GB/T 20190-2025 (GB/T 20190-2006) PDF English
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GB/T 20190-2006: Detection of bovine, sheep and goat-derived material in feeds - Qualitative polymerase chain reaction (PCR) method
---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GBT20190-2006
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 65.120
B 20
Detection of bovine, sheep and goat-derived material in feeds
- Qualitative polymerase chain reaction (PCR) method
Issued on. MAY 17, 2006
Implemented on. SEPTEMBER 01, 2006
Issued by. General Administration of Quality Supervision, Inspection and
Quarantine of PRC;
Standardization Administration of PRC.
Table of Contents
Foreword... 3
Introduction... 4
1 Scope... 5
2 Normative references... 5
3 Principles... 5
4 Reagents and materials... 6
5 Instruments... 8
6 Operation steps... 8
7 Results analysis and presentation... 12
Appendix A (Normative) Bovine, sheep, goat specific DNA sequences... 13
Foreword
Appendix A of this standard is a normative appendix.
This standard was proposed by the National Feed Industry Standardization Technical
Committee.
Drafting organizations of this standard. National Feed Quality Supervision and
Inspection Center (Beijing), Institute of Agricultural Resources and Agricultural Zoning
of Chinese Academy of Agricultural Sciences.
The main drafters of this standard. Yang Shuming, Song Rong, Cheng Xianguo, Gao
Sheng, Lai Weihua, Wang Tong.
1 Scope
This standard specifies the PCR method for the qualitative detection of bovine, sheep
and goat-derived material in feeds.
This standard is applicable to the qualitative detection of bovine, sheep and goat-
derived material in feeds. The minimum detection limit of this method is 0.25%.
2 Normative references
The provisions in following documents become the provisions of this Standard through
reference in this Standard. For the dated references, the subsequent amendments
(excluding corrections) or revisions do not apply to this Standard; however, parties who
reach an agreement based on this Standard are encouraged to study if the latest versions
of these documents are applicable. For undated references, the latest edition of the
referenced document applies.
GB/T 6682 Water for analytical laboratory use - Specification and test methods
3 Principles
According to the specificity of the genetic material of bovine, sheep and goat, the DNA
sequence specific to bovine, sheep and goat is selected by searching the gene bank or
patent library. The sequence must be highly conserved, in similar animals (no matter
what breed), whilst other animals do not contain.
4 Reagents and materials
Unless otherwise specified, only analytical reagents are used in the analysis; the water
shall meet the requirements of grade-1 water in GB/T 6682.
4.1 Trimethylaminomethane hydrochloric acid (Tris-HCl) solution, 1 mol/L, pH 8.0.
Dissolve 121.1 g of Tris in 800 mL of deionized water. Cool to room temperature. Use
concentrated hydrochloric acid to adjust the pH value of the solution to 8.0.Add water
to make the volume reach to 1 L. Divide-contain it. Autoclave it.
4.3 Sodium chloride solution, 5 mol/L. Dissolve 29.22 g of sodium chloride in 80 mL
of water. Add water to make the volume reach to 100 mL.
4.4 Ethylenediaminetetraacetic acid disodium salt (EDTA) solution, 500 mmol/L.
Weigh 186.1 g of ethylenediaminetetraacetic acid disodium dihydrate (EDTA-Na2 •
2H2O). Add it in 700 mL of water. Stir vigorously on a magnetic stirrer. Use 10 mol/L
sodium hydroxide solution, to adjust the pH value to 8.0.Use water to make the volume
reach to 1 L. Divide-contain it. Autoclave it.
4.7 Ribonuclease A (RNase A) stock solution. Dissolve 10 mg of RNase A in 987 µL of
water. Add 10 µL of Tris-HCl solution (4.2). Add 3 µL of sodium chloride solution (4.3).
Incubate in a 100 °C water bath for 15 mm. Cool to room temperature. Divide it into
small parts and contain it. Preserve it at -20 °C.
4.8 Mixture of Tris saturated phenol and chloroform, V (Tris saturated phenol) + V
(chloroform) = 1 + 1.
4.9 Mixture of chloroform and isoamyl alcohol, V (chloroform) + V (isoamyl alcohol)
= 24 + 1.
4.10 Isopropanol.
4.14 N-hexane.
4.15 Taq DNA polymerase (5 U/µL) and 10 X PCR reaction buffer (containing 25
mmol/L Mg2+).
4.16 The sequences of primers (pairs) for the detection of bovine-derived components
are.
4.19 Mixed solutions of four deoxyribonucleotides (dATP, dCTP, dGTP, dTTP), at 10
mmol/L each.
4.20 10 mg/mL ethidium bromide solution.
Note. Ethidium bromide (EB) has carcinogenic effect, so wear disposable gloves when using
it; dispose of it, according to safety requirements after use.
4.21 DNA molecular weight marker (50 bp ~ 300 bp).
4.24 Agarose.
4.25 PCR product recovery and purification kit. Operate according to the instructions
for use.
4.26 Restrictive endonuclease Sau3AI and reaction buffer.
4.27 Gel recovery and purification kit. Operate according to the instructions for use.
4.28 Paraffin oil.
4.29 DNA sequencing reagents.
4.30 95% ethanol by volume.
4.31 Formamide.
5 Instruments
5.1 Commonly used laboratory equipment.
5.2 PCR amplification instrument.
5.3 Electrophoresis apparatus.
5.6 Autoclave.
6 Operation steps
6.1 Pretreatment of the specimen
Pulverize 50 g solid sample, to keep its particle size below 0.125 mm.
6.2 Extraction and purification of template DNA
6.2.1 Extraction of template DNA by CTAB method
Weigh 100 mg of the pretreated specimen.
6.2.2 Extraction of template DNA from oily feed
Take an appropriate volume of oily feed (30 mL of liquid oil, 5 g of phospholipids and
solid oils), into a 250 mL conical flask. Add 25 mL of n-hexane (4.14). Shake and mix
on a magnetic stirrer for 2 hours. Add 25 mL of CTAB extraction buffer II (4.6).
Continue to shake and mix on a magnetic stirrer, for 2 h. Transfer the solution into a
100 mL centrifuge tube. Centrifuge at 8000 r/min for 10 min, to separate the organic
phase from the water phase. Take the water phase. Add the isopropyl alcohol (4.10),
which has the same volume as the water phase solution, AND the sodium acetate
solution (4.11), which has 1/10 volume of the aqueous solution. Gently invert and mix
well.
6.2.3 Other methods for template DNA extraction
It may use an equivalent DNA extraction (kit), to extract the template DNA.
6.3 PCR reaction of specimens
In a 200 µL or 500 µL PCR reaction tube, add 5 µL of 10 X PCR buffer, 1 µL of each
10 mmol/L mixed solution of four kinds of deoxyribonucleic acid (dATP, dCTP, dGTP,
dTTP), 1 µL of primer solution (containing forward and reverse primer), 10 µL of
template DNA (25 ng ~ 50 ng), 1 µL of Taq DNA polymerase. Add sterilized water, to
make the PCR reaction system reach 50 µL. Add about 50 µL of paraffin oil (the
paraffin oil may not be added, for the PCR instrument which has a hot-lid equipment).
Repeat twice for each specimen.
6.4 Electrophoresis detection of PCR products
Add an appropriate volume of agarose into the electrophoresis buffer. Heat to dissolve
it. Prepare the agarose solution, which has a volume fraction of 1.5%. Then according
to the proportion of adding 5 µL of ethidium bromide solution to each 100 mL of
agarose solution, add the ethidium bromide solution. Mix well.
6.5 Endonuclease digestion reaction of PCR products
The electrophoresis detection of PCR amplification product is positive. Carry out the
restrictive endonuclease digestion reaction.
6.6 Sequencing of PCR amplification products
When necessary, the PCR amplification products shall be sequenced, for the positive
results of the PCR product digestion detection.
7 Results analysis and presentation
7.1 Electrophoresis results of PCR amplification products
The PCR amplification product of bovine-derived components is 271 bp; the PCR
amplification products of sheep-derived components are 295 bp for sheep and 294 bp
for goat.
7.2 Electrophoresis results of restrictive endonuclease digestion products
PCR amplification products Sau3AI restrictive endonuclease digestion products.
7.3 Sequence comparison
The sequencing results of PCR amplification products are compared with the specific
DNA sequences of bovine, sheep, goat, in Appendix A.
7.4 Presentation of results
If the results of electrophoresis of PCR amplification products are negative, no bovine
or sheep-derived components are detected.
Appendix A
(Normative)
Bovine, sheep, goat specific DNA sequences
A.1 PCR amplification product sequence of bovine-derived components
A.2 PCR amplification product sequence of sheep & goat-derived components
A.2.1 Sheep
A.2.2 Goat
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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