high specificity and low detection cost.
Description
Technical field
The present invention relates to a kind of test kit and a kind of method that detects streptococcus suis 2-type.
Background technology
Streptococcus suis 2-type is claimed pod membrane 2 type swine streptococcus (Streptococcus suis type 2 again; SS2); The R crowd who belongs to the gram classification usually causing that weanling pig sacroiliitis, meningitis, septicemia and bronchitis are characteristic, and can cause zoonosis.Streptococcus suis 2-type extensively is present in the respiratory tract of normal pig usually, but bacterial bearing rate also can be up to 76% in the tonsilla of health pig.Streptococcus suis 2-type repeatedly breaks out in China in recent years, not only causes the massive losses of aquaculture, also infects and gives the people, has caused significant damage.
At present, it is widely-used that the PCR method detects streptococcus suis 2-type, and still, its susceptibility and specificity are still waiting to improve.
For example, CN 1896279A discloses the method that a kind of PCR method detects streptococcus suis 2-type, has wherein used 3 pairs of primers to carry out pcr amplification simultaneously, this method can only bacteria concentration be 450/have positive findings when ml is above.
Summary of the invention
In order to overcome the defective that is difficult to reach highly sensitive and high specific in the present streptococcus suis 2-type detection, the invention provides the detection method of a kind of test kit and a kind of streptococcus suis 2-type.
According to test kit provided by the present invention, this test kit comprises upstream primer, downstream primer and probe, and 5 ' end of said upstream primer and/or 5 ' end of said downstream primer are by biotin modification; 5 ' the end or the 3 ' end of said probe are modified by digoxin; Said upstream primer and said downstream primer can obtain the double-stranded DNA amplified production through the fragment of PCR amplification dna molecular D or dna molecular D, and the base sequence of dna molecular D is shown in sequence table 1; Said probe can with said double-stranded DNA amplified production in have the single stranded DNA hybridization of biotin modification.
According to the method for detection streptococcus suis 2-type provided by the present invention, this method may further comprise the steps:
(1) in the presence of dNTP, heat-stable DNA polymerase and PCR damping fluid, make upstream primer under the PCR reaction conditions, carry out first and contact with downstream primer and determined nucleic acid sample, obtain first the contact after product; Said upstream primer and said downstream primer can obtain amplified production through the fragment of PCR amplification dna molecular D or dna molecular D, and the base sequence of dna molecular D is shown in sequence table 1; 5 ' end of said upstream primer and/or 5 ' end of said downstream primer are by biotin modification;
(2) down, the product after contacting first carries out second with probe and contacts under hybridization conditions, obtains second the product after contacting at sealing nucleic acid; Said probe can with said double-stranded DNA amplified production in have the single stranded DNA hybridization of biotin modification; 5 ' the end and/or the 3 ' end of said probe are modified by digoxin;
(3) product after second contact successively can contacted with the 4th and contacting with being marked with horseradish peroxidase and can carrying out the 3rd with the albumen of digoxin specific combination with immobilised respectively an anti-action condition under with under the two anti-action conditions with the albumen of vitamin H specific combination, obtain the 4th product after contacting;
(4) product after the 4th contact is successively carried out the 5th with developer and terminator respectively under color condition and end condition and contact with the 6th and contact, obtain the 6th product after contacting;
(5) measure product after the 6th contact at the OD value at 490-494nm place, and judge that whether this OD value is greater than threshold value.
If OD value greater than threshold value, is then thought under this threshold value condition, contain streptococcus suis 2-type in the sample of acquisition determined nucleic acid sample.
At present, fluorescent quantitative PCR technique has been applied to the detection of streptococcus suis 2-type, and this method has not only been inherited conventional PCR method rapid amplifying, and makes specificity further improve, and can realize the rapid detection to sample.
But fluorescence quantifying PCR method need use special quantitative real time PCR Instrument, and the purchasing of quantitative real time PCR Instrument, operation and maintenance cost are all higher, have therefore limited promoting the use of of this method.
Test kit provided by the invention and streptococcus suis 2-type detection method; Can in 2.5 hours, accomplish detection; And obtain positive findings in the time of can bacteria concentration is merely 10/ml in sample, have, also have the specificity height, detect advantage with low cost than high sensitivity.
Description of drawings
Fig. 1 is the nucleic acid electrophoresis figure of the amplified production of Comparative Examples 1-6.
Embodiment
According to test kit provided by the present invention, this test kit comprises upstream primer A, downstream primer B and probe C, and the 5 ' end of said upstream primer A and/or the 5 ' end of said downstream primer B are by biotin modification; 5 ' end and/or the 3 ' end of said probe C are modified by digoxin; Said upstream primer A and said downstream primer B can obtain the double-stranded DNA amplified production through the fragment of PCR amplification dna molecular D or dna molecular D, and the base sequence of dna molecular D is shown in sequence table 1; Said probe C can with said double-stranded DNA amplified production in have the single stranded DNA hybridization of biotin modification.
Wherein, Sequence table 1 is capsular polysaccharide gene (the capsular polysaccharide gene of streptococcus suis 2-type; Cps2J) nucleotide coding sequence, therefore, dna molecular D one or more in can coding strand dna molecular, antisence strand dna molecule and the double chain DNA molecule of cps2J gene.
Wherein, The sequence of said upstream primer A and said downstream primer B and length is restriction especially not; Get final product so long as can obtain amplified production through the fragment of PCR amplification dna molecular D or dna molecular D; For example; Can from the sequence of the coding strand dna molecular of cps2J gene, select the sequence of one section sequence of 17-25 base length as upstream primer A, simultaneously can be from the sequence of the coding strand dna molecular of cps2J gene the downstream 100-500 base of this upstream primer A sequence select 17-25 base length the reverse complementary sequence of one section sequence as the sequence of downstream primer B.
Wherein, The sequence of said sequence E and length is restriction especially not; So long as the single stranded DNA hybridization that has biotin modification in probe C and the said double-stranded DNA amplified production is got final product; One section sequence for example can from the sequence of the coding strand dna molecular of cps2J gene, selecting 17-25 base length is as upstream primer A, simultaneously can be from the sequence of the coding strand dna molecular of cps2J gene the downstream 100-500 base of this upstream primer A sequence select 17-25 base length the reverse complementary sequence of one section sequence as downstream primer B; And between the reverse complementary sequence of the sequence of the sequence middle and upper reaches primer A of the coding strand dna molecular of cps2J gene and downstream primer B sequence, select the sequence of one section sequence of 17-25 base length as sequence E.
Need to prove that the sequence of the sequence of upstream primer A, downstream primer B and the sequence of sequence E can be identical with corresponding sequence in the double chain DNA molecule of cps2J gene, also can allow the mispairing of 1-3 base.
According to test kit of the present invention, wherein, under the preferable case; The sequence of said upstream primer A is 5 '-CAAACGCAAGGAATTACGGTATC-3 '; The sequence of said downstream primer B is 5 '-GAGTATCTAAAGAATGCCTATTG-3 ', and the sequence of said probe C is the reverse complementary sequence of sequence E or sequence E, and said sequence E is 5 '-AAGTAGCAAGTAACCCTCCCGACA-3 '; And when the sequence of probe C was sequence E, the 5 ' end of upstream primer A was by biotin modification; When the sequence of probe C was the reverse complementary sequence of sequence E, the 5 ' end of downstream primer B was by biotin modification, and the test kit under this preferable case can be obtained best detection effect.
Need to prove that the upstream primer A among the present invention, downstream primer B and probe C all can obtain through the mode that is purchased customization.
According to test kit provided by the present invention, wherein, use and guarantee the consistence of testing conditions for the convenience of the users, under the preferable case, said test kit also contains one or more in PCR reaction reagent, nucleic acid hybridization reagent and the ELISA reagent; Said PCR reaction reagent is selected from one or more in dNTP, heat-stable DNA polymerase and the PCR damping fluid; Said nucleic acid hybridization reagent comprises sealing nucleic acid; Said ELISA reagent be selected from immobilised can with the albumen of vitamin H specific combination, be marked with horseradish peroxidase and can with the albumen of digoxin specific combination, can with horseradish peroxidase carry out coupling reaction reagent, can stop in the reagent of horseradish peroxidase coupling reaction one or more.
According to test kit provided by the present invention, wherein, use, guarantee the consistence of testing conditions for the convenience of the users and reduce the production cost of said test kit that said heat-stable DNA polymerase is the rTaq-DNA polysaccharase; Said sealing nucleic acid is salmon sperm dna; Said immobilised can be immobilised streptavidin with the albumen of vitamin H specific combination; Saidly be marked with horseradish peroxidase and can be the antibody that is marked with the anti-digoxin of horseradish peroxidase with the albumen of digoxin specific combination; Said can be O-Phenylene Diamine with the reagent that horseradish peroxidase carries out coupling reaction; The said reagent that can stop the horseradish peroxidase coupling reaction is sulfuric acid.
According to the method for detection streptococcus suis 2-type provided by the present invention, these method following steps:
(1) in the presence of dNTP, heat-stable DNA polymerase and PCR damping fluid, make upstream primer A under the PCR reaction conditions, carry out first with the determined nucleic acid sample and contact with downstream primer B, obtain first the contact after product; Said upstream primer A and said downstream primer B can obtain amplified production through the fragment of PCR amplification dna molecular D or dna molecular D, and the base sequence of dna molecular D is shown in sequence table 1; 5 ' the end of said upstream primer A and/or the 5 ' end of said downstream primer B are by biotin modification;
(2) down, the product after contacting first carries out second with probe C and contacts under hybridization conditions, obtains second the product after contacting at sealing nucleic acid; Said probe C can with said double-stranded DNA amplified production in have the single stranded DNA hybridization of biotin modification; 5 ' end and/or the 3 ' end of said probe C are modified by digoxin;
(3) product after second contact successively can contacted with the 4th and contacting with being marked with horseradish peroxidase and can carrying out the 3rd with the albumen of digoxin specific combination with immobilised respectively an anti-action condition under with under the two anti-action conditions with the albumen of vitamin H specific combination, obtain the 4th product after contacting;
(4) product after the 4th contact is successively carried out the 5th with developer and terminator respectively under color condition and end condition and contact with the 6th and contact, obtain the 6th product after contacting;
(5) product after mensuration the 6th contact is at the OD value at 490-494nm place.
Recording under the situation of above-mentioned OD value, can judge that whether this OD value is greater than threshold value.
If OD value greater than threshold value, is then thought under this threshold value condition, contain streptococcus suis 2-type in the sample of acquisition determined nucleic acid sample.
Wherein, said upstream primer A and said downstream primer B can obtain amplified production through the fragment of PCR amplification dna molecular D or dna molecular D, and the base sequence of dna molecular D is shown in sequence table 1; Said probe C can carry out nucleic acid hybridization with said amplified production, it is characterized in that, wherein, the 5 ' end of said upstream primer A or the 5 ' end of said downstream primer B are by biotin modification; 5 ' end or the 3 ' end of said probe C are modified by digoxin.
Wherein, said heat-stable DNA polymerase has no particular limits, and gets final product so long as can be used for the polymerase chain reaction, for example can be commercially available various Taq-DNA polysaccharases.
Wherein, said PCR damping fluid has no particular limits, and gets final product so long as can be used for the polymerase chain reaction, for example can be commercially available various PCR damping fluids.
Wherein, In said first contact; The consumption of dNTP, heat-stable DNA polymerase, PCR damping fluid, water, upstream primer A, downstream primer B and determined nucleic acid sample is restriction especially not; Get final product so long as can be used for the polymerase chain reaction, for example can be the ratio described in " molecular cloning experiment guide " (Science Press published in 2003).
Wherein, said PCR reaction conditions has no particular limits, and gets final product so long as can be used for the polymerase chain reaction, for example can be the condition described in " molecular cloning experiment guide " (Science Press published in 2003); Said PCR reaction conditions comprises under the preferable case: 94 ℃ of preparatory sex change 4min, and circulation 25 is taken turns under the cycling condition of 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ of extension 50s then, and 72 ℃ are extended 10min then.
Need to prove; Among the present invention; Said determined nucleic acid sample refers to from the doubtful sample of nucleic acid that obtains the sample of streptococcus suis 2-type that contains of various needs detections; The preparation method of said determined nucleic acid sample does not have special requirement, uses known method to get final product, the sample of nucleic acid of respiratory secretions of the pig after for example will butchering with boiling method or the culture samples that tonsilla is organized cleaning piece preparation.
Method according to detection streptococcus suis 2-type provided by the present invention; Wherein, Sequence table 1 is capsular polysaccharide gene (the capsular polysaccharide gene of streptococcus suis 2-type; Cps2J) nucleotide coding sequence, therefore, dna molecular D one or more in can coding strand dna molecular, antisence strand dna molecule and the double chain DNA molecule of cps2J gene.
Method according to detection streptococcus suis 2-type provided by the present invention; Wherein, The sequence of said upstream primer A and said downstream primer B and length is restriction especially not; Get final product so long as can obtain amplified production through the fragment of PCR amplification dna molecular D or dna molecular D; For example; Can from the sequence of the coding strand dna molecular of cps2J gene, select the sequence of one section sequence of 17-25 base length as upstream primer A, simultaneously can be from the sequence of the coding strand dna molecular of cps2J gene the downstream 100-500 base of this upstream primer A sequence select 17-25 base length the reverse complementary sequence of one section sequence as the sequence of downstream primer B.
Method according to detection streptococcus suis 2-type provided by the present invention; Wherein, The sequence of said sequence E and length is restriction especially not; So long as can be that probe C and the hybridization of above-mentioned amplified production get final product; One section sequence for example can from the sequence of the coding strand dna molecular of cps2J gene, selecting 17-25 base length is as upstream primer A, simultaneously can be from the sequence of the coding strand dna molecular of cps2J gene the downstream 100-500 base of this upstream primer A sequence select 17-25 base length the reverse complementary sequence of one section sequence as downstream primer B; And between the reverse complementary sequence of the sequence of the sequence middle and upper reaches primer A of the coding strand dna molecular of cps2J gene and downstream primer B sequence, select the sequence of one section sequence of 17-25 base length as sequence E.
Need to prove that the sequence of the sequence of upstream primer A, downstream primer B and the sequence of sequence E can be identical with the sequence of respective segments in the cps2J gene, also can allow the mispairing of 1-3 base.
Method according to detection streptococcus suis 2-type provided by the present invention; Wherein, the sequence of said upstream primer A is 5 '-CAAACGCAAGGAATTACGGTATC-3 ', and the sequence of said downstream primer B is 5 '-GAGTATCTAAAGAATGCCTATTG-3 '; The sequence of said probe C is the reverse complementary sequence of sequence E or sequence E; Said sequence E is 5 '-AAGTAGCAAGTAACCCTCCCGACA-3 ', and when the sequence of probe C was sequence E, the 5 ' end of upstream primer A was by biotin modification; When the sequence of probe C was the reverse complementary sequence of sequence E, the 5 ' end of downstream primer B was by biotin modification.
Method according to detection streptococcus suis 2-type provided by the present invention; Wherein, in second contact, the consumption of probe C, sealing nucleic acid and said amplified production does not have special demands; Can be the consumption described in " molecular cloning experiment guide " (Science Press published in 2003); Under the preferable case, with respect to every micromolar probe C, the consumption of sealing nucleic acid is the 28-36 microgram; The consumption of amplified production is 20 microlitres, and amplified production and probe C are the 55-65 microlitre with the TV of the mixed material of sealing nucleic acid.
According to the method for detection streptococcus suis 2-type provided by the present invention, wherein, said hybridization conditions does not have special requirement, can be the condition described in " molecular cloning experiment guide " (Science Press published in 2003); Under the preferable case, said hybridization conditions comprises: said sealing nucleic acid is salmon sperm dna; Said hybridization conditions comprises to be kept amplified production and probe C 0.5-1.5 minute after keeping 1.5-2.5 minute under 93.5-94.5 ℃ with the mixed material of sealing nucleic acid under 54.5-55.5 ℃.
According to the method for detection streptococcus suis 2-type provided by the present invention, wherein, under the preferable case, with respect to the hybridization product of every microlitre, said immobilised can be the 1-2 microgram with the proteic consumption of vitamin H specific combination; Said one anti-action condition comprises: be 0.5-1.5 hour action time, and operative temperature is 20-40 ℃.
Wherein, immobilised can be for being coated on the Streptavidin in the microwell plate with the albumen of vitamin H specific combination.The said method that encapsulates can be method well known in the art.
According to the method for detection streptococcus suis 2-type provided by the present invention, wherein, under the preferable case,, saidly be marked with horseradish peroxidase and can be the 3-10 nanogram with the proteic consumption of digoxin specific combination with respect to the hybridization product of every microlitre; Said two anti-action conditions comprise: be 35-45 minute action time, and operative temperature is 20-40 ℃.
According to the method for detection streptococcus suis 2-type provided by the present invention, wherein, said color condition and end condition do not have special requirement, can carry out with reference to known enzyme linked immunological colour developing and terminated condition.
Need to prove that OD value described in the present invention is greater than threshold value, then think the judgement that contains streptococcus suis 2-type in the sample, can be under confidence level, obtains after the experimental result of known sample is calculated according to the statistical test method.Through method of the present invention 83 routine known samples being detected, is that calculating an available threshold value according to T inspection statistics method is 0.198 under 0.01 the situation in degree of confidence.
Below further illustrate the present invention through embodiment, but scope of the present invention is not limited in following examples.
Embodiment 1
To buy sequence be that 5 '-CAAACGCAAGGAATTACGGTATC-3 ' (seeing sequence table 2) and 5 ' end are that downstream primer B1 and the sequence of 5 '-GAGTATCTAAAGAATGCCTATTG-3 ' (seeing sequence table 3) is that 5 '-AAGTAGCAAGTAACCCTCCCGACA-3 ' (seeing sequence table 4) and 3 ' end are by the probe C1 of digoxin modification by the upstream primer A1 of biotin modification, sequence to the customization of precious biotech firm from Dalian.
Take by weighing Carnis Bovis seu Bubali cream 10g, Tryptones 20g, glucose 2g, sodium hydrogencarbonate 2g, sodium-chlor 2g, Sodium phosphate, dibasic 0.4g; Be dissolved in the 1000ml zero(ppm) water; 121 ℃ of autoclavings 15 minutes; When treating that substratum is chilled to 45-50 ℃, add many sticking plain E 10mg and nalidixic acid 15mg, obtain the THB substratum behind the mixing.
With the 0.5ml bacteria concentration be 10/ml contain the streptococcus suis 2-type reference strain (available from ATCC company; Article No. is 43765) the THB substratum as sample; In boiling water bath, placed 5 minutes, the abandoning supernatant after under the speed of 12000g centrifugal 2 minutes of the sample behind the boiling water bath obtained deposition, with the deposition that obtains with 150 μ l deionized water resuspensions after placement 10 minutes in boiling water bath again; Obtain once more the product behind the boiling water bath; Product behind the boiling water bath is once more placed centrifugal (12000g, 2 minutes) after 3 minutes on ice, draw the supernatant that obtains 100 μ l and be the determined nucleic acid sample.
With the above-mentioned determined nucleic acid sample of 2 μ l and the dNTP aqueous solution (dATP, dTTP, dCTP and the dGTP of 1.6 μ l; Total concn 100mM; Available from the precious biotech firm in Dalian; Article No. is BG4001A), 0.5 μ l heat-stable DNA polymerase (the rTaq-DNA polysaccharase, concentration 2.5U/ μ l is available from the precious biotech firm in Dalian; Article No. is CK5901AA), PCR damping fluid (10 times of liquid concentrators of 2 μ l; Available from the precious biotech firm in Dalian, article No. A6401A), the aqueous solution (the concentration 10ng/ μ l) mixing of the downstream primer B1 of the aqueous solution (concentration 10ng/ μ l) of the upstream primer A1 of the aseptic deionized water of 12.4 μ l, 0.5 μ l and 0.5 μ l is placed on the PCR appearance, carries out pcr amplification by following condition: keep 4min for 94 ℃; Then 94 ℃ keep cycling condition that 30s, 56 ℃ of annealing 30s, 72 ℃ extend 50s down circulation 25 take turns 72 ℃ of extension 10min then; Obtain amplified production 20 μ l.
With the aqueous solution (concentration 5 μ mol/ μ l) of the probe C1 of the above-mentioned amplified production of 10 μ l, 10 μ l and the aqueous solution (the concentration 3.2mg/ml of 10 μ l salmon sperm dnas; Available from the precious biotech firm in Dalian) mix post-heating to 94 and ℃ keep certain sex change time (5 minutes), be cooled to hybridization temperature (56 ℃) then rapidly and keep certain hybridization time (20 minutes); Obtain hybridizing product 30 μ l.
Obtain hybridizing product and all add in the hole (every hole package amount is 45 μ g) of the microwell plate that has encapsulated streptavidin above-mentioned, after the room temperature held one anti-action time (5 minutes), discard the liquid in the hole, with phosphate buffer soln (PBST solution, KH
2PO
41.4mM, Na
2HPO
44.3mM KCl 2.7mM, NaCl 137mM, tween 20 concentration is 0.05 volume %) washing micropore inwall 3 times (each consumption 200 μ l, each washing time 3 minutes), obtain the product after the anti-effect.
(solute of PBST and concentration are as stated for the PBST solution of adding 50 μ l horseradish peroxidase-labeled DigiTAbs in the product after above-mentioned one anti-effect; The concentration of horseradish peroxidase-labeled DigiTAb is 5ng/ μ l); After the room temperature held two anti-action times (40 minutes), discard the liquid in the hole, with 3 (each consumption 200 μ l of PBST solution washing micropore inwall; Each washing time 3 minutes), obtain product after the two anti-effects.
Add the developer (O-Phenylene Diamine is available from the precious biotech firm in Dalian) of 50 μ l in the product after above-mentioned two anti-effects, the room temperature held adds 50 μ l terminators (aqueous sulfuric acid of 2M), the product after obtaining behind the mixing developing the color after 10 minutes again.
The OD value at the 492nm place (OD value) that uses enzyme-linked immunosorbent assay instrument to measure the product after developing the color is 0.269; Contain streptococcus suis 2-type in the judgement sample thus, with sample for the 0.5ml bacteria concentration be 10/ml contain the true consistent of streptococcus suis 2-type reference strain.
Embodiment 2-.8
Detect according to embodiment 1 identical method, different is, and to use bacteria concentration according to table 1 be that to substitute bacteria concentration be the THB substratum that contains streptococcus suis 2-type of 10/ml for the corresponding substratum of the thalline that contains non-streptococcus suis 2-type of 10/ml.
The OD value that uses enzyme-linked immunosorbent assay instrument to measure the product after developing the color is listed in the table 1.
Table 1
| Bacterial classification | Substratum | The OD value | |
| Embodiment 1 | Streptococcus suis 2-type | ?THB | 0.269 |
| Embodiment 2 | Do not have | ?THB | 0.009 |
| Embodiment 3 | Swine streptococcus C1 | Nutrient broth * | 0.013 |
| |
Pneumococcus | Nutrient broth | 0.016 |
| Embodiment 5 | Staphylococcus | Nutrient broth | 0.025 |
| Embodiment 6 | Have a liking for the water Zymomonas mobilis | Nutrient broth | 0.012 |
| Embodiment 7 | Pasteurellosis bacillus | Nutrient broth | 0.011 |
| Embodiment 8 | Intestinal bacteria | Nutrient broth | 0.014 |
* nutrient broth medium encircles triumphant bio tech ltd (article No. 022011) available from Guangdong
Comparative Examples 1-6
0.5ml is contained the THB substratum of streptococcus suis 2-type reference strain, and (bacteria concentration is respectively 10
5Individual/ml, 10
4Individual/ml, 10
3Individual/ml, 10
2Individual/ml, 10/ml and 1/ml) as sample; In boiling water bath, placed 5 minutes, the abandoning supernatant after under the speed of 12000g centrifugal 2 minutes of the sample behind the boiling water bath obtained deposition, with the deposition that obtains with 150 μ l deionized water resuspensions after placement 10 minutes in boiling water bath again; Obtain once more the product behind the boiling water bath; Product behind the boiling water bath is once more placed centrifugal (12000g, 2 minutes) after 3 minutes on ice, draw the supernatant that obtains 100 μ l and be the determined nucleic acid sample.
With the above-mentioned determined nucleic acid sample of 2 μ l and the dNTP aqueous solution (dATP, dTTP, dCTP and the dGTP of 1.6 μ l; Total concn 100mM; Available from the precious biotech firm in Dalian; Article No. is identical with embodiment 1), 0.5 μ l heat-stable DNA polymerase (the rTaq-DNA polysaccharase, concentration is identical with embodiment 1, available from the precious biotech firm in Dalian; Article No. is identical with embodiment 1), PCR damping fluid (10 times of liquid concentrators of 2 μ l; Available from the precious biotech firm in Dalian, article No. is identical with embodiment 1), the aseptic deionized water of 12.4 μ l, 0.5 μ l the aqueous solution (the concentration 10ng/ μ l) mixing of downstream primer B1 of the aqueous solution (concentration 10ng/ μ l) and 0.5 μ l of upstream primer A1 be placed on the PCR appearance, carry out pcr amplification by following condition: keep 4min for 94 ℃; Then 94 ℃ keep cycling condition that 30s, 56 ℃ of annealing 30s, 72 ℃ extend 50s down circulation 25 take turns 72 ℃ of extension 10min then; Obtain each 20 μ l of amplified production respectively.
Above-mentioned amplified production is carried out electrophoresis detection, and the result sees Fig. 1, finds to work as bacteria concentration smaller or equal to 10
2Individual/as to be difficult to observe the purpose band during ml, promptly can't whether contain streptococcus suis 2-type in the judgement sample.
Embodiment 9
Precious biotech firm customization purchase sequence is that upstream primer A2, the sequence of 5 '-CGTATACTAATCTAGAGATTC-3 ' (seeing sequence table 5) are that 5 '-CTTATAAAGTTTGCAAC-3 ' (seeing sequence table 6) and 5 ' end are the probe C2 that 5 '-GTTTAAAAGAGAATGATAG-3 ' (seeing sequence table 7) and 5 ' end are modified by digoxin by the downstream primer B2 of biotin modification and sequence from Dalian.
Detect according to embodiment 1 identical method, different is to use upstream primer A2, downstream primer B2 and probe C2 to substitute upstream primer A1, downstream primer B1 and probe C1.
The OD value at the 492nm place (OD value) that uses enzyme-linked immunosorbent assay instrument to measure the product after developing the color is 0.232.
Embodiment 10
Precious biotech firm custom sequence is that upstream primer A3, the sequence of 5 '-GTTGACGGCAACATTGTTG-3 ' (seeing sequence table 8) are that 5 '-CTGTTCAGTGTCAAAACC-3 ' (seeing sequence table 9) and 5 ' end are the probe C3 that 5 '-GGAAATTATCAAGAATCTGAG-3 ' (seeing sequence table 10) and 3 ' end are modified by digoxin by the downstream primer B3 of biotin modification and sequence from Dalian.
Detect according to embodiment 1 identical method, different is to use upstream primer A3, downstream primer B3 and probe C3 to substitute upstream primer A1, downstream primer B1 and probe C1.
The OD value at the 492nm place (OD value) that uses enzyme-linked immunosorbent assay instrument to measure the product after developing the color is 0.221.
Embodiment 11-12
Detect according to embodiment 1 identical method, different is that the concentration of the salmon sperm dna aqueous solution is respectively 1.6mg/ml and 0.16mg/ml.
The OD value at the 492nm place (OD value) that uses enzyme-linked immunosorbent assay instrument to measure the product after developing the color is respectively 0.250 and 0.245.
Embodiment 13-17
Detect according to embodiment 1 identical method, different is that hybridization temperature is respectively 35 ℃, 40 ℃, 45 ℃, 50 ℃ and 60 ℃.
The OD value at the 492nm place (OD value) that uses enzyme-linked immunosorbent assay instrument to measure the product after developing the color is respectively 0.223,0.241,0.246,0.252,0.259.
Embodiment 18-21
Detect according to embodiment 1 identical method, different is that hybridization time was respectively 1 minute, 10 minutes, 15 minutes and 60 minutes.
The OD value at the 492nm place (OD value) that uses enzyme-linked immunosorbent assay instrument to measure the product after developing the color is respectively 0.215,0.241,0.255,0.240.
Embodiment 22-24
Detect according to embodiment 1 identical method, different is that the sex change time was respectively 30 seconds, 1 minute and 2 minutes.
The OD value at the 492nm place (OD value) that uses enzyme-linked immunosorbent assay instrument to measure the product after developing the color is respectively 0.215,0.234,0.247.
Embodiment 25-27
Detect according to embodiment 1 identical method, different is that the concentration of the aqueous solution of probe C1 is respectively 0.1 μ mol/ μ l, 0.5 μ mol/ μ l and 1 μ mol/ μ l.
The OD value at the 492nm place (OD value) that uses enzyme-linked immunosorbent assay instrument to measure the product after developing the color is respectively 0.225,0.236,0.243.
Embodiment 28-30
Detect according to embodiment 1 identical method, different is to be respectively an anti-action time 15 minutes, 30 minutes and 45 minutes.
The OD value at the 492nm place (OD value) that uses enzyme-linked immunosorbent assay instrument to measure the product after developing the color is respectively 0.247,0.235,0.229.
Embodiment 31-33
Detect according to embodiment 1 identical method, different is to be respectively for two anti-action times 10 minutes, 20 minutes and 80 minutes.
The OD value at the 492nm place (OD value) that uses enzyme-linked immunosorbent assay instrument to measure the product after developing the color is respectively 0.201,0.226,0.237.
Embodiment 34-38
Detect according to embodiment 1 identical method, different is that bacteria concentration is respectively 10
5Individual/ml, 10
4Individual/ml, 10
3Individual/ml, 10
2Individual/ml and 1/ml.
The OD value at the 492nm place (OD value) that uses enzyme-linked immunosorbent assay instrument to measure the product after developing the color is respectively 2.224,2.095,1.654,0.488,0.113.
Claims (10)
1. test kit, this test kit comprises upstream primer, downstream primer and probe, 5 ' end of said upstream primer and/or 5 ' end of said downstream primer are by biotin modification; 5 ' the end and/or the 3 ' end of said probe are modified by digoxin; Said upstream primer and said downstream primer can obtain the double-stranded DNA amplified production through the fragment of PCR amplification dna molecular D or dna molecular D, and the base sequence of dna molecular D is shown in sequence table 1; Said probe can with said double-stranded DNA amplified production in have the single stranded DNA hybridization of biotin modification.
2. test kit according to claim 1; Wherein, The sequence of said upstream primer is 5 '-CAAACGCAAGGAATTACGGTATC-3 '; The sequence of said downstream primer is 5 '-GAGTATCTAAAGAATGCCTATTG-3 ', and the sequence of said probe is the reverse complementary sequence of sequence E or sequence E, and said sequence E is 5 '-AAGTAGCAAGTAACCCTCCCGACA-3 '.
3. test kit according to claim 2, wherein, said test kit also contains one or more in PCR reaction reagent, nucleic acid hybridization reagent and the ELISA reagent; Said PCR reaction reagent is selected from one or more in dNTP, heat-stable DNA polymerase and the PCR damping fluid; Said nucleic acid hybridization reagent comprises sealing nucleic acid; Said ELISA reagent be selected from immobilised can with the albumen of vitamin H specific combination, be marked with horseradish peroxidase and can with the albumen of digoxin specific combination, can with horseradish peroxidase carry out coupling reaction reagent, can stop in the reagent of horseradish peroxidase coupling reaction one or more.
4. test kit according to claim 3, wherein, said heat-stable DNA polymerase is the rTaq-DNA polysaccharase; Said sealing nucleic acid is salmon sperm dna; Said immobilised can be immobilised streptavidin with the albumen of vitamin H specific combination; Saidly be marked with horseradish peroxidase and can be the antibody that is marked with the anti-digoxin of horseradish peroxidase with the albumen of digoxin specific combination; Said can be O-Phenylene Diamine with the reagent that horseradish peroxidase carries out coupling reaction; The said reagent that can stop the horseradish peroxidase coupling reaction is sulfuric acid.
5. method that detects streptococcus suis 2-type, this method may further comprise the steps:
(1) in the presence of dNTP, heat-stable DNA polymerase and PCR damping fluid, make upstream primer under the PCR reaction conditions, carry out first and contact with downstream primer and determined nucleic acid sample, obtain first the contact after product; Said upstream primer and said downstream primer can obtain amplified production through the fragment of PCR amplification dna molecular D or dna molecular D, and the base sequence of dna molecular D is shown in sequence table 1; 5 ' end of said upstream primer and/or 5 ' end of said downstream primer are by biotin modification;
(2) down, the product after contacting first carries out second with probe and contacts under hybridization conditions, obtains second the product after contacting at sealing nucleic acid; Said probe can with said double-stranded DNA amplified production in have the single stranded DNA hybridization of biotin modification; 5 ' the end and/or the 3 ' end of said probe are modified by digoxin;
(3) product after second contact successively can contacted with the 4th and contacting with being marked with horseradish peroxidase and can carrying out the 3rd with the albumen of digoxin specific combination with immobilised respectively an anti-action condition under with under the two anti-action conditions with the albumen of vitamin H specific combination, obtain the 4th product after contacting;
(4) product after the 4th contact is successively carried out the 5th with developer and terminator respectively under color condition and end condition and contact with the 6th and contact, obtain the 6th product after contacting;
(5) measure product after the 6th contact at the OD value at 490-494nm place, and judge that whether this OD value is greater than threshold value.
6. method according to claim 5; Wherein, The sequence of said upstream primer is 5 '-CAAACGCAAGGAATTACGGTATC-3 '; The sequence of said downstream primer is 5 '-GAGTATCTAAAGAATGCCTATTG-3 ', and the sequence of said probe is the reverse complementary sequence of sequence E or sequence E, and said sequence E is 5 '-AAGTAGCAAGTAACCCTCCCGACA-3 '.
7. method according to claim 5, wherein, with respect to every micromolar probe, the consumption of sealing nucleic acid is the 28-36 microgram, and the consumption of amplified production is 20 microlitres, and amplified production and probe are the 55-65 microlitre with the TV of the mixed material of sealing nucleic acid.
8. method according to claim 7, wherein, said sealing nucleic acid is salmon sperm dna; Said hybridization conditions comprises to be kept amplified production and probe 0.5-1.5 minute after keeping 1.5-2.5 minute under 93.5-94.5 ℃ with the mixed material of sealing nucleic acid under 54.5-55.5 ℃.
9. method according to claim 5, wherein, with respect to the hybridization product of every microlitre, said immobilised can be the 1-2 microgram with the proteic consumption of vitamin H specific combination; Said one anti-action condition comprises that be 4-6 minute action time, and operative temperature is 20-40 ℃.
10. method according to claim 5 wherein, with respect to the hybridization product of every microlitre, saidly is marked with horseradish peroxidase and can is the 3-10 nanogram with the proteic consumption of digoxin specific combination; Said two anti-action conditions comprise that be 35-45 minute action time, and operative temperature is 20-40 ℃.
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Cited By (4)
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| CN103323585A (en) * | 2013-06-06 | 2013-09-25 | 广东海大畜牧兽医研究院有限公司 | ELISA kit used for detecting fish streptococcus agalactiae IgM antibody, and preparation method thereof |
| CN106198952A (en) * | 2016-07-01 | 2016-12-07 | 清华大学 | A kind of suppress the nucleic acid molecules enclosure method to sensing interface non-specific adsorption |
| CN108315401A (en) * | 2018-04-26 | 2018-07-24 | 华南农业大学 | Triple PCR primer, method and kit for detecting streptococcus suis type 2, swine pasteurella multocida and haemophilus parasuis |
| CN108384833A (en) * | 2018-04-02 | 2018-08-10 | 李佳萌 | RPA methods, its primer special and the probe and purposes of a kind of 2 type Streptococcus suis of detection |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103323585A (en) * | 2013-06-06 | 2013-09-25 | 广东海大畜牧兽医研究院有限公司 | ELISA kit used for detecting fish streptococcus agalactiae IgM antibody, and preparation method thereof |
| CN106198952A (en) * | 2016-07-01 | 2016-12-07 | 清华大学 | A kind of suppress the nucleic acid molecules enclosure method to sensing interface non-specific adsorption |
| CN108384833A (en) * | 2018-04-02 | 2018-08-10 | 李佳萌 | RPA methods, its primer special and the probe and purposes of a kind of 2 type Streptococcus suis of detection |
| CN108315401A (en) * | 2018-04-26 | 2018-07-24 | 华南农业大学 | Triple PCR primer, method and kit for detecting streptococcus suis type 2, swine pasteurella multocida and haemophilus parasuis |
| CN108315401B (en) * | 2018-04-26 | 2021-08-27 | 华南农业大学 | Triple PCR primer, method and kit for detecting streptococcus suis type 2, swine pasteurella multocida and haemophilus parasuis |
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