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Method and kit for detecting ox and sheep components in feed

A technology for feed, cattle and sheep, applied in the field of detection, can solve the problems of large quantitative error, cumbersome analysis process, inability to meet the requirements of rapidity and high sensitivity, etc.

Inactive Publication Date: 2006-08-02
浙江迪恩生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, methods such as enzyme chain immunosorbent assay (ELISA), ordinary PCR, nested PCR, and immuno-PCR are mainly used to detect cattle and sheep-derived components in feed (Multiplex polymerase chain reaction method for detection of bovine materials in foodstuffs, Gao HW, ZhangDB , Pan AH, Liang WQ, Liang CZ. J AOAC Int. 2003 Jul-Aug; 86(4): 764-7; Development of a PCR assay for the detection of animal tissues inruminant feeds. Bottero MT, Dalmasso IA, Nucera D , Turi RM, Rosati S, Squadrone S, Goria M, Civera T.J Food Prot.2003 Dec; 66(12): 2307-12.), this type of method, the follow-up analysis process is cumbersome; the sensitivity is not high enough; it is easy to produce false positive results , causing misreading; and the quantitative error is large
For example, the common PCR method to detect cattle and sheep components is not only time-consuming, but also requires electrophoresis, staining, enzyme digestion, sequencing and other methods for subsequent analysis, which cannot meet the daily detection requirements of rapidity and high sensitivity.

Method used

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  • Method and kit for detecting ox and sheep components in feed
  • Method and kit for detecting ox and sheep components in feed
  • Method and kit for detecting ox and sheep components in feed

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Design and Synthesis of Primer Pairs and Probes Specific to Bovine / Ovine Components in Feed

[0053] According to the characteristic sequences of various cattle, sheep, pigs, chickens, fish, horses, etc. listed in the NCBI database, after sequence comparison, select a suitable fragment from the D-Loop region, and then use ABI's PrimerExpress software, The following primers and probes with very high specificity were designed for cattle and sheep respectively:

[0054] Upstream primer: 5'GAG CTT AAT TAC CAT GCC G 3' (SEQ ID NO: 3);

[0055] Downstream primer: 5'TTA TGT GTG AGC ATG GGC 3' (SEQ ID NO: 4);

[0056] Bovine probe: FAM-5'TTT CTT CAG GGC CAT C 3'-TAMRA (SEQ ID NO: 5);

[0057] Sheep probe: (FAM-5'AGG GAT CCC TCT TC 3'-TAMRA) (SEQ ID NO: 6);

[0058] Universal probe: (FAM-5'AGG GAT CCC TCT TC 3'-TAMRA) (SEQ ID NO: 7);

[0059] Among them, FAM is a fluorescent reporter group, and TAMRA is a fluorescent quencher group.

[0060] The bovine D-Loop region sequenc...

Embodiment 2

[0063] Detection of ingredients of bovine / sheep origin in feed

[0064] First, extract DNA samples from various feed samples by conventional methods, or use the following extraction kits to extract DNA samples from various feed samples:

[0065] A. Buffer 1 (100mmol / L Tris-HCl, pH=8.0, 0.20mmol / L EDTA, 500mmol / L NaCl)

[0066] B. Buffer 2 (diethyl ether, phenol: chloroform: isoamyl alcohol = 24: 25: 1, 10% SDS)

[0067] C. Wash buffer (70% ethanol, 3mol / L sodium acetate)

[0068] D. Proteinase K (dry powder, 10mg / tube, mixed with 10mg / mL when used)

[0069] E. DNA adsorption column.

[0070] Next, add primers (0.25ul each, concentration 20μM / L) and extracted DNA to a PCR reaction system (40uL) containing the following components:

[0071] A. 20% glycerol (10uL)

[0072] B. 10×TaqMan Buffer A (5uL)

[0073] C. 25uM MgCl 2 (10uL),

[0074] D. dATP(1uL), dCTP(1uL), dGTP(1uL), dUTP(1uL)

[0075] E. Gold Taq polymerase (5U / uL, purchased from ABI company) 0.5uL

[0076] F....

Embodiment 3

[0090] Comparison of experimental results of negative samples and positive samples

[0091] In order to verify the method of the present invention, the same method as in Example 2 was used to detect 20 blind samples respectively. That is, the DNA was first extracted from the finely ground feed samples, and then on the 7000 SequenceDetection System (Applied Biosystems, USA) real-time fluorescence quantitative instrument, the samples were detected with universal probes to determine whether the samples were positive or negative. If the sample is positive, the bovine probe and the sheep probe are further used for detection to determine the composition and content of the sample.

[0092] The test results are shown in Table 3 and Table 4, which are consistent with the actual composition.

[0093]

[0094] Note: % is the mass percentage of feed; Ct value is cycle threshold

[0095]

[0096] Note: % is the mass percentage of feed; Ct value is cycle threshold

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Abstract

The present invention provides genetic marker, method and reagent kit for detecting ox originated / sheep originated components in feed. Specifically, the present invention provides the D-loop doze nucleotide sequence of the ox originated / sheep originated components in feed and the primer and probe designed based on the sequence. The method may be used in convenient, fast and accurate qualitative and quantitative detection of ox originated / sheep originated components in feed.

Description

technical field [0001] The invention relates to the field of detection, and more specifically relates to a nucleotide sequence that can be used to detect bovine / goat-derived components in feed and a method for detecting bovine / goat-derived components in feed based on the sequence. Background technique [0002] In recent years, the international community has paid more and more attention to the safety of animal-derived foods. The Ministry of Agriculture of my country has also successively issued some regulations prohibiting the import of ruminant meat and meat and bone meal from the source countries of mad cow disease such as the United Kingdom, and prohibiting the use of protein feed derived from ruminants. [0003] At present, methods such as enzyme chain immunosorbent assay (ELISA), ordinary PCR, nested PCR, and immuno-PCR are mainly used to detect cattle and sheep-derived components in feed (Multiplex polymerase chain reaction method for detection of bovine materials in f...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 张苏华沈富林黄士新李洁周文骏王蓓孙亚云曹莹
Owner 浙江迪恩生物科技股份有限公司
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