CN106442353A - Detection method of pig heparin sodium crude product mixed with ruminant source - Google Patents
Detection method of pig heparin sodium crude product mixed with ruminant source Download PDFInfo
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- CN106442353A CN106442353A CN201610853008.1A CN201610853008A CN106442353A CN 106442353 A CN106442353 A CN 106442353A CN 201610853008 A CN201610853008 A CN 201610853008A CN 106442353 A CN106442353 A CN 106442353A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
- G01N2021/3148—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths using three or more wavelengths
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Abstract
The invention discloses a detection method of a pig heparin sodium crude product mixed with a ruminant source. The detection method is characterized by comprising the following steps: step (1): taking pig livers, cattle livers and sheep livers as samples and extracting heparin sodium DNAs (Deoxyribonucleic Acid); step (2): determining absorbance at a 265nm part and a specific value of the absorbance at the 265nm part and absorbance at a 285nm part of the heparin sodium DNAs extracted from the pig livers, the cattle livers and the sheep livers, so as to judge the purity of the extracted DNAs, wherein the purity meets the requirements when being in a range of 1.8-1.9; taking the DNAs as a DNA template; step (3): preparing a PCR (Polymerase Chain Reaction) template, designing a primer and preparing a PCR amplification reaction system; step (4): obtaining a qPCR result by adopting a fluorescent probe, wherein when an average Ct value of an amplification curve is 14.566+/-0.624, the pig heparin sodium DNA is doped with bovine derived heparin sodium DNA; and when the average Ct value of the amplification curve is 15.271+/-0.256, the pig heparin sodium DNA is doped with sheep derived heparin sodium DNA. The pig heparin sodium crude product disclosed by the invention has good specificity, high sensitivity and high accuracy and is used for qualitative determination of the heparin sodium crude products with different sources; and technical supports are provided for guaranteeing medication safety.
Description
Technical field
The present invention relates to a kind of detection method of pork liver element crude product sodium is and in particular to a kind of be mixed into the pig that ruminant is originated
The detection method of heparin sodium crude.
Background technology
Heparin finds from liver first and gains the name, and is concurrently present in the tissue such as lung, vascular wall, intestinal mucosa, is animal body
A kind of interior natural anticoagulative substance, primarily now extracts from intestinal mucosa, heparin as conventional anticoagulation, clinically often
It is used for myocardial infarction, haemodialysis etc..The ruminant such as ox and sheep may carry pathogenic PrPC, thus suffering from infectiousness sea
Continuous shape encephalopathic（It is commonly called as scrapie and rabid ox disease）.The mankind may be carried the animal infection of virus and suffer from new Keyashi's syndrome.
Therefore, for guaranteeing the safety of heparin supply chain, heparin bulk drug does not allow to come from ox and Yang Fanchudongwuyuan pollution is extensively deposited
How effectively to differentiate that in source, the liquaemin containing ruminant is the problem needing to solve.
Content of the invention
In order to solve the above problems, the invention provides a kind of detection of the porcine heparin crude product being mixed into ruminant source
Method.
The technical scheme is that：A kind of detection method of the porcine heparin crude product being mixed into ruminant source, including
Following steps：
Step one：With pork liver, beef liver and sheep liver are sample extraction liquaemin DNA；
Step 2：By pork liver, liquaemin DNA that beef liver and sheep liver are extracted absorbance and 265 nm at 265 nm after measured
With the ratio of absorbance at 285nm thus judging the purity of extracted DNA, purity conforms between 1.8 ~ 1.9
Ask, in this, as DNA profiling；
Step 3：By the preparation of PCR template, design of primers, pcr amplification reaction system and PCR response procedures, expanded
Product；
Step 4：QPCR result is obtained using fluorescence probe, amplification curve average Ct value is 14.566 ± 0.624, pig source liver
Doped with ox source liquaemin DNA in plain sodium DNA；Amplification curve average Ct value is 15.271 ± 0.256, in the liquaemin DNA of pig source
Doped with sheep source liquaemin DNA.
Further, in described step one, the step of extraction liquaemin DNA is：
A () each takes 100g pork liver, beef liver and sheep liver respectively and adds isopyknic water to carry out high-speed homogenization, and then add
1mol/L NaCl, 45mmol/L sodium citrate, 12mmol/L EDTA, papain is 1.6g/L, salt adding acid for adjusting pH
It is worth to 5.5 about, heating water bath to 60 DEG C and stirs 3.5h, respectively obtain the enzymolysis just liquid of pork liver, beef liver and sheep liver；
B () just liquid is heated to 90 DEG C and maintains 25min to carry out enzyme-deactivating by the enzymolysis of pork liver, beef liver and sheep liver, add matter
The diatomite of amount fraction 8%, uses Buchner funnel every filter paper suction filtration while hot, washs filter with the NaCl hot solution of 1 a small amount of mol/L
Slag, merging filtrate, obtain the leaching liquor of the DNA of pork liver, beef liver and sheep liver；
15 mmol/L sodium citrates, 1.2mmol/L is added in the leaching liquor of (c) DNA in pork liver, beef liver and sheep liver
EDTA, adjust pH value to 8.0 about, carry out filter, peritoneal effluent dropping mass fraction is 3% cetylpyridinium chloride to not having
Precipitation is had to occur；
D () is slowly added to CaCl in above-mentioned three kinds of filtrates2, and be sufficiently stirred for, to precipitating completely, adjust pH value left to 3
The right side, stands 12h, extracts precipitation, with the CaCl for 3.0 for the 0.1mol/L pH value2Solution abundant filter wash precipitation, obtain pork liver,
The DNA calcium salt of beef liver and sheep liver；
E () is slow added into 1mol/LNaCl, be slowly stirred and be 12% with mass fraction NaOH solution adjust pH value to 8.0,
It is completely dissolved to precipitation and obtains colourless or slightly flaxen solution, add volume fraction 90% ethanol solution of double amount to sink
Form sediment 10～12 h, and precipitation dehydrates the liquaemin DNA obtaining pork liver, beef liver and sheep liver.
Further, use Epoch ultramicron nucleic acid determination instrument mensuration absorbance in described step 2.
Further, the PCR template in described step 3 be prepared as trained on YEPD flat board with sterile toothpick picking
Support the heparin of 48 h, add 250 μ L ddH2In O, boil 5min, -20 DEG C of freezing 5min at 100 DEG C, circulate 2 times,
Draw the template that a certain amount of supernatant expands as PCR.
Further, the design of primers in described step 3 is：
Pig derived component upstream primer:GCCTAAATCTCCCCTCAATGGTA
Downstream primer:ATGAAAGAGGCAAATAGATTTTCG
Calf-derived Cyclospora upstream primer:GCCATATACTCTCCTTGGTGACA
Downstream primer:GTAGGCTTGGGAATAGTACGA
Sheep derived material upstream primer:TATTAGGCCTCCCCCTTGTT
Downstream primer:CCCTGCTCATAAGGGAATAGCC.
Further, described pig source property primer amplification fragment is 150bp, and described ox source property primer amplification fragment is
271bp, described sheep source property primer amplification fragment is 294bp.
Further, the amplification reaction system in described step 3 is：DNA profiling 5 μ l, buffer solution 18 μ l, upstream primer 1
μ l, downstream primer 1 μ l, sterilized water 25 μ l.
Further, the PCR response procedures in described step 3 are：
（a）Denaturation：Template DNA denaturation 2min at 95 DEG C, single-stranded formation；
B () is annealed：Template DNA by denaturation formed single-stranded after, when temperature reaches about 80 DEG C, by primer and template list
The complementary series pairing of chain combines, and keeps 20s；
C () extends：55 DEG C of renaturation 30 s, 60 DEG C plus extension 50s, one new chain complementary with template DNA chain of synthesis, will
Above three process regards one cycle, repetitive cycling 35 times as, enters performing PCR for above-mentioned DNA profiling and negative control sample
Amplification, obtains amplified production.
Further, in described step 4, the proportioning of the pig source liquaemin DNA doped with ox source liquaemin DNA is：To
Add the liquaemin DNA extract of the Niu Laiyuan of 20pg/ μ l after the 5 times of dilutions of the liquaemin DNA extract in 5ng/ μ l pig source.
Further, in described step 4, the proportioning of the pig source liquaemin DNA doped with sheep source liquaemin DNA is：To
Add the liquaemin DNA extract in 20pg/ μ l sheep source after the 5 times of dilutions of the liquaemin DNA extract in 5ng/ μ l pig source.
Beneficial effects of the present invention are：Specificity is good, and sensitivity is high, accuracy is high, can be used for the thick of different genera source
The qualitative detection of product liquaemin, supervision medicine specification produces it is ensured that drug safety provides technical support.
Brief description
Fig. 1 is the amplification curve of pig source liquaemin DNA.
Fig. 2 is the amplification curve of the pig source liquaemin DNA of doping ox source liquaemin DNA.
Fig. 3 is the amplification curve of the pig source liquaemin DNA of doping sheep source liquaemin DNA.
Specific embodiment
Example below is used for further describing the present invention, but embodiment does not do any type of limit to the present invention
Fixed.
First, the extraction of liquaemin DNA
Choose pork liver, beef liver and sheep liver are sample extraction liquaemin DNA.
1st, each take 100g pork liver, beef liver and sheep liver respectively and add isopyknic water to carry out high-speed homogenization, and then add 1
Mol/L NaCl, 45 mmol/L sodium citrates, 12mmol/L EDTA, papain is 1.6g/L, salt adding acid for adjusting pH
It is worth to 5.5 about, heating water bath to 60 DEG C and stirs 3.5h, respectively obtain the enzymolysis just liquid of pork liver, beef liver and sheep liver；
2nd, by the enzymolysis of pork liver, beef liver and sheep liver, just liquid is heated to 90 DEG C and maintains 25min to carry out enzyme-deactivating, adds quality
The diatomite of fraction 8%, uses Buchner funnel while hot every filter paper suction filtration.Wash filter residue with the NaCl hot solution of 1 a small amount of mol/L,
Merging filtrate, obtains the leaching liquor of the DNA of pork liver, beef liver and sheep liver；
3rd, 15 mmol/L sodium citrates, 1.2 mmol/L are added in the leaching liquor of the DNA of pork liver, beef liver and sheep liver
EDTA, adjust pH value to 8.0 about, carry out filter, peritoneal effluent dropping mass fraction is 3% cetylpyridinium chloride to not having
Precipitation is had to occur；
4th, it is slowly added to CaCl2 in above-mentioned three kinds of filtrates, and is sufficiently stirred for, to precipitating completely, adjust pH value left to 3
The right side, stands 12h, extracts precipitation, with the abundant filter wash of solution of the CaCl2 for 3.0 for 0.1mol/L pH value precipitation, obtain pork liver,
The DNA calcium salt of beef liver and sheep liver；
5th, be slow added into 1 mol/LNaCl, be slowly stirred and be 12% with mass fraction NaOH solution adjust pH value to 8.0,
It is completely dissolved to precipitation and obtains colourless or slightly flaxen solution, add volume fraction 90% ethanol solution of double amount to sink
Form sediment 10～12 h, and precipitation dehydrates the liquaemin DNA obtaining pork liver, beef liver and sheep liver.
2nd, the liquaemin DNA of three kinds of materials is measured absorbance at 265 nm through Epoch ultramicron nucleic acid determination instrument
And 265 nm and 285nm locate the ratio of absorbance thus the purity of the extracted DNA of judgement, purity is 1.8 ~ 1.9
Between meet the requirements, in this, as DNA profiling.
3rd, PCR DNA amplification template
Because heparin has the effect of suppression PCR reaction, so need to pre-process to the DNA extracting, it is dissolved in heparinase
Buffer solution.
（1）The preparation of PCR template
The heparin of 48 h is cultivated with sterile toothpick picking on YEPD flat board, adds in 250 μ L dd H2O, at 100 DEG C
Boil 5min, -20 DEG C of freezing 5min, circulate 2 times, draw the template that a certain amount of supernatant expands as PCR.
（2）Design of primers
Using gene database, the genome sequence of pig, ox, sheep is compared, the respective characteristic sequence of selection, selected pig,
Ox, sheep derived material primer sequence as follows：
Pig derived component upstream primer:GCCTAAATCTCCCCTCAATGGTA amplified fragments size（bp）150
Downstream primer:ATGAAAGAGGCAAATAGATTTTCG
Calf-derived Cyclospora upstream primer:GCCATATACTCTCCTTGGTGACA amplified fragments size（bp271
Downstream primer:GTAGGCTTGGGAATAGTACGA
Sheep derived material upstream primer:TATTAGGCCTCCCCCTTGTT amplified fragments size（bp）294
Downstream primer:CCCTGCTCATAAGGGAATAGCC .
（3）PCR expands
Set up the amplification reaction system of 50 μ l, add following reagent：
DNA profiling 5 μ l, buffer solution 18 μ l, upstream primer 1 μ l, downstream primer 1 μ l, sterilized water 25 μ l.
The reaction of PCR is divided into three steps：Denaturation, initiation（Annealing）And extension,
Denaturation：Template DNA denaturation 2min at 95 DEG C, single-stranded formation；
Cause（Annealing）：Template DNA by denaturation formed single-stranded after, when temperature reaches about 80 DEG C, by primer and template
Single-stranded complementary series pairing combines, and keeps 20s；
Extend：When selective polymerization enzyme is as catalyzing enzyme, 55 DEG C of renaturation 30 s, 60 DEG C plus extension 50s, synthesize one and template
The complementary new chain of DNA chain；
Above three process is regarded as one cycle, repetitive cycling 35 times, above-mentioned DNA profiling and negative control sample are entered
Performing PCR expands, and obtains amplified production.
4th, adopt the qPCR result of fluorescence probe
PCR reaction directly reads testing result, with the peak just above normal negative control group amplification curve after completing
Fluorescence intensity be threshold value, the foundation as result judgement for the period (i.e. Ct value) needed for threshold value is reached with sample.
If positive reaction comparison is compareed with negative reaction, positive control is with containing that fluorescence PCR detection reagent kit carries
The plasmid of the animal derived characteristic sequence being detected replaces template DNA, and negative control replaces template DNA with pure water.
（1）5 times of dilutions are carried out with the liquaemin DNA extract in 5 ng/ μ l pig sources, Fig. 1 show pig source liquaemin
DNA cloning curve, amplification curve average Ct value is 29.504 ± 0.485.
（2）Add the liquaemin of the Niu Yuan of 20pg/ μ l to after 5 times of dilutions of liquaemin DNA extract in 5ng/ μ l pig source
DNA extract, as shown in Fig. 2 amplification curve average Ct value is 14.566 ± 0.624, according to cow genome on calibration curve
Template amount, effective detection can go out bovine material.
（3）Add the liquaemin of 20pg/ μ l Yang Yuan to after 5 times of dilutions of liquaemin DNA extract in 5ng/ μ l pig source
DNA extract, as shown in figure 3, amplification curve average Ct value is 15.271 ± 0.256, according to sheep gene on calibration curve
Template amount, effective detection can go out sheep material.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not subject to described embodiment
Limit, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplify,
All should be equivalent substitute mode, be included within protection scope of the present invention.
Claims (10)
1. a kind of be mixed into ruminant source porcine heparin crude product detection method it is characterised in that：Comprise the following steps：
Step one：With pork liver, beef liver and sheep liver are sample extraction liquaemin DNA；
Step 2：By pork liver, liquaemin DNA that beef liver and sheep liver are extracted absorbance and 265 nm at 265 nm after measured
With the ratio of absorbance at 285nm thus judging the purity of extracted DNA, purity conforms between 1.8 ~ 1.9
Ask, in this, as DNA profiling；
Step 3：By the preparation of PCR template, design of primers, pcr amplification reaction system and PCR response procedures, expanded
Product；
Step 4：QPCR result is obtained using fluorescence probe, amplification curve average Ct value is 14.566 ± 0.624, pig source
Doped with ox source liquaemin DNA in liquaemin DNA；Amplification curve average Ct value is 15.271 ± 0.256, pig source liquaemin DNA
In doped with sheep source liquaemin DNA.
2. the detection method of a kind of porcine heparin crude product being mixed into ruminant source according to claim 1, its feature
It is：In described step one, the step of extraction liquaemin DNA is：
A () each takes 100g pork liver, beef liver and sheep liver respectively and adds isopyknic water to carry out high-speed homogenization, and then add
1mol/L NaCl, 45mmol/L sodium citrate, 12mmol/L EDTA, papain is 1.6g/L, salt adding acid for adjusting pH
It is worth to 5.5 about, heating water bath to 60 DEG C and stirs 3.5h, respectively obtain the enzymolysis just liquid of pork liver, beef liver and sheep liver；
B () just liquid is heated to 90 DEG C and maintains 25min to carry out enzyme-deactivating by the enzymolysis of pork liver, beef liver and sheep liver, add matter
The diatomite of amount fraction 8%, uses Buchner funnel every filter paper suction filtration while hot, washs filter with the NaCl hot solution of 1 a small amount of mol/L
Slag, merging filtrate, obtain the leaching liquor of the DNA of pork liver, beef liver and sheep liver；
15 mmol/L sodium citrates, 1.2 mmol/L are added in the leaching liquor of (c) DNA in pork liver, beef liver and sheep liver
EDTA, adjust pH value to 8.0 about, carry out filter, peritoneal effluent dropping mass fraction is 3% cetylpyridinium chloride to not having
Precipitation is had to occur；
D () is slowly added to CaCl2 in above-mentioned three kinds of filtrates, and be sufficiently stirred for, and to precipitating completely, adjusts pH value left to 3
The right side, stands 12h, extracts precipitation, with the abundant filter wash of solution of the CaCl2 for 3.0 for 0.1mol/L pH value precipitation, obtain pork liver,
The DNA calcium salt of beef liver and sheep liver；
E () is slow added into 1 mol/LNaCl, be slowly stirred and be 12% with mass fraction NaOH solution adjust pH value extremely
8.0, it is completely dissolved to precipitation and obtains colourless or slightly flaxen solution, add volume fraction 90% ethanol of double amount molten
Liquid precipitate 10～12 h, precipitation dehydrates the liquaemin DNA obtaining pork liver, beef liver and sheep liver.
3. the detection method of a kind of porcine heparin crude product being mixed into ruminant source according to claim 1, its feature
It is：Epoch ultramicron nucleic acid determination instrument mensuration absorbance is used in described step 2.
4. the detection method of a kind of porcine heparin crude product being mixed into ruminant source according to claim 1, its feature
It is：Being prepared as of PCR template in described step 3 cultivates the liver of 48 h on YEPD flat board with sterile toothpick picking
Element, adds in 250 μ L dd H2O, boils 5min, -20 DEG C of freezing 5min, circulate 2 times, draw certain at 100 DEG C
The template that amount supernatant expands as PCR.
5. the detection method of a kind of porcine heparin crude product being mixed into ruminant source according to claim 1, its feature
It is：Design of primers in described step 3 is：
Pig derived component upstream primer:GCCTAAATCTCCCCTCAATGGTA
Downstream primer:ATGAAAGAGGCAAATAGATTTTCG
Calf-derived Cyclospora upstream primer:GCCATATACTCTCCTTGGTGACA
Downstream primer:GTAGGCTTGGGAATAGTACGA
Sheep derived material upstream primer:TATTAGGCCTCCCCCTTGTT
Downstream primer:CCCTGCTCATAAGGGAATAGCC.
6. the detection method of a kind of porcine heparin crude product being mixed into ruminant source according to claim 5, its feature
It is：Described pig source property primer amplification fragment is 150bp, and described ox source property primer amplification fragment is 271bp, described sheep source property
Primer amplification fragment is 294bp.
7. the detection method of a kind of pig liver sodium crude product being mixed into ruminant source according to claim 1, its feature
It is：Amplification reaction system in described step 3 is：DNA profiling 5 μ l, buffer solution 18 μ l, upstream primer 1 μ l, downstream primer 1
μ l, sterilized water 25 μ l.
8. the detection method of a kind of porcine heparin crude product being mixed into ruminant source according to claim 1, its feature
It is：PCR response procedures in described step 3 are：
(a) denaturation：Template DNA denaturation 2min at 95 DEG C, single-stranded formation；
B () is annealed：Template DNA by denaturation formed single-stranded after, when temperature reaches about 80 DEG C, by primer and template list
The complementary series pairing of chain combines, and keeps 20s；
C () extends：55 DEG C of renaturation 30 s, 60 DEG C plus extension 50s, one new chain complementary with template DNA chain of synthesis, will
Above three process regards one cycle, repetitive cycling 35 times as, enters performing PCR for above-mentioned DNA profiling and negative control sample
Amplification, obtains amplified production.
9. the detection method of a kind of porcine heparin crude product being mixed into ruminant source according to claim 1, its feature
It is：In described step 4, the proportioning of the pig source liquaemin DNA doped with ox source liquaemin DNA is：To 5ng/ μ l pig source
Add the liquaemin DNA extract of the Niu Laiyuan of 20pg/ μ l after the 5 times of dilutions of liquaemin DNA extract.
10. the detection method of a kind of porcine heparin crude product being mixed into ruminant source according to claim 1, its feature
It is：In described step 4, the proportioning of the pig source liquaemin DNA doped with sheep source liquaemin DNA is：To 5ng/ μ l pig source
Add the liquaemin DNA extract in 20pg/ μ l sheep source after the 5 times of dilutions of liquaemin DNA extract.
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Application publication date: 20170222 |