GB/T 20190-2006 English PDF (GBT20190-2006)

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GB/T 20190-2006: Detection of bovine, sheep and goat-derived material in feeds -- Qualitative polymerase chain reaction (PCR) method
GB/T 20190-2006
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 
Detection of bovine, sheep and goat-derived material in feeds
- Qualitative polymerase chain reaction (PCR) method
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. Use species-specific primers to
amplify the specific DNA sequence by PCR, separate the PCR products by
electrophoresis, use the standard-length PCR product as a control, to detect the specific
DNA fragment, which is amplified by PCR, to determine whether it contains bovine,
sheep and goat-derived ingredients. In addition, the results are further judged, by
restricting the endonuclease digestion reaction. Test results are confirmed, by
sequencing specific DNA fragments, which are amplified by PCR, AND comparing
with standard sequences.
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.
4.2 Trimethylaminomethane hydrochloric acid (Tris-HCl) solution, 1 mol/L, pH 7.5:
Dissolve 12.11 g of Tris in 80 mL of deionized water. Cool to room temperature. Use
concentrated hydrochloric acid, to adjust the pH of the solution to 7.5. Add water to
make the volume reach to 100 mL. 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.
4.5 Cetyltrimethyl ammonium bromide (CTAB) extraction buffer I: Add 46.75 g of
sodium chloride to 800 mL of deionized water. Shake the container to completely
dissolve the solute. Then add 50 mL of Tris-HCl solution (4.1) and 20 mL of EDTA
solution (4.4). Make the volume reach to 1 L. Divide-contain it. Autoclave it.
4.6 Cetyltrimethyl ammonium bromide (CTAB) extraction buffer II: Add 46.75 g of
sodium chloride and 20 g of cetyltrimethyl ammonium bromide (CTAB) to 800 mL of
deionized water. Shake the container to completely dissolve the solute. Then add 50 mL
of Tris-HCl solution (4.1) and 20 mL of EDTA solution (4.4). 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.
(4.5), which was pre-chilled on ice. Add 500 µL of CTAB extraction buffer II (4.6),
which was preheated at 65 °C. Mix well. Keep at 65 °C for 30 ~ 90 minutes, during
which invert and mix gently from time to time. After cooling to room temperature, add
5 µL of RNase A stock solution (4.7). Place it at room temperature, for 30 min.
Centrifuge it at 12000 r/min for 10 min. Take the supernatant. Add the same volume of
Tris saturated phenol + chloroform (4.8) as the supernatant. Gently invert to mix the
solution well. Centrifuge at 12000 r/min for 10 min. Take the supernatant. Add the equal
volume of chloroform + isoamyl alcohol (4.9). Gently invert to mix the solution well.
Centrifuge at 12000 r/min for 10 min. Transfer the supernatant into a clean centrifugal
tube. Add equal volume of isopropanol (4.10) and 1/10 volume of sodium acetate
solution (4.11) in turn. Gently invert to mix the solution well. Centrifuge at 12000 r/min
for 10 min. Discard the supernatant. Add 800 µL of ethanol (4.12), which has a volume
fraction of 75%, to wash the precipitate. Centrifuge at 12000 r/min for 5 min. Discard
the supernatant. Then add 100 µL of ethanol, which has a volume fraction of 75%, to
wash the precipitate. Centrifuge at 12000 r/min for 5 min. Discard the supernatant.
Remove the residual ethanol. After the precipitate is dried, dissolve the DNA precipitate
in 100 µL of TE buffer (4.13), to prepare for testing, OR store it at -20 °C for later use.
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. After standing for 10 minutes at room temperature. Centrifuge at 12000 r/min for
10 minutes. Discard the supernatant. After the precipitate is dry, use 200 µL of TE buffer