GB/T 20190-2006 PDF in English
GB/T 20190-2006 (GB/T20190-2006, GBT 20190-2006, GBT20190-2006)
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Detection of bovine, sheep and goat-derived material in feeds -- Qualitative polymerase chain reaction (PCR) method
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Standards related to (historical): GB/T 20190-2006
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GB/T 20190-2006: PDF in English (GBT 20190-2006) 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
(4.13), to dissolve the precipitate. Add 200 µL of Tris saturated phenol + chloroform
(4.8). Gently invert to mix the solution. Centrifuge at 12000 r/min for 10 min. Take the
supernatant. Add the chloroform + Isoamyl alcohol (4.9), which has the same volume
as supernatant. Gently invert to mix the solution. Centrifuge at 12000 r/min for 2 min,
to separate the phases. Transfer the supernatant to a clean centrifuge tube. Add the
isopropanol (4.10), which has the same volume as the solution, AND the sodium acetate
solution (4.11), which has a volume of 1/10 of the solution. Gently invert to mix the
solution. After standing at room temperature for 10 min, centrifuge at 12000 r/min for
10 min. After discarding the supernatant, add 800 µL of 75% ethanol and 100 µL of 75%
ethanol (4.12) in turn, to wash the precipitate. Centrifuge at 12000 r/min for 5 min.
Discard the ethanol solution. After the precipitation is dried, dissolve the DNA
precipitate in 100 µL of TE buffer (4.13), to prepare for testing OR store at -20 °C for
later use.
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.
After centrifugation at 4000 r/min for 10 s, insert the PCR tube into the PCR instrument.
Let it be subject to 95 °C constant temperature for 1 min ~ 3 min. Carry out 30
amplification reaction cycles (95 °C constant temperature for 30 s ~ 60 s, 56 °C constant
temperature for 30 s ~ 60 s, 72 °C constant temperature for 30 s ~ 60 s). Then let it be
subject to 72 °C constant temperature for 5 min. Take out the PCR reaction tube. Carry
out electrophoresis detection for the reactant. OR store it at 4 °C.
During the PCR reaction of the specimen, it shall set a negative control, a positive
control, a blank control.
Negative control refers to the use of DNA, which is extracted from the feed containing
no bovine or sheep & goat, as the DNA template of the PCR reaction system. Positive
control refers to the use of DNA, which is extracted from the feed containing bovine or
sheep & goat, as the template of the PCR reaction system. The blank control refers to
the use of sterile double-distilled water or a reagent without target DNA as the DNA
template of the PCR reaction system. In the above-mentioned control PCR reaction
system, the other components are the same, except for the template.
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. Cool slightly. Pour it
into electrophoresis plate. Solidify into gel at room temperature. Put it into
electrophoresis buffer. Add 5 µL ~ 8 µL of PCR product (need to be mixed with loading
buffer) to each electrophoresis path. Add DNA molecular weight marker, to one of the
electrophoresis paths. Turn on the power. At 9 V/cm voltage, perform electrophoresis
for 20 min ~ 30 min.
After electrophoresis, place the agarose gel on a gel imager or UV transilluminator, for
imaging. The size of the amplified target band is judged, according to the DNA
molecular weight markers. Achieve the electrophoresis results in electronic files OR
use a camera system for photographing.
Add 170 µL of formamide solution to the purified product, at 95 °C for 5 min. Quickly
transfer it onto ice. Hold for 2 min. Dispense the samples into the sample loading tank
of the sequencer, to perform automatic sequencing.
6.6.5 Splicing of sequencing product
Use the forward and reverse primers, for sequencing amplification reaction, purification,
sequencing, respectively. Splice the two measured sequences, to obtain the final PCR
product sequencing result.
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:
bovine-derived components are 57 bp and 214 bp; sheep are 91 bp and 204 bp; goat are
92 bp and 202 bp.
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.
If the electrophoresis test result of PCR amplification product is positive, AND the size
of the restrictive endonuclease-digested product is correct, it is confirmed that it
contains bovine or sheep-derived components; however, if the size of the endonuclease-
digested product is incorrect, it is confirmed that it does not contain bovine or sheep-
derived components.
If the enzyme digestion result is confirmed by sequencing, AND the consistency
between the sequencing result and the specific DNA sequence of bovine, sheep and goat
in Appendix A is more than 90% (including 90%), then, it is confirmed that it contains
bovine or sheep-derived components; however, if the consistency of the result is below
90%, then it is confirmed that it does not contain bovine or sheep-derived ingredients
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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