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US$335.00 · In stock · Download in 9 secondsGB/T 20190-2025: Determination of bovine, ovine and goat-derived materials in feeds Delivery: 9 seconds. True-PDF full-copy in English & invoice will be downloaded + auto-delivered via email. See step-by-step procedureStatus: Valid GB/T 20190: Evolution and historical versions
| Standard ID | Contents [version] | USD | STEP2 | [PDF] delivery | Name of Chinese Standard | Status |
| GB/T 20190-2025 | English | 335 |
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Determination of bovine, ovine and goat-derived materials in feeds
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| GB/T 20190-2006 | English | 140 |
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Detection of bovine, sheep and goat-derived material in feeds - Qualitative polymerase chain reaction (PCR) method
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GB/T 20190-2025: Determination of bovine, ovine and goat-derived materials in feeds---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/GBT20190-2025
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 65.120
CCS B 46
Replacing GB/T 20190-2006
Determination of bovine, ovine and goat-derived materials
in feeds
Issued on: JUNE 30, 2025
Implemented on: JANUARY 01, 2026
Issued by. State Administration for Market Regulation;
Standardization Administration of the People’s Republic of China.
Table of Contents
Foreword... 3
1 Scope... 5
2 Normative references... 5
3 Terms and definitions... 5
4 Abbreviated terms... 6
5 Real-time fluorescent polymerase chain reaction... 6
6 Polymerase chain reaction... 10
7 Laboratory pollution prevention and control measures... 17
8 Hazardous waste disposal... 17
Appendix A (Informative) Real-time fluorescence PCR detection of internal reference
genes and target gene sequences in bovine, ovine and goat... 18
Appendix B (Normative) PCR detection of internal reference genes and target gene
sequences in bovine, ovine, and goat... 19
Bibliography... 20
Determination of bovine, ovine and goat-derived materials
in feeds
1 Scope
This document describes methods for the determination of bovine, ovine and goat-
derived materials in feeds using real-time fluorescent polymerase chain reaction and
polymerase chain reaction.
This document applies to the detection of bovine, ovine and goat-derived materials in
compound feeds, concentrated feeds, concentrate supplements, feed ingredients,
compound premixes and mixed feed additives.
The detection limit for this document is 0.1%.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this
document and are indispensable for its application. For dated references, only the
edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
GB/T 6682, Water for analytical laboratory use - Specification and test methods
GB/T 19495.3-2004, Detection of genetically modified organisms and derived
products - Nucleic acid extraction
GB/T 20195, Animal feeding stuffs. Preparation of test samples
GB/T 27403-2008, Criterion on quality control of laboratories - Molecular
biological testing of food
GB/T 35918-2018, Identification of animal origin in animal products by DNA
barcoding - Sanger sequencing
3 Terms and definitions
No terms and definitions need to be defined in this document.
4 Abbreviated terms
For the purposes of this document, the following abbreviated terms apply.
bp. base pair
Ct. cycle threshold
CTAB. cetyltrimethylammonium bromide
DNA. deoxyribonucleic acid
EDTA. ethylene diamine tetraacetic acid
PCR. polymerase chain reaction
Tris. trihydroxymethyl aminomethane
5 Real-time fluorescent polymerase chain reaction
5.1 Principle
Design primers and probes based on the specific sequences of the bovine β-actin gene,
the ovine prolactin receptor gene, and the goat chromosome 9 gene. Use real-time
fluorescence PCR technology for amplification, and detect the bovine, ovine, and goat-
derived DNA components based on the fluorescent signal and Ct value generated in the
amplification reaction.
5.2 Reagents or materials
Unless otherwise specified, use analytical reagents only. All reagents shall be stored or
aliquoted in containers free of DNA enzyme contamination.
5.2.1 Water. GB/T 6682, grade 1.
5.2.2 2× real-time PCR premix. Containing Taq DNA polymerase, real-time
fluorescence PCR buffer, MgCl2, and dNTPs; shall not contain bovine serum albumin.
5.2.3 Sodium hydroxide solution (10 mol/L). Weigh 400 g of sodium hydroxide; add
water to fix the volume to 1 L.
5.2.4 Tris-hydrochloric acid solution (1 mol/L). Weigh 121.1 g of Tris and dissolve it
in 800 mL of water; use hydrochloric acid to adjust the pH to 8.0; add water to dilute to
1 L. Sterilize at 103.4 kPa and 121 °C for 20 min and store at room temperature.
5.2.5 EDTA solution (0.5 mol/L). Weigh 186.1 g of Na2EDTA·2H2O and dissolve it in
800 mL of water; stir vigorously on a magnetic stirrer; add approximately 20 g of
5.3.2 Analytical balance. precision 0.1 mg.
5.3.3 Centrifuge. centrifugal speed not less than 14 000 r/min.
5.3.4 Ultraviolet spectrophotometer or micro-nucleic acid protein analyzer.
5.3.5 Micropipettes. 2.5 μL, 10 μL, 100 μL, 200 μL, 1 000 μL, etc.
5.4 Test sample
Prepare a sample of at least 200 g according to GB/T 20195; crush or grind it so that it
passes through a 0.25 mm test sieve; mix it thoroughly; place it in a sealed container
for later use. Other crushing or grinding methods that have been shown to be equivalent
may also be used.
Prepare the positive control sample (5.2.8) and negative control sample (5.2.9) in the
same manner and set aside.
Note. After crushing or grinding a sample, clean the crusher or mill container and
knives to prevent contamination.
5.5 Procedure
5.5.1 DNA extraction
Perform two tests in parallel. Weigh 100 mg ~ 200 mg of the sample; extract DNA
according to the method in C.6.2 of Appendix C of GB/T 19495.3-2004.Alternatively,
use a verified and reliable DNA extraction kit for DNA extraction. Refer to the
instructions for specific use. The extraction of blank control is the same as that of
sample except that no sample is added. If necessary, extract DNA from positive and
negative control samples. If the extracted DNA needs to be stored, store it below -18°C.
5.5.2 DNA solution concentration determination
Measure the concentration of the DNA solution (5.5.1) using an ultraviolet
spectrophotometer (wavelength. 260 nm) or a micro-nucleic acid protein analyzer
(5.3.4). The mass concentration of DNA solution should be controlled at 5 ng/μL ~ 50
ng/μL.
5.5.3 Control settings
During the real-time fluorescence detection process, positive controls, negative controls,
amplification blank controls, and extraction blank controls shall be set up (5.5.1). Use
the DNA from the positive sample (5.2.8) as the positive control, and the DNA from
the negative sample (5.2.9) as the negative control. Use an equal volume of water to
replace the DNA solution as the amplification blank control.
5.5.4 Reaction system preparation
5.6.2 Amplification of bovine, ovine and goat-derived specific genes
Real-time fluorescence PCR detection of bovine, ovine and goat-derived specific genes
shall meet the following conditions.
a) Extraction of blank control. Ct value = 40.0 or no Ct value;
b) Amplification of blank control. Ct value = 40.0 or no Ct value;
c) Negative control. Ct value = 40.0 or no Ct value;
d) Positive control. Fluorescent signal is detected in the fluorescence channel and
a typical amplification curve appears, with a Ct value ≤ 35.0.
5.7 Result judgment and expression
5.7.1 Result judgment
When compliance with 5.6 is achieved, the test results of the test samples shall be
determined as follows.
a) If the Ct value is ≤35.0, the test sample is considered positive;
b) If the Ct value is =40.0 or there is no Ct value, the sample is considered
negative;
c) If 35.0< Ct value< 40.0, re-measurement is required. If the Ct value after further
amplification is still less than 40.0, the sample is judged to be positive; if the
Ct value after further amplification is = 40 or there is no Ct value, the sample
is judged to be negative.
5.7.2 Result expression
A positive result is expressed as "××-derived DNA component detected".
A negative result is expressed as “no ××-derived DNA component detected”.
6 Polymerase chain reaction
6.1 Principle
Design primers based on the specific sequences of mitochondrial genes of bovine, ovine
and goat, and use PCR technology for amplification. Separate PCR products by
electrophoresis; compare with DNA molecular weight markers; sequence, and compare
with species reference sequences in the gene library to detect DNA components derived
from bovine, ovine, and goat.
6.2 Reagents and materials
Unless otherwise specified, use analytical reagents only. All reagents shall be stored or
aliquoted in containers free of DNA enzyme contamination.
6.2.1 Water. GB/T 6682, grade 1.
6.2.2 n-Hexane.
6.2.3 Isopropyl alcohol.
6.2.4 2× PCR reaction premix. Containing Taq DNA polymerase, PCR buffer, MgCl2
and dNTPs; shall not contain bovine serum albumin.
6.2.5 Ethanol with a volume fraction of 75%.
6.2.6 Agarose.
6.2.7 Nucleic acid dye Goldview solution (10 000×).
6.2.8 DNA molecular weight markers (covering the range of 100 bp ~ 500 bp).
6.2.9 PCR product recovery and purification kit. follow the instructions.
6.2.10 DNA sequencing reagents.
6.2.11 Ethanol with a volume fraction of 95%.
6.2.12 Formamide.
6.2.13 Tris-hydrochloric acid solution (1 mol/L). same as 5.2.4.
6.2.14 EDTA solution (0.5 mol/L). same as 5.2.5.
6.2.15 Cetyltrimethylammonium bromide (CTAB) extraction buffer. Add 46.75 g of
sodium chloride and 20 g of cetyltrimethylammonium bromide (CTAB) to 800 mL of
deionized water; shake the container to completely dissolve the solutes; then add 50 mL
of Tris-hydrochloric acid solution (6.2.13) and 20 mL of E-TTA solution (6.2.14); use
water to fix the volume to 1 L; after aliquoting, sterilize at 103.4 kPa and 121°C for 20
min; set aside.
6.2.16 Mixture of Tris-saturated phenol and trichloromethane, V (Tris-saturated phenol)
+ V (trichloromethane) = 1 + 1.
6.2.17 Mixture of trichloromethane and isoamyl alcohol, V (trichloromethane) + V
(isoamyl alcohol) = 24 + 1.
6.2.18 Sodium acetate solution (3 mol/L). Add 40.81 g of sodium acetate trihydrate to
80 mL of water; dissolve, and use glacial acetic acid to adjust the pH to 5.2; use water
to fix the volume to 100 mL; aliquot, and sterilize at 103.4 kPa and 121°C for 20 min;
set aside.
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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