CN102323400A - Method for detecting and identifying variety of snake venom by utilizing adaptor technology - Google Patents
Method for detecting and identifying variety of snake venom by utilizing adaptor technology Download PDFInfo
- Publication number
- CN102323400A CN102323400A CN201110152346A CN201110152346A CN102323400A CN 102323400 A CN102323400 A CN 102323400A CN 201110152346 A CN201110152346 A CN 201110152346A CN 201110152346 A CN201110152346 A CN 201110152346A CN 102323400 A CN102323400 A CN 102323400A
- Authority
- CN
- China
- Prior art keywords
- stranded dna
- snake venom
- purifying
- dna
- screening
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention discloses a method for detecting and identifying the variety of snake venom by utilizing an adaptor technology, belonging to the field of biomedicine verification and relating to a method for detecting snake venom by utilizing the adaptor technology. Specific to the problems complex compositions of snake venom and poor specificity and sensitivity of the traditional snake bite detection method, the invention adopts a depletion SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology, and an adaptor with high-degree snake venom species specificity is obtained through screening and is converted into a report adaptor so as to establish a novel method for quickly detecting snake venom.
Description
Technical field
The invention belongs to biomedical check field, the snakebite that relates to domestic common poisonous snake is differentiated detection method, and the triage techniques that adaptive sub-technology is carried out the special adaptive son of snake venom kind is subdued in particularly a kind of utilization.
Background technology
The poisonous snake injury is a global public health problem, can cause respiratory paralysis, mortality hueppe's disease, nonreversibility renal failure and cause the local organization of permanent disability seriously downright bad.According to statistics China have known poisonous snake 4 sections 28 belong to 63 surplus kind, common poisonous snake comprises Chinese cobra, king cobra, banked krait, Bungarus multicinctus, agkistrodon acutus, circle spot adder, pallas pit viper, green bamboo snake, sea snake etc.Antivenin is the specific medicament of at present domestic treatment venomous snake bite commonly used, and the common antivenin of China comprises agkistrodon halys antivenin, agkistrodon acutus antivenin, bungarus multicinctus antivenin, naja antivenin etc. at present.But before using antivenin treatment snakebite, must select the antivenin of correct kind according to accident poisonous snake kind; Could reduce anaphylactoid risk; Improve toxin antagonism efficient and obtain desirable curative effect; Otherwise will cause the generation of severe allergic reactions such as serum sickness, miss the Best Times of treatment.China's snakebite clinical diagnosis at present mainly relies on medical history and clinical manifestation, lacks the method that the snake venom kind is differentiated in quick, special laboratory, often causes the blindness of treatment.Tracing it to its cause, is because the snake venom complicated component, and there are many homologous proteins in the nearer ophiotoxin of sibship, has a large amount of identical antigen sites, causes to seek the species specificity monoclonal antibody very difficulty that seems.And the adaptive sub-triage techniques that development in recent years is got up, can be from capacity up to 10
14 The few nucleic acid random library of strand in obtain the adaptive son that combines with the high special high-affinity of target through the screening of number wheel.
Summary of the invention
For solving above technical matters, the object of the present invention is to provide a kind of adaptive son of snake venom species specificity that utilizes to detect the method for differentiating snake venom.
The present invention seeks to realize like this:
A kind of method of utilizing adaptive sub-technology for detection discriminating snake venom kind, its key is to carry out as follows:
(1) make up earlier random single-stranded DNA banks and primer, single-stranded DNA banks through pcr amplification be double-stranded DNA,
Reach through asymmetric pcr againPurifying obtains single-stranded DNA banks;
(2) filter out the single stranded DNA that the single-stranded DNA banks of purifying combines with the target raw venin; Once more pcr amplification be double-stranded DNA,
Asymmetric pcr reachesPurifying obtains the single stranded DNA that combines with the target raw venin;
(3) single stranded DNA that step (2) is obtained instead sieves the single stranded DNA that is not combined with 4 ~ 8 kinds of snake venom that needs are differentiated again;
(4) intersect Cycle Screening according to step (2) and step (3), obtain combining with the echidnotoxin high-affinity single-strand DNA aptamer that do not combine simultaneously, pcr amplification with the discriminating snake venom be double-stranded DNA,
Asymmetric pcr reachesPurifying obtains the single-strand DNA aptamer storehouse;
(5) the compatibility detection is carried out in screening terminal cistern single-strand DNA aptamer storehouse and filtered out the adaptive son of target;
(6) the adaptive son that obtains is modified and is strengthened its stability, carries out mark with luciferin, biotin, radioactive isotope or collaurum then and transfers the adaptive son of report to
(7) adopt ELISA from snakebite victim blood, urine, wound tissue and secretion, to detect corresponding snake venom material, thereby judge the snakebite kind, carry out corresponding antivenin treatment.
Take turns above according to step (2) and step (3) intersection Cycle Screening 12 in the above-mentioned steps (4).
Step (1), (2) and (4) adopt asymmetric PCR method or biotin streptavidin magnesphere legal system to be equipped with single-stranded DNA banks.
Be separating medium with nitrocellulose filter, affine resin or microwell plate in step (2), (3) and (4) screening process.
A kind of method of utilizing adaptive sub-technology for detection discriminating snake venom kind, specifically carry out as follows:
One, the screening of adaptive son
1), make up random single chain DNA (ssDNA) library and primer: making up sequence length is single stranded DNA (ssDNA) library: the 5 '-GGGAGCTCAGAATAAACGCTCAA-N35-TTCGACATGAGGCCCGGATC-3 ' of 78 bases; Wherein N represents base A; G; C, among the T any one, the capacity in this library is about 10
14-10
15, make up upstream primer: 5 '-GGGAGCTCAGAATAAACGCTCAA-3 ', make up downstream primer: 5 '-TTCGACATGAGGCCCGGATC-3 '.Random single-stranded DNA banks and primer are synthetic by biotech firm.
2), single-stranded DNA banks double chain DNA library synthesis: with single-stranded DNA banks 0.1 μ g; The archaeal dna polymerase of upstream primer 100pmol, downstream primer 10pmol, 15mmol/l Mgcl2,0.2mmol/l dNTP, 1 * dna polymerase reaction damping fluid and 1 active unit; Add distilled water, making cumulative volume is 20 μ l; Put into the PCR appearance then, carry out 94 ℃ of reactions preparatory sex change in 5 minutes earlier, then by following condition circulation 18 times: 94 ℃ were reacted 30 seconds; 65 ℃ were reacted 30 seconds, and 72 ℃ were reacted 30 seconds, last 72 ℃ of reactions 5 minutes; Obtain the pcr amplification product in double-stranded DNA library, and with phenol chloroform method purifying and recovering.
3), the asymmetric PCR legal system is equipped with single-stranded DNA banks: the DNA that reclaims step 2) is masterplate 0.1 μ g; The archaeal dna polymerase of upstream primer 0.1pmol, downstream primer 10pmol, 7.5mmol/l MgCl2,0.2mmol/l dNTP, 1 * dna polymerase reaction damping fluid and 1 active unit; Add distilled water, making cumulative volume is 20 μ l; Put into the PCR appearance then; Carry out 94 ℃ of reactions preparatory sex change in 5 minutes earlier, then by following condition circulation 18 times: 94 ℃ were reacted 30 seconds, and 65 ℃ were reacted 30 seconds; 72 ℃ were reacted 30 seconds; Last 72 ℃ of reactions 5 minutes obtain the pcr amplification product of single-stranded DNA banks and with phenol chloroform method purifying and recovering, obtain the single-stranded DNA banks of purifying.
4), screening circulation:
1. 10 μ g target raw venins after will dialysing encapsulate on elisa plate with the carbonic acid buffer of pH value 9.6, and 37 ℃ act on 3 hours, establish the blank hole simultaneously.Sample aperture and control wells were sealed 2 hours with 3% BSA.The single-stranded DNA banks 1ng of purifying in the step 5) is acted on 40 minutes for 37 ℃ with the blank hole earlier in SELEX binding buffer liquid; Anti-sieve is removed the single stranded DNA that combines with BSA, transfers to echidnotoxin then and encapsulates the hole and combine 40 minutes for 37 ℃ with echidnotoxin, washs 6 times with SELEX dcq buffer liquid; Add the SELEX eluent again in 80 ℃ of effects 10 minutes; The ssDNA that wash-out combines with echidnotoxin down through phenol-chloroform extracting, precipitation with alcohol, obtains the single stranded DNA of purifying.According to step 2) ssDNA behind the purifying is carried out conventional PCR reaction amplification, then carry out the asymmetric pcr reaction according to step 3), phenol chloroform method purifying and recovering obtains the enrichment single stranded DNA with hangar, and this library is as the library of lower whorl screening.
2. 15 μ g after the PBS dialysis are differentiated that snake venom amalgam (needing the 4-8 kind snake venom potpourri of discriminating with it) encapsulates on elisa plate with the carbonic acid buffer of pH value 9.6,37 ℃ act on 3 hours, establish the blank hole simultaneously.Sample aperture and control wells were sealed 2 hours with 3% BSA.With step 1. in the single-stranded DNA banks 1ng of purifying transfer to echidnotoxin and encapsulate the hole and combines 40 minutes for 37 ℃ with echidnotoxin, wash 6 times the ssDNA that collection is washed, the single stranded DNA that purifying obtains with SELEX dcq buffer liquid.The single stranded DNA that obtains according to step 2) conventional PCR reaction amplification, then carry out the asymmetric pcr reaction according to step 3), phenol chloroform method purifying and recovering obtains the enrichment single stranded DNA with hangar, this library is as the library of lower whorl screening.
5), repeating step 4) 12 take turns: the screening of odd number wheel as the screening target, is collected the target snake venom combination wash-out ssDNA and is used for next circulation; Even numbers wheel screening use needs 4-8 kind snake venom amalgam conduct the subduing screening target of discriminating with it, collects not combine wash-out ssDNA to be used for next circulation.The concentration of the echidnotoxin that encapsulates constantly reduces along with the increase of screening wheel number with the single stranded DNA concentration that is used to combine; When the 12nd takes turns; The concentration of coating protein reaches every hole 0.05 μ g; The concentration of corresponding combination single stranded DNA is every hole 0.02ng, obtains the single-strand DNA aptamer storehouse that can combine the while not combine with the discriminating snake venom with the echidnotoxin high-affinity.
6), with the final high-affinity single-strand DNA aptamer storehouse that obtains of step 5) according to step 2) amplification, obtain the pcr amplification product in double-stranded DNA library.With 2% Ago-Gel that contains concentration 0.5 μ g/ml ethidium bromide; The pcr amplification product in double-stranded DNA library is carried out electrophoresis; Behind the electrophoresis Ago-Gel is seated on the fluoroscopic examination plate; The pcr amplification product that will be the double-stranded DNA library of Chinese red band downcuts, and with the DNA purifying and recovering kit purifying that TaKaRa company provides, obtains the double-stranded DNA of purifying.
7), with the double-stranded DNA that step 6) obtains, the pMD18-T Simple Vector kit that provides with TaKaRa company is connected on the pMD18-T carrier, is transformed into escherichia coli DH5a, ammonia benzyl resistance screening, the single growth bacterium colony of picking checks order.
8) the adaptive son of the pairing DNA of each single growth bacterium colony is used biotin labeling, amount is 0.1 μ g, the echidnotoxin that encapsulates with every hole 10 μ g in SELEX binding buffer liquid 37 ℃ combine 40 minutes; With SELEX dcq buffer liquid washing 6 times, add the horseradish peroxidase mark Streptavidin of 1:1000,37 ℃; Act on 30 minutes, with PBST damping fluid washing 4 times, flush away not with echidnotoxin on the enzyme that combines of DNA biotin mark streptavidin; Add the effect in 20 minutes of tetramethyl benzidine color development at room temperature then; Add the reaction of 2M concentrated sulphuric acid color development stopping, the 450nm enzyme joins appearance and measures the OD value, chooses the highest adaptive son of DNA of OD value; This adaptive son is the adaptive son of DNA that can combine with echidnotoxin, should adaptive son order-checking obtain sequence.
Two, the foundation of detection method
To modify by the adaptive son that said process obtains and strengthen its stability; Carry out mark with luciferin, biotin, radioactive isotope or collaurum then and transfer the adaptive son of report to; Just can adopt ELISA from snakebite victim blood, urine, wound tissue and secretion, to detect corresponding snake venom material; Thereby judge the snakebite kind, carry out corresponding antivenin treatment.
Beneficial effect: the snake venom that the present invention is directed to complicated component; Adaptive sub-triage techniques (depletion SELEX) is subdued in employing; Screening obtains to have the adaptive son of height snake venom species specificity, and transfers adaptive son to report adaptive son and set up snake venom fast detecting new method.The snakebite detection specificity is strong good etc. with susceptibility, and detection is rapid, after snake bite, can treat targetedly at once.
Embodiment
Embodiment 1:
A kind of NNAV discrimination method based on adaptive son technology, carry out as follows:
1), the screening of the adaptive son of NNAV species specificity; Make up random single chain DNA (ssDNA) library and primer; Single-stranded DNA banks is passed through the PCR double chain DNA library synthesis; Utilize asymmetric PCR method DNA amplification library again, pcr amplification product obtains the single-stranded DNA banks of large-scale purification with phenol chloroform method purifying and recovering.
2), Chinese cobra snake venom screening circulation: with encapsulating on elisa plate after the NNAV dialysis, 37 ℃ act on 3 hours, establish the blank hole simultaneously.Sample aperture and control wells were sealed 2 hours with 3% BSA.The single-stranded DNA banks 1ng of purifying is acted on 40 minutes for 37 ℃ with the blank hole earlier in SELEX binding buffer liquid; Anti-sieve is removed the single stranded DNA that combines with BSA; Transferring to the Chinese cobra echidnotoxin then encapsulates the hole and combines 40 minutes for 37 ℃ with the Chinese cobra echidnotoxin; With SELEX dcq buffer liquid washing 6 times, add the SELEX eluent again in 80 ℃ of effects 10 minutes, the ssDNA that wash-out combines with the Chinese cobra echidnotoxin down; Through phenol-chloroform extracting, precipitation with alcohol, obtain the single stranded DNA of purifying.SsDNA behind the purifying is carried out conventional PCR reaction amplification, then carry out the asymmetric pcr reaction, phenol chloroform method purifying and recovering obtains the enrichment single stranded DNA with hangar, and this library is as the library of lower whorl screening.
3), differentiating that snake venom is counter sieves circulation: will differentiate that snake venom amalgam (Ahylysantinfarctase, Jiangsu and Zhejiang Provinces cobra-venom, solder horn snake venom) encapsulates on elisa plate with the carbonic acid buffer of pH value 9.6,37 ℃ act on 3 hours, establish the blank hole simultaneously.Sample aperture and control wells were sealed 2 hours with 3% BSA.With step 2) in the single-stranded DNA banks 1ng of purifying transfer to the snake venom amalgam and encapsulate the hole and mix echidnotoxin and combines 40 minutes for 37 ℃, wash 6 times the ssDNA that collection is washed, the single stranded DNA that purifying obtains with SELEX dcq buffer liquid.Carrying out the single stranded DNA that obtains conventional PCR reaction amplification, then carry out the asymmetric pcr reaction, phenol chloroform method purifying and recovering obtains the enrichment single stranded DNA with hangar, and this library is as the library of lower whorl screening.
4), repeating step 2)-3) 12 take turns: the screening of odd number wheel as the screening target, is collected NNAV combination wash-out ssDNA and is used for next circulation; Even numbers wheel screening use needs snake venom amalgam (Ahylysantinfarctase, Jiangsu and Zhejiang Provinces cobra-venom, solder horn snake venom) conduct the subduing screening target of discriminating with it, collects not combine wash-out ssDNA to be used for next circulation.The concentration of the echidnotoxin that encapsulates constantly reduces along with the increase of screening wheel number with the single stranded DNA concentration that is used to combine; When the 12nd takes turns; The concentration of coating protein reaches every hole 0.05 μ g; The concentration of corresponding combination single stranded DNA is every hole 0.02ng, obtains the single-strand DNA aptamer storehouse that can combine the while not combine with discriminating snake venom (Ahylysantinfarctase, Jiangsu and Zhejiang Provinces cobra-venom, solder horn snake venom) with Chinese cobra echidnotoxin high-affinity.
5) pcr amplification is carried out, the pcr amplification product in acquisition double-stranded DNA library in the high-affinity single-strand DNA aptamer storehouse that, step 4) is obtained.With 2% Ago-Gel that contains concentration 0.5 μ g/ml ethidium bromide; The pcr amplification product in double-stranded DNA library is carried out electrophoresis; Behind the electrophoresis Ago-Gel is seated on the fluoroscopic examination plate; The pcr amplification product that will be the double-stranded DNA library of Chinese red band downcuts, and with the DNA purifying and recovering kit purifying that TaKaRa company provides, obtains the double-stranded DNA of purifying.
6), with the double-stranded DNA that step 5) obtains, the pMD18-T Simple Vector kit that provides with TaKaRa company is connected on the pMD18-T carrier, is transformed into escherichia coli DH5a, ammonia benzyl resistance screening, the single growth bacterium colony of picking checks order.
7), the adaptive son of the pairing DNA of each single growth bacterium colony is used biotin labeling, amount is 0.1 μ g, the Chinese cobra echidnotoxin that encapsulates with every hole 10 μ g in SELEX binding buffer liquid 37 ℃ combine 40 minutes; With SELEX dcq buffer liquid washing 6 times, add the horseradish peroxidase mark Streptavidin of 1:1000,37 ℃; Act on 30 minutes, with PBST damping fluid washing 4 times, flush away not with the Chinese cobra echidnotoxin on the enzyme that combines of DNA biotin mark streptavidin; Add the effect in 20 minutes of tetramethyl benzidine color development at room temperature then; Add the reaction of 2M concentrated sulphuric acid color development stopping, the 450nm enzyme joins appearance and measures the OD value, chooses the highest adaptive son of DNA of OD value; This adaptive son is the adaptive son of DNA that can combine with the Chinese cobra echidnotoxin, should adaptive son order-checking obtain sequence.
8), will modify by the adaptive son that said process obtains and strengthen its stability, carry out mark with luciferin, biotin, radioactive isotope or collaurum then and transfer to and report adaptive son,
9), from snakebite victim blood, urine, wound tissue and secretion, detect corresponding snake venom material, thereby judge the snakebite kind, carry out corresponding antivenin treatment.
Embodiment 2:
A kind of Ahylysantinfarctase discrimination method based on adaptive son technology, carry out as follows:
1), the screening of the adaptive son of Ahylysantinfarctase species specificity; Make up random single chain DNA (ssDNA) library and primer; Single-stranded DNA banks is passed through the PCR double chain DNA library synthesis; Utilize asymmetric PCR method DNA amplification library again, pcr amplification product obtains the single-stranded DNA banks of large-scale purification with phenol chloroform method purifying and recovering.
2), agkistrodon acutus snake venom screening circulation: with encapsulating on elisa plate after the Ahylysantinfarctase dialysis, 37 ℃ act on 3 hours, establish the blank hole simultaneously.Sample aperture and control wells were sealed 2 hours with 3% BSA.The single-stranded DNA banks 1ng of purifying is acted on 40 minutes for 37 ℃ with the blank hole earlier in SELEX binding buffer liquid; Anti-sieve is removed the single stranded DNA that combines with BSA; Transferring to the agkistrodon acutus echidnotoxin then encapsulates the hole and combines 40 minutes for 37 ℃ with the agkistrodon acutus echidnotoxin; With SELEX dcq buffer liquid washing 6 times, add the SELEX eluent again in 80 ℃ of effects 10 minutes, the ssDNA that wash-out combines with the agkistrodon acutus echidnotoxin down; Through phenol-chloroform extracting, precipitation with alcohol, obtain the single stranded DNA of purifying.SsDNA behind the purifying is carried out conventional PCR reaction amplification, then carry out the asymmetric pcr reaction, phenol chloroform method purifying and recovering obtains the enrichment single stranded DNA with hangar, and this library is as the library of lower whorl screening.
3), differentiating that snake venom is counter sieves circulation: will differentiate that snake venom amalgam (Chinese cobra, Jiangsu and Zhejiang Provinces cobra-venom, solder horn snake venom) encapsulates on elisa plate with the carbonic acid buffer of pH value 9.6,37 ℃ act on 3 hours, establish the blank hole simultaneously.Sample aperture and control wells were sealed 2 hours with 3% BSA.With step 2) in the single-stranded DNA banks 1ng of purifying transfer to the snake venom amalgam and encapsulate the hole and mix echidnotoxin and combines 40 minutes for 37 ℃, wash 6 times the ssDNA that collection is washed, the single stranded DNA that purifying obtains with SELEX dcq buffer liquid.Carrying out the single stranded DNA that obtains conventional PCR reaction amplification, then carry out the asymmetric pcr reaction, phenol chloroform method purifying and recovering obtains the enrichment single stranded DNA with hangar, and this library is as the library of lower whorl screening.
4), repeating step 2)-3) 12 take turns: the screening of odd number wheel as the screening target, is collected Ahylysantinfarctase combination wash-out ssDNA and is used for next circulation; Even numbers wheel screening use needs snake venom amalgam (NNAV, Jiangsu and Zhejiang Provinces cobra-venom, solder horn snake venom) conduct the subduing screening target of discriminating with it, collects not combine wash-out ssDNA to be used for next circulation.The concentration of the echidnotoxin that encapsulates constantly reduces along with the increase of screening wheel number with the single stranded DNA concentration that is used to combine; When the 12nd takes turns; The concentration of coating protein reaches every hole 0.05 μ g; The concentration of corresponding combination single stranded DNA is every hole 0.02ng, obtains the single-strand DNA aptamer storehouse that can combine the while not combine with discriminating snake venom (NNAV, Jiangsu and Zhejiang Provinces cobra-venom, solder horn snake venom) with agkistrodon acutus echidnotoxin high-affinity.
5) pcr amplification is carried out, the pcr amplification product in acquisition double-stranded DNA library in the high-affinity single-strand DNA aptamer storehouse that, step 4) is obtained.With 2% Ago-Gel that contains concentration 0.5 μ g/ml ethidium bromide; The pcr amplification product in double-stranded DNA library is carried out electrophoresis; Behind the electrophoresis Ago-Gel is seated on the fluoroscopic examination plate; The pcr amplification product that will be the double-stranded DNA library of Chinese red band downcuts, and with the DNA purifying and recovering kit purifying that TaKaRa company provides, obtains the double-stranded DNA of purifying.
6), with the double-stranded DNA that step 5) obtains, the pMD18-T Simple Vector kit that provides with TaKaRa company is connected on the pMD18-T carrier, is transformed into escherichia coli DH5a, ammonia benzyl resistance screening, the single growth bacterium colony of picking checks order.
7), the adaptive son of the pairing DNA of each single growth bacterium colony is used biotin labeling, amount is 0.1 μ g, the agkistrodon acutus echidnotoxin that encapsulates with every hole 10 μ g in SELEX binding buffer liquid 37 ℃ combine 40 minutes; With SELEX dcq buffer liquid washing 6 times, add the horseradish peroxidase mark Streptavidin of 1:1000,37 ℃; Act on 30 minutes, with PBST damping fluid washing 4 times, flush away not with the agkistrodon acutus echidnotoxin on the enzyme that combines of DNA biotin mark streptavidin; Add the effect in 20 minutes of tetramethyl benzidine color development at room temperature then; Add the reaction of 2M concentrated sulphuric acid color development stopping, the 450nm enzyme joins appearance and measures the OD value, chooses the highest adaptive son of DNA of OD value; This adaptive son is the adaptive son of DNA that can combine with the agkistrodon acutus echidnotoxin, should adaptive son order-checking obtain sequence.
8), will modify by the adaptive son that said process obtains and strengthen its stability, carry out mark with luciferin, biotin, radioactive isotope or collaurum then and transfer to and report adaptive son,
9), from snakebite victim blood, urine, wound tissue and secretion, detect corresponding snake venom material, thereby judge the snakebite kind, carry out corresponding antivenin treatment.
Embodiment 3:
A kind of Jiangsu and Zhejiang Provinces cobra-venom discrimination method based on adaptive son technology, carry out as follows:
1), the screening of the adaptive son of Jiangsu and Zhejiang Provinces cobra-venom species specificity; Make up random single chain DNA (ssDNA) library and primer; Single-stranded DNA banks is passed through the PCR double chain DNA library synthesis; Utilize asymmetric PCR method DNA amplification library again, pcr amplification product obtains the single-stranded DNA banks of large-scale purification with phenol chloroform method purifying and recovering.
2), Jiangsu and Zhejiang Provinces viper venom screening circulation: with encapsulating on elisa plate after the dialysis of Jiangsu and Zhejiang Provinces cobra-venom, 37 ℃ act on 3 hours, establish the blank hole simultaneously.Sample aperture and control wells were sealed 2 hours with 3% BSA.The single-stranded DNA banks 1ng of purifying is acted on 40 minutes for 37 ℃ with the blank hole earlier in SELEX binding buffer liquid; Anti-sieve is removed the single stranded DNA that combines with BSA; Transferring to Jiangsu and Zhejiang Provinces viper venom albumen then encapsulates hole and Jiangsu and Zhejiang Provinces viper venom protein 37 and ℃ combines 40 minutes; With SELEX dcq buffer liquid washing 6 times, add the SELEX eluent again in 80 ℃ of effects 10 minutes, the following and protein bound ssDNA of Jiangsu and Zhejiang Provinces viper venom of wash-out; Through phenol-chloroform extracting, precipitation with alcohol, obtain the single stranded DNA of purifying.SsDNA behind the purifying is carried out conventional PCR reaction amplification, then carry out the asymmetric pcr reaction, phenol chloroform method purifying and recovering obtains the enrichment single stranded DNA with hangar, and this library is as the library of lower whorl screening.
3), differentiating that snake venom is counter sieves circulation: will differentiate that snake venom amalgam (Chinese cobra, Ahylysantinfarctase, solder horn snake venom) encapsulates on elisa plate with the carbonic acid buffer of pH value 9.6,37 ℃ act on 3 hours, establish the blank hole simultaneously.Sample aperture and control wells were sealed 2 hours with 3% BSA.With step 2) in the single-stranded DNA banks 1ng of purifying transfer to the snake venom amalgam and encapsulate the hole and mix echidnotoxin and combines 40 minutes for 37 ℃, wash 6 times the ssDNA that collection is washed, the single stranded DNA that purifying obtains with SELEX dcq buffer liquid.Carrying out the single stranded DNA that obtains conventional PCR reaction amplification, then carry out the asymmetric pcr reaction, phenol chloroform method purifying and recovering obtains the enrichment single stranded DNA with hangar, and this library is as the library of lower whorl screening.
4), repeating step 2)-3) 12 take turns: the screening of odd number wheel as the screening target, is collected the Jiangsu and Zhejiang Provinces cobra-venom combination wash-out ssDNA and is used for next circulation; Even numbers wheel screening use needs snake venom amalgam (NNAV, Ahylysantinfarctase, solder horn snake venom) conduct the subduing screening target of discriminating with it, collects not combine wash-out ssDNA to be used for next circulation.The concentration of the echidnotoxin that encapsulates constantly reduces along with the increase of screening wheel number with the single stranded DNA concentration that is used to combine; When the 12nd takes turns; The concentration of coating protein reaches every hole 0.05 μ g; The concentration of corresponding combination single stranded DNA is every hole 0.02ng, obtains the single-strand DNA aptamer storehouse that can combine the while not combine with discriminating snake venom (NNAV, Ahylysantinfarctase, solder horn snake venom) with Jiangsu and Zhejiang Provinces viper venom albumen high-affinity.
5) pcr amplification is carried out, the pcr amplification product in acquisition double-stranded DNA library in the high-affinity single-strand DNA aptamer storehouse that, step 4) is obtained.With 2% Ago-Gel that contains concentration 0.5 μ g/ml ethidium bromide; The pcr amplification product in double-stranded DNA library is carried out electrophoresis; Behind the electrophoresis Ago-Gel is seated on the fluoroscopic examination plate; The pcr amplification product that will be the double-stranded DNA library of Chinese red band downcuts, and with the DNA purifying and recovering kit purifying that TaKaRa company provides, obtains the double-stranded DNA of purifying.
6), with the double-stranded DNA that step 5) obtains, the pMD18-T Simple Vector kit that provides with TaKaRa company is connected on the pMD18-T carrier, is transformed into escherichia coli DH5a, ammonia benzyl resistance screening, the single growth bacterium colony of picking checks order.
7), the adaptive son of the pairing DNA of each single growth bacterium colony is used biotin labeling, amount is 0.1 μ g, the Jiangsu and Zhejiang Provinces viper venom albumen that encapsulates with every hole 10 μ g in SELEX binding buffer liquid 37 ℃ combine 40 minutes; With SELEX dcq buffer liquid washing 6 times, add the horseradish peroxidase mark Streptavidin of 1:1000,37 ℃; Act on 30 minutes, with PBST damping fluid washing 4 times, flush away not with Jiangsu and Zhejiang Provinces viper venom albumen on the enzyme that combines of DNA biotin mark streptavidin; Add the effect in 20 minutes of tetramethyl benzidine color development at room temperature then; Add the reaction of 2M concentrated sulphuric acid color development stopping, the 450nm enzyme joins appearance and measures the OD value, chooses the highest adaptive son of DNA of OD value; This adaptive son be can with the adaptive son of the protein bound DNA of Jiangsu and Zhejiang Provinces viper venom, should be adaptive the son order-checking obtain sequence.
8), will modify by the adaptive son that said process obtains and strengthen its stability, carry out mark with luciferin, biotin, radioactive isotope or collaurum then and transfer to and report adaptive son,
9), from snakebite victim blood, urine, wound tissue and secretion, detect corresponding snake venom material, thereby judge the snakebite kind, carry out corresponding antivenin treatment.
Embodiment 4:
A kind of solder horn snake venom discrimination method based on adaptive son technology, carry out as follows:
1), the Trimeresurus mucrosquamatus seed culture of viruses belongs to the screening of the adaptive son of specificity; Make up random single chain DNA (ssDNA) library and primer; Single-stranded DNA banks is passed through the PCR double chain DNA library synthesis; Utilize asymmetric PCR method DNA amplification library again, pcr amplification product obtains the single-stranded DNA banks of large-scale purification with phenol chloroform method purifying and recovering.
2), mountain habu venom screening circulation: with encapsulating on elisa plate after the dialysis of solder horn snake venom, 37 ℃ act on 3 hours, establish the blank hole simultaneously.Sample aperture and control wells were sealed 2 hours with 3% BSA.The single-stranded DNA banks 1ng of purifying is acted on 40 minutes for 37 ℃ with the blank hole earlier in SELEX binding buffer liquid; Anti-sieve is removed the single stranded DNA that combines with BSA; Transferring to mountain habu venom albumen then encapsulates hole and Jiangsu and Zhejiang Provinces viper venom protein 37 and ℃ combines 40 minutes; With SELEX dcq buffer liquid washing 6 times, add the SELEX eluent again in 80 ℃ of effects 10 minutes, the following and protein bound ssDNA of mountain habu venom of wash-out; Through phenol-chloroform extracting, precipitation with alcohol, obtain the single stranded DNA of purifying.SsDNA behind the purifying is carried out conventional PCR reaction amplification, then carry out the asymmetric pcr reaction, phenol chloroform method purifying and recovering obtains the enrichment single stranded DNA with hangar, and this library is as the library of lower whorl screening.
3), differentiating that snake venom is counter sieves circulation: will differentiate that snake venom amalgam (Chinese cobra, Ahylysantinfarctase, Jiangsu and Zhejiang Provinces pallas pit viper) encapsulates on elisa plate with the carbonic acid buffer of pH value 9.6,37 ℃ act on 3 hours, establish the blank hole simultaneously.Sample aperture and control wells were sealed 2 hours with 3% BSA.With step 2) in the single-stranded DNA banks 1ng of purifying transfer to the snake venom amalgam and encapsulate the hole and mix echidnotoxin and combines 40 minutes for 37 ℃, wash 6 times the ssDNA that collection is washed, the single stranded DNA that purifying obtains with SELEX dcq buffer liquid.Carrying out the single stranded DNA that obtains conventional PCR reaction amplification, then carry out the asymmetric pcr reaction, phenol chloroform method purifying and recovering obtains the enrichment single stranded DNA with hangar, and this library is as the library of lower whorl screening.
4), repeating step 2)-3) 12 take turns: the screening of odd number wheel as the screening target, is collected the Jiangsu and Zhejiang Provinces cobra-venom combination wash-out ssDNA and is used for next circulation; Even numbers wheel screening use needs snake venom amalgam (NNAV, Ahylysantinfarctase, Jiangsu and Zhejiang Provinces cobra-venom) conduct the subduing screening target of discriminating with it, collects not combine wash-out ssDNA to be used for next circulation.The concentration of the echidnotoxin that encapsulates constantly reduces along with the increase of screening wheel number with the single stranded DNA concentration that is used to combine; When the 12nd takes turns; The concentration of coating protein reaches every hole 0.05 μ g; The concentration of corresponding combination single stranded DNA is every hole 0.02ng, obtains the single-strand DNA aptamer storehouse that can combine the while not combine with discriminating snake venom (NNAV, Ahylysantinfarctase, Jiangsu and Zhejiang Provinces cobra-venom) with mountain habu venom albumen high-affinity.
5) pcr amplification is carried out, the pcr amplification product in acquisition double-stranded DNA library in the high-affinity single-strand DNA aptamer storehouse that, step 4) is obtained.With 2% Ago-Gel that contains concentration 0.5 μ g/ml ethidium bromide; The pcr amplification product in double-stranded DNA library is carried out electrophoresis; Behind the electrophoresis Ago-Gel is seated on the fluoroscopic examination plate; The pcr amplification product that will be the double-stranded DNA library of Chinese red band downcuts, and with the DNA purifying and recovering kit purifying that TaKaRa company provides, obtains the double-stranded DNA of purifying.
6), with the double-stranded DNA that step 5) obtains, the pMD18-T Simple Vector kit that provides with TaKaRa company is connected on the pMD18-T carrier, is transformed into escherichia coli DH5a, ammonia benzyl resistance screening, the single growth bacterium colony of picking checks order.
7), the adaptive son of the pairing DNA of each single growth bacterium colony is used biotin labeling, amount is 0.1 μ g, the mountain habu venom albumen that encapsulates with every hole 10 μ g in SELEX binding buffer liquid 37 ℃ combine 40 minutes; With SELEX dcq buffer liquid washing 6 times, add the horseradish peroxidase mark Streptavidin of 1:1000,37 ℃; Act on 30 minutes, with PBST damping fluid washing 4 times, flush away not with mountain habu venom albumen on the enzyme that combines of DNA biotin mark streptavidin; Add the effect in 20 minutes of tetramethyl benzidine color development at room temperature then; Add the reaction of 2M concentrated sulphuric acid color development stopping, the 450nm enzyme joins appearance and measures the OD value, chooses the highest adaptive son of DNA of OD value; This adaptive son be can with the adaptive son of the protein bound DNA of mountain habu venom, should be adaptive the son order-checking obtain sequence.
8), will modify by the adaptive son that said process obtains and strengthen its stability, carry out mark with luciferin, biotin, radioactive isotope or collaurum then and transfer to and report adaptive son,
9), from snakebite victim blood, urine, wound tissue and secretion, detect corresponding snake venom material, thereby judge the snakebite kind, carry out corresponding antivenin treatment.
Claims (4)
1. one kind is utilized adaptive sub-technology for detection to differentiate the method for snake venom kind, it is characterized in that carrying out as follows:
(1) make up random single-stranded DNA banks and primer earlier, single-stranded DNA banks is double-stranded DNA, obtains single-stranded DNA banks through asymmetric pcr amplification and purifying again through pcr amplification;
(2) filter out the single stranded DNA that the single-stranded DNA banks of purifying combines with the target raw venin; Conventional once more PCR and asymmetric pcr DNA amplification, purifying obtain the single stranded DNA that combines with the target raw venin;
(3) single stranded DNA that step (2) is obtained instead sieves the single stranded DNA that is not combined with 4 ~ 8 kinds of snake venom that needs are differentiated again;
(4) according to step (2) and step (3) intersection Cycle Screening; Obtain combining simultaneously not and the single-strand DNA aptamer of differentiating that snake venom combines with the echidnotoxin high-affinity, pcr amplification be double-stranded DNA, asymmetric pcr amplification also purifying obtain the single-strand DNA aptamer storehouse;
(5) the compatibility detection is carried out in screening terminal cistern single-strand DNA aptamer storehouse and filtered out the adaptive son of target;
(6) the adaptive son that obtains is modified and is strengthened its stability, carries out mark with luciferin, biotin, radioactive isotope or collaurum then and transfers the adaptive son of report to;
(7) adopt ELISA from snakebite victim blood, urine, wound tissue and secretion, to detect corresponding snake venom material, thereby judge the snakebite kind, carry out corresponding antivenin treatment.
2. according to the said a kind of method of utilizing adaptive sub-technology for detection discriminating snake venom kind of claim 1, it is characterized in that: take turns above according to step (2) and step (3) intersection Cycle Screening 12 in the said step (4).
3. according to the said a kind of method of utilizing adaptive sub-technology for detection discriminating snake venom kind of claim 1, it is characterized in that: step (1), (2) and (4) adopt asymmetric pcr method or biotin streptavidin magnesphere legal system to be equipped with single-stranded DNA banks.
4. according to the said a kind of method of utilizing adaptive sub-technology for detection to differentiate the snake venom kind of claim 1, it is characterized in that: be separating medium with nitrocellulose filter, affine resin or microwell plate in step (2), (3) and (4) screening process.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110152346XA CN102323400B (en) | 2011-06-08 | 2011-06-08 | Method for detecting and identifying variety of snake venom by utilizing adaptor technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110152346XA CN102323400B (en) | 2011-06-08 | 2011-06-08 | Method for detecting and identifying variety of snake venom by utilizing adaptor technology |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102323400A true CN102323400A (en) | 2012-01-18 |
| CN102323400B CN102323400B (en) | 2013-11-27 |
Family
ID=45451183
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110152346XA Active CN102323400B (en) | 2011-06-08 | 2011-06-08 | Method for detecting and identifying variety of snake venom by utilizing adaptor technology |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102323400B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110396549A (en) * | 2018-04-25 | 2019-11-01 | 锦州奥鸿药业有限责任公司 | Utilize the method for specific molecular genetic marker detection snake venom Species origin |
| CN110396550A (en) * | 2018-04-25 | 2019-11-01 | 锦州奥鸿药业有限责任公司 | Utilize the method for cytochrome genes detection snake venom Species origin |
| CN110426522A (en) * | 2019-08-13 | 2019-11-08 | 上海赛伦生物技术股份有限公司 | A kind of identification method of Vipera russelli venom and its application |
| CN113801939A (en) * | 2020-06-16 | 2021-12-17 | 上海赛伦生物技术股份有限公司 | Sea snake venom specificity detection method and kit |
| CN114354525A (en) * | 2022-01-11 | 2022-04-15 | 云南大学 | Alpha-bungarotoxin detection probe and method for detecting alpha-bungarotoxin in non-diagnosis purpose |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060257914A1 (en) * | 2005-05-13 | 2006-11-16 | Pronucleotein Biotechnologies, Llc | Methods of producing interachain fluorophore-quencher FRET-aptamers and assays |
| US20060257915A1 (en) * | 2005-05-13 | 2006-11-16 | Pronucleotein Biotechnologies, Llc | Methods of producing competitive aptamer fret reagents and assays |
| EP1730158A2 (en) * | 2003-08-06 | 2006-12-13 | Cygene, Inc. | Methods and compositions for in vitro and in vivo use of parallel stranded hairpins and triplex structures as nucleic acid ligands |
| CN1958809A (en) * | 2006-09-12 | 2007-05-09 | 上海市肺科医院 | Method for detecting mycobacterium tuberculosis by using adaptor technique |
| CN101144814A (en) * | 2007-10-22 | 2008-03-19 | 中国人民解放军第三军医大学第一附属医院 | Method for detecting, identifying and/ or quantifying compound using adapter type reagent |
| CN101665821A (en) * | 2008-09-01 | 2010-03-10 | 中国人民解放军军事医学科学院基础医学研究所 | Oligonucleotide aptamer group for specifically identifying staphylococcus aureus and use thereof |
-
2011
- 2011-06-08 CN CN201110152346XA patent/CN102323400B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1730158A2 (en) * | 2003-08-06 | 2006-12-13 | Cygene, Inc. | Methods and compositions for in vitro and in vivo use of parallel stranded hairpins and triplex structures as nucleic acid ligands |
| US20060257914A1 (en) * | 2005-05-13 | 2006-11-16 | Pronucleotein Biotechnologies, Llc | Methods of producing interachain fluorophore-quencher FRET-aptamers and assays |
| US20060257915A1 (en) * | 2005-05-13 | 2006-11-16 | Pronucleotein Biotechnologies, Llc | Methods of producing competitive aptamer fret reagents and assays |
| CN1958809A (en) * | 2006-09-12 | 2007-05-09 | 上海市肺科医院 | Method for detecting mycobacterium tuberculosis by using adaptor technique |
| CN101144814A (en) * | 2007-10-22 | 2008-03-19 | 中国人民解放军第三军医大学第一附属医院 | Method for detecting, identifying and/ or quantifying compound using adapter type reagent |
| CN101665821A (en) * | 2008-09-01 | 2010-03-10 | 中国人民解放军军事医学科学院基础医学研究所 | Oligonucleotide aptamer group for specifically identifying staphylococcus aureus and use thereof |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110396549A (en) * | 2018-04-25 | 2019-11-01 | 锦州奥鸿药业有限责任公司 | Utilize the method for specific molecular genetic marker detection snake venom Species origin |
| CN110396550A (en) * | 2018-04-25 | 2019-11-01 | 锦州奥鸿药业有限责任公司 | Utilize the method for cytochrome genes detection snake venom Species origin |
| CN110396550B (en) * | 2018-04-25 | 2021-04-06 | 锦州奥鸿药业有限责任公司 | Method for detecting snake venom species source by using cytochrome gene |
| CN110396549B (en) * | 2018-04-25 | 2021-07-23 | 锦州奥鸿药业有限责任公司 | Method for detecting snake venom species source by utilizing specific molecular genetic marker |
| CN110426522A (en) * | 2019-08-13 | 2019-11-08 | 上海赛伦生物技术股份有限公司 | A kind of identification method of Vipera russelli venom and its application |
| CN110426522B (en) * | 2019-08-13 | 2021-01-12 | 上海赛伦生物技术股份有限公司 | Identification method of Vipera cirrhosa snake venom and application thereof |
| CN113801939A (en) * | 2020-06-16 | 2021-12-17 | 上海赛伦生物技术股份有限公司 | Sea snake venom specificity detection method and kit |
| CN114354525A (en) * | 2022-01-11 | 2022-04-15 | 云南大学 | Alpha-bungarotoxin detection probe and method for detecting alpha-bungarotoxin in non-diagnosis purpose |
| CN114354525B (en) * | 2022-01-11 | 2023-09-05 | 云南大学 | Alpha-bungarotoxin detection probe and method for detecting alpha-bungarotoxin for non-diagnostic purpose |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102323400B (en) | 2013-11-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102323400B (en) | Method for detecting and identifying variety of snake venom by utilizing adaptor technology | |
| CN113151282B (en) | Aptamer binding to novel coronavirus (SARS-CoV-2) nucleocapsid protein and use thereof | |
| CN104830866A (en) | ssDNA aptamer for recognizing ofloxacin with specificity and application of ssDNA aptamer | |
| CN103289990A (en) | Rapid extraction method of blood deoxyribonucleic acid (DNA) | |
| CN104031135B (en) | Rhacophorus defense peptides PopuDef and gene thereof and application | |
| CN101816789B (en) | Lyophilized viper antivenin and preparation method thereof | |
| KR101891406B1 (en) | DNA Aptamer Specifically Binding to Surface of Living Cell of Salmonella typhimurium and Uses Thereof | |
| CN112029771B (en) | Aptamer specifically binding to meperidine and application thereof | |
| RU2388819C2 (en) | Method for production of horseradish peroxidase | |
| CN111019940A (en) | Extracting solution for directly extracting whole blood genome DNA and extracting method thereof | |
| CN100381560C (en) | Process for synthesizing recombined human intestine trilobate factor using GS115 microzyme | |
| CN107365384A (en) | A kind of targeting Reg3A single-chain antibody | |
| CN105087531B (en) | A kind of method preparing urokinase | |
| CN103305521B (en) | The sequence of the aptamer of a kind of stomach cancer cell and application | |
| CN110438120A (en) | A kind of kit and its method extracting microbe genome DNA from blood | |
| CN106636105B (en) | The aptamer C203 and its screening technique of staphylococcus aureus enterotoxin C 2 and application | |
| CN104450628A (en) | Escherichia coli resistance O157:H7 monoclonal antibody and application thereof | |
| KR101540444B1 (en) | Probe for Concentration and Detection of Norovirus or Hepatitis A Virus(HAV) | |
| CN102199191A (en) | Sealing system composed of small anaerobic operation bags and chromatography column in series | |
| CN111778255A (en) | Screening and identifying method of pepsin ssDNA aptamer | |
| CN105483133A (en) | Staphylococcus aureus toxic shock syndrome toxin-1(TSST-1) aptamer T-7 and preparation method and application thereof | |
| CN100383239C (en) | Process for extracting oxalate oxidase from bran of wheat | |
| CN110387370A (en) | It is a kind of to extract micro adder gene and its method for detecting specificity | |
| CN111217905A (en) | Preparation method of recombinant rabbit monoclonal antibody | |
| CN108950024A (en) | A kind of LAMP detection kit and detection primer of the bacillary parotitis pathogen of Shelled Turtle Trionyx Sinensis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |