CN104250667A - Detecting method for horse source elements in meat and meat products and detecting method for donkey source element in meat and meat products - Google Patents

Abstract

The invention provides a detecting method and kit for detecting horse source elements in meat and meat products. The kit for detecting horse source elements in meat and meat products comprises an upstream primer as shown in SEQ ID No.1 (in the Specification) and a downstream primer as shown in SEQ ID No.2 (in the Specification). The invention further provides a detecting method and kit for detecting donkey source elements in the meat and meat products. The kit for detecting donkey source elements in the meat and meat products comprises an upstream primer as shown in SEQ ID No.3 (in the Specification) and a downstream primer as shown in SEQ ID No.4 (in the Specification). The upstream primer group is selected to perform PCR amplification on DNA templates extracted from the meat and meat products, so that high specificity is achieved, and the high sensitivity and accuracy of the detection methods can be guaranteed.

Description

The detection method of horse derived components and the detection method of donkey derived components in meat quail

Technical field

The invention belongs to technical field of food detection, particularly relate to the detection method of horse derived components in a kind of meat quail and the detection method of donkey derived components.

Background technology

Along with the development that deepens continuously of market economy, the illegal retailer of part is in order to seek illegitimate benefits, and a kind of phenomenon of adulterating, mixing the spurious with the genuine, has appearred in other low-cost meat kinds of adulterating in meat or meat product in SDS in broiler chickens market.Other the low-cost animal derived materials beyond nominal are mixed with in meat or meat product, what have is even all other low-cost animal derived materials beyond nominal, this greatly compromises the interests of human consumer, upset circulation market order and sound development, also certain potential conflict has been caused to ethnic and religious faith simultaneously.

The general parameter such as color and luster, smell, tissue profile of sense organ method to meat or meat product that adopt of tradition judges, but its reliability is poor, especially with the addition of the additive such as pigment and perfume compound or the SDS in broiler chickens through other processing treatment, sense organ method judgment accuracy is lower.

Summary of the invention

In view of this, the object of the present invention is to provide the detection method of horse derived components in a kind of meat quail and the detection method of donkey derived components, detection method result provided by the invention accurately, reliably.

The invention provides a kind of test kit for detecting horse derived components in meat quail, comprising: the upstream primer as shown in SEQ ID No.1 and the downstream primer as shown in SEQ ID No.2.

The present invention has logged in based on the Cytb gene order of all horses of announcement on Genbank, biosoftware DnaStar is used to carry out sequence alignment analysis, find out from country variant, area, kind horse, Cytb gene kind in conserved sequence region, for designing upstream and downstream amplimer, by designed go out each species pcr amplification primer carry out comparison again, get rid of intercrossing between planting, ensure the specificity of amplification.Meanwhile, find out homologous sequence total in the Cytb gene order of horse, design Mammals detects primer, in order to monitor each detection system.Finally, submit each species upstream and downstream amplimer sequence obtained to Gembank, carry out Blast confirmation, to guarantee that designed primer sequence does not have non-specific detection to increase to the DNA sequence dna announced at present, and selected upstream primer and downstream primer.The present invention selects specific upstream primer and downstream primer for detecting the horse derived components in meat quail, and it has good specificity, thus can realize the detection to horse derived components.

Carry out pcr amplification with the such as upstream primer shown in SEQ ID No.1 and the downstream primer as shown in SEQ ID No.2, its amplification site and amplified fragments size as shown in table 1:

The amplification site of table 1 horse derived components and amplified fragments size

The concentration of described upstream primer is preferably 0.1 μM ~ 0.5 μM, is more preferably 0.5 μM; The concentration of described downstream primer is preferably 0.1 μM ~ 0.5 μM, is more preferably 0.5 μM.

Also comprise archaeal dna polymerase, 10 × PCR Buffer solution and deionized water in described test kit, the concentration of described archaeal dna polymerase is preferably 0.5-1u/ μ L.Also comprise in described test kit, described 10 × PCR Buffer solution specifically comprises: 100mM Tris-Hcl (pH8.3), 500mM KCl, 15mM MgCl ₂.

Present invention also offers the detection method of horse derived components in a kind of meat quail, comprise the following steps:

Extract the DNA of meat sample or meat product sample;

With described DNA for template carries out pcr amplification, the upstream primer in described pcr amplification reaction system is as shown in SEQ ID No.1, and downstream primer is as shown in SEQ ID No.2;

Electrophoresis is carried out to described pcr amplification product, identifies described amplified production.

First the present invention extracts the DNA in meat sample or meat product sample, the extracting method of the present invention to described DNA is not particularly limited, commercial reagents box can be used to extract when extracting in a small amount, can extract according to CTAB-NaCl method during a large amount of extraction, specifically:

When extracting in a small amount, its extracting method is:

A. the meat gruel will taken, puts into mortar, adds liquid nitrogen, until sample freezing completely after fast, be firmly ground to Powdered, should be interrupted during grinding and add liquid nitrogen to prevent material melts;

B. mortar is moved into 65 DEG C of water-baths, when sample powder has just started to melt, in mortar, added the Solution A of 700 μ l and the Rnase A of 1.2 μ l, grind 30 seconds;

C. the ground tissue homogenate of 650 μ l is collected in Collection Tube.If homogenate volume is less than 650 μ l, supplement Solution A to 650 μ l.65 DEG C are incubated 10 minutes;

D. the Solution B of 400 μ l is added, vibration mixing;

E. the Solution C of 4 DEG C of precoolings of 1ml is added, fully after mixing, centrifugal 2 minutes of 12000rpm;

F. discard upper organic phase, then add the Solution C of 4 DEG C of precoolings of 1ml, fully after mixing, centrifugal 2 minutes of 12000rpm;

G. discard upper organic phase, then aqueous phase solution (colourless lower floor) is transferred to the Filter Cup be placed on Collection Tube, centrifugal 1 minute of 12000rpm;

H. abandon Filter Cup, in filtrate, add the DB Buffer of 400 μ l, mix;

I. the Spin Column in test kit is placed on Collection Tube.Be transferred to by the mixing solutions of aforesaid operations in Spin Column, centrifugal 1 minute of 12000rpm, abandons filtrate;

J. be added in Spin Column by the Rinse A of 500 μ l, centrifugal 30 seconds of 12000rpm, abandons filtrate;

K. be added in Spin Column by the Rinse B of 700 μ l, centrifugal 30 seconds of 12000rpm, abandons filtrate;

L. work of drilling is repeated;

M. be placed in by Spin Column on the centrifuge tube of new 1.5ml, add sterile purified water or the Elution Buffer of 50-200 μ l in the centre of Spin Column film, room temperature leaves standstill 1 minute;

N.12000rpm centrifugal 1 minute wash-out collects DNA.

During a large amount of extraction, its extracting method can be:

A. get the sample muscle tissue that 2g Subcommittee-to is broken, in 50mL centrifuge tube, add 10mL CTAB-Nacl lysate, 65 DEG C of shaking baths 1 hour;

B. add 10 μ L Proteinase K (20mg/ml), 65 DEG C of shaking baths, spend the night;

C. get 1ml and spend the night treatment solution in Eppendorf centrifuge tube, the centrifugal 10min of 12000rpm;

D. get in the centrifugal supernatant of 800 μ L to new centrifuge tube, add 600 μ L chloroforms, fully mix, the centrifugal 10min of 12000rpm;

E. get 600 μ L upper strata aqueous phases in new centrifuge tube, add the pre-cold isopropanol of 500 μ L, turn upside down centrifuge tube, precipitation at room temperature DNA, 30min;

F.12000rpm centrifugal 10min, abandons Virahol, and add 1ml 75% ice ethanol and carry out washing DNA, the centrifugal 2min of 12000rpm, abandons most ethanol, air-dry under room temperature, adds appropriate ddw dissolving DNA, and measures OD ₂₆₀value.

After obtaining the DNA in meat sample or meat product, carry out pcr amplification using it as template, the upstream primer in described pcr amplification reaction system is as shown in SEQ ID No.1, and downstream primer is as shown in SEQID No.2.

The concentration of described upstream primer is preferably 0.1 μM ~ 0.5 μM, is more preferably 0.5 μM; The concentration of described downstream primer is preferably 0.1 μM ~ 0.5 μM, is more preferably 0.5 μM.

Also comprise archaeal dna polymerase, 10 × PCR Buffer solution and deionized water in described pcr amplification reaction system, the concentration of described archaeal dna polymerase is preferably: 0.5-1u/ μ L.Described 10 × PCR Buffer solution specifically comprises: 100mM Tris-Hcl (pH8.3), 500mM KCl, 15mM MgCl ₂.In described PCR amplification system, the concentration of described template DNA is preferably 10 ~ 100ng/ μ L.

In a specific embodiment, described pcr amplification reaction system can comprise:

Described pcr amplification parameter is:

After pcr amplification terminates, electrophoresis is carried out to the pcr amplification product obtained, identify described amplified production, concrete grammar is as follows: prepare 1% concentration agarose gel, gets 8 μ L amplified productions, carry out electrophoresis, electrophoresis terminates, and EB dyes, and observes with or without non-specific amplification band under ultraviolet, illustrate if having containing horse derived component in meat or meat product, if do not contain horse derived component without in explanation meat or meat product.

Whether in order to confirm detected result further, the present invention can also carry out enzyme to the amplified production obtained further and cut, qualification digestion products, thus judge in meat sample or meat product containing horse derived components.The present invention preferably carries out enzyme with restriction enzyme xhoI to described amplified production and cuts, and its correlation parameter is as follows:

Detection system	Expection size (bp)	Checking restriction enzyme	Endonuclease bamhi size (bp)
				Horse derived component detection system	395	xhoI	142+253

After enzyme is cut, if the endonuclease bamhi obtained meets target fragment, can confirm in detected sample containing horse derived component; If do not meet target fragment, can check order further to endonuclease bamhi.

Compared with prior art, based on the polymerase chain reaction of the present invention in molecular biology (PCR) method, from gene level, qualification is carried out to meat quail DNA and differentiate, first extract the DNA of meat sample or meat product sample; Then with described DNA for template carries out pcr amplification, the upstream primer in described pcr amplification reaction system is as shown in SEQ ID No.1, and downstream primer is as shown in SEQ ID No.2; Carry out enzyme to described pcr amplification product to cut, identify described digestion products, thus comprehensively analyze according to the length scale of amplified production and restriction enzyme site information, judge the horse derived components in meat quail.The present invention selects above-mentioned primer sets to carry out pcr amplification to the DNA profiling extracted in meat quail, has higher specificity, can ensure sensitivity and the accuracy of detection method.Experimental result shows, method provided by the invention is 0.01ng/ μ L to the Monitoring lower-cut of horse derived components in meat quail, and accuracy rate can reach 100%.

The invention provides a kind of test kit for detecting donkey derived components in meat quail, comprising: the upstream primer as shown in SEQ ID No.3 and the downstream primer as shown in SEQ ID No.4.

The present invention has logged in based on the Cytb gene order of all donkeys of announcement on Genbank, biosoftware DnaStar is used to carry out sequence alignment analysis, find out conserved sequence region in the Cytb gene kind of the donkey of country variant, area, kind, for designing upstream and downstream amplimer, by designed go out each species pcr amplification primer carry out comparison again, get rid of intercrossing between planting, ensure the specificity of amplification.Meanwhile, find out homologous sequence total in the Cytb gene order of donkey, design Mammals detects primer, in order to monitor each detection system.Finally, submit each species upstream and downstream amplimer sequence obtained to Gembank, carry out Blast confirmation, to guarantee that designed primer sequence does not have non-specific detection to increase to the DNA sequence dna announced at present, and selected upstream primer and downstream primer.The present invention selects specific upstream primer and downstream primer for detecting the donkey derived components in meat quail, and it has good specificity, thus can realize the detection to donkey derived components.

Carry out pcr amplification with the such as upstream primer shown in SEQ ID No.3 and the downstream primer as shown in SEQ ID No.4, its amplification site and amplified fragments size as shown in table 2:

The amplification site of table 2 donkey derived components and amplified fragments size

The concentration of described upstream primer is preferably 0.1 μM ~ 0.5 μM, is more preferably 0.5 μM; The concentration of described downstream primer is preferably 0.1 μM ~ 0.5 μM, is more preferably 0.5 μM.

Also comprise archaeal dna polymerase, 10 × PCR Buffer solution and deionized water in described test kit, the concentration of described archaeal dna polymerase is preferably 0.5-1u/ μ L.Also comprise in described test kit, described 10 × PCR Buffer solution specifically comprises: 100mM Tris-Hcl (pH8.3), 500mM KCl, 15mM MgCl ₂.

Present invention also offers the detection method of donkey derived components in a kind of meat quail, comprise the following steps:

Extract the DNA of meat sample or meat product sample;

With described DNA for template carries out pcr amplification, the upstream primer in described pcr amplification reaction system as shown in SEQ ID No.3, shown in downstream primer SEQ ID No.4;

Electrophoresis is carried out to described pcr amplification product, identifies described amplified production.

First the present invention extracts the DNA in meat sample or meat product sample, the extracting method of the present invention to described DNA is not particularly limited, commercial reagents box can be used to extract when extracting in a small amount, can extract according to CTAB-NaCl method during a large amount of extraction, specifically:

When extracting in a small amount, its extracting method is:

A. the meat gruel will taken, puts into mortar, adds liquid nitrogen, until sample freezing completely after fast, be firmly ground to Powdered, should be interrupted during grinding and add liquid nitrogen to prevent material melts;

B. mortar is moved into 65 DEG C of water-baths, when sample powder has just started to melt, in mortar, added the Solution A of 700 μ l and the Rnase A of 1.2 μ l, grind 30 seconds;

C. the ground tissue homogenate of 650 μ l is collected in Collection Tube.If homogenate volume is less than 650 μ l, supplement Solution A to 650 μ l.65 DEG C are incubated 10 minutes;

D. the Solution B of 400 μ l is added, vibration mixing;

E. the Solution C of 4 DEG C of precoolings of 1ml is added, fully after mixing, centrifugal 2 minutes of 12000rpm;

F. discard upper organic phase, then add the Solution C of 4 DEG C of precoolings of 1ml, fully after mixing, centrifugal 2 minutes of 12000rpm;

G. discard upper organic phase, then aqueous phase solution (colourless lower floor) is transferred to the Filter Cup be placed on Collection Tube, centrifugal 1 minute of 12000rpm;

H. abandon Filter Cup, in filtrate, add the DB Buffer of 400 μ l, mix;

I. the Spin Column in test kit is placed on Collection Tube.Be transferred to by the mixing solutions of aforesaid operations in Spin Column, centrifugal 1 minute of 12000rpm, abandons filtrate;

J. be added in Spin Column by the Rinse A of 500 μ l, centrifugal 30 seconds of 12000rpm, abandons filtrate;

K. be added in Spin Column by the Rinse B of 700 μ l, centrifugal 30 seconds of 12000rpm, abandons filtrate;

L. work of drilling is repeated;

M. be placed in by Spin Column on the centrifuge tube of new 1.5ml, add sterile purified water or the Elution Buffer of 50-200 μ l in the centre of Spin Column film, room temperature leaves standstill 1 minute;

N.12000rpm centrifugal 1 minute wash-out collects DNA.

During a large amount of extraction, its extracting method can be:

A. get the sample muscle tissue that 2g Subcommittee-to is broken, in 50mL centrifuge tube, add 10mL CTAB-Nacl lysate, 65 DEG C of shaking baths 1 hour;

B. add 10 μ L Proteinase K (20mg/ml), 65 DEG C of shaking baths, spend the night;

C. get 1ml and spend the night treatment solution in Eppendorf centrifuge tube, the centrifugal 10min of 12000rpm;

D. get in the centrifugal supernatant of 800 μ L to new centrifuge tube, add 600 μ L chloroforms, fully mix, the centrifugal 10min of 12000rpm;

E. get 600 μ L upper strata aqueous phases in new centrifuge tube, add the pre-cold isopropanol of 500 μ L, turn upside down centrifuge tube, precipitation at room temperature DNA, 30min;

F.12000rpm centrifugal 10min, abandons Virahol, and add 1ml 75% ice ethanol and carry out washing DNA, the centrifugal 2min of 12000rpm, abandons most ethanol, air-dry under room temperature, adds appropriate ddw dissolving DNA, and measures OD ₂₆₀value.

After obtaining the DNA in meat sample or meat product, carry out pcr amplification using it as template, the upstream primer in described pcr amplification reaction system is as shown in SEQ ID No.3, and downstream primer is as shown in SEQ ID No.4.

The concentration of described upstream primer is preferably 0.1 μM ~ 0.5 μM, is more preferably 0.5 μM; The concentration of described downstream primer is preferably 0.1 μM ~ 0.5 μM, is more preferably 0.5 μM.

Also comprise archaeal dna polymerase, 10 × PCR Buffer solution and deionized water in described pcr amplification reaction system, the concentration of described archaeal dna polymerase is preferably 0.5-1u/ μ L.Also comprise in described test kit, described 10 × PCR Buffer solution specifically comprises: 100mM Tris-Hcl (pH8.3), 500mM KCl, 15mM MgCl ₂.In described PCR amplification system, the concentration of described template DNA is preferably 10 ~ 100ng/ μ L.

In a specific embodiment, described pcr amplification reaction system can comprise:

Described pcr amplification parameter is:

After pcr amplification terminates, electrophoresis is carried out to the pcr amplification product obtained, identify described amplified production, concrete grammar is as follows: prepare 1% concentration agarose gel, gets 8 μ L amplified productions, carry out electrophoresis, electrophoresis terminates, and EB dyes, and observes with or without non-specific amplification band under ultraviolet, illustrate if having containing donkey derived component in meat or meat product, if do not contain donkey derived component without in explanation meat or meat product.

Whether in order to confirm detected result further, the present invention can also carry out enzyme to the amplified production obtained further and cut, qualification digestion products, thus judge in meat sample or meat product containing donkey derived components.The present invention preferably carries out enzyme with restriction enzyme xhoI to described amplified production and cuts, and its correlation parameter is as follows:

Detection system	Expection size (bp)	Checking restriction enzyme	Endonuclease bamhi size (bp)
				Donkey derived component detection system	229	XhoI	103+126

After enzyme is cut, if the endonuclease bamhi obtained meets target fragment, can confirm in detected sample containing horse derived component; If do not meet target fragment, can check order further to endonuclease bamhi.

Compared with prior art, based on the polymerase chain reaction of the present invention in molecular biology (PCR) method, from gene level, qualification is carried out to meat quail DNA and differentiate, first extract the DNA of meat sample or meat product sample; Then with described DNA for template carries out pcr amplification, the upstream primer in described pcr amplification reaction system is as shown in SEQ ID No.3, and downstream primer is as shown in SEQ ID No.4; Carry out enzyme to described pcr amplification product to cut, identify described digestion products, thus comprehensively analyze according to the length scale of amplified production and restriction enzyme site information, judge the donkey derived components in meat quail.The present invention selects above-mentioned primer sets to carry out pcr amplification to the DNA profiling extracted in meat quail, has higher specificity, can ensure sensitivity and the accuracy of detection method.Experimental result shows, method provided by the invention is 1.0ng/ μ L to the Monitoring lower-cut of donkey derived components in meat quail, and accuracy rate can reach 100%.

Accompanying drawing explanation

In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is only embodiments of the invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to the accompanying drawing provided.

Fig. 1 is the electrophoresis result of the amplified production that the embodiment of the present invention 1 obtains;

Fig. 2 is the electrophoresis result of the amplified production that the embodiment of the present invention 1 and comparative example 1 ~ 8 obtain;

The electrophoresis result of the horse that Fig. 3 provides for the embodiment of the present invention and donkey standard endonuclease bamhi;

Fig. 4 is the electrophoresis result of the donkey amplified production that the embodiment of the present invention 4 obtains;

Fig. 5 is the electrophoresis result of the amplified production that the embodiment of the present invention 4 and comparative example 9 ~ 17 obtain;

Fig. 6 is the electrophoresis result of the amplified production that the embodiment of the present invention 7 ~ 16 obtains;

Fig. 7 is the electrophoresis result of the amplified production that the embodiment of the present invention 17 ~ 26 obtains.

Embodiment

Below in conjunction with the embodiment in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.

Embodiment 1

The method extracted according to a small amount of mentioned above extracts the DNA of Mongolian horse;

To extract the DNA that obtains as template, increase according to following Amplification in following pcr amplification reaction system, obtain amplified production;

Pcr amplification reaction system can comprise:

Described pcr amplification parameter is:

Wherein, upstream primer as shown in SEQ ID No.1, shown in downstream primer SEQ ID No.2;

After pcr amplification terminates, electrophoresis is carried out to the pcr amplification product obtained, identify described amplified production, concrete grammar is as follows: prepare 1% concentration agarose gel, gets 8 μ L amplified productions, carries out electrophoresis, electrophoresis terminates, EB dyes, and observe under ultraviolet, result is see Fig. 1, Fig. 1 is the electrophoresis result of the amplified production that the embodiment of the present invention 1 obtains, wherein, the electrophoresis result of 1 amplified production obtained for embodiment, M is DL2000.As shown in Figure 1, method provided by the invention can increase and obtain goal gene, therefore, it is possible to for the detection of horse derived components.

Embodiment 2

Detect horseflesh according to the method identical with embodiment 1, difference is, extracts the DNA of Yili horse, result is the electrophoresis result of the amplified production that the embodiment of the present invention 1 obtains see Fig. 1, Fig. 1, wherein, the electrophoresis result of 2 amplified productions obtained for embodiment 2, M is DL2000.As shown in Figure 1, method provided by the invention can realize the detection of the horse derived components to different varieties.

Comparative example 1 ~ 8

Respectively rabbit meat, goose, duck, chicken, mutton, donkey meat, beef and pork are detected according to the method identical with embodiment 1, result is see Fig. 2, Fig. 2 is the electrophoresis result of the amplified production that the embodiment of the present invention 1 and comparative example 1 ~ 8 obtain, and wherein M is DL2000.As shown in Figure 2, method provided by the invention has good specificity.

Embodiment 3

After obtaining amplified production according to the method identical with embodiment 1, adopt restriction enzyme xhoI to carry out enzyme to it and cut, result is see Fig. 3, and wherein M is the electrophoresis result of the endonuclease bamhi that DL2000, Fig. 3 provide for the embodiment of the present invention.

Embodiment 4

The method extracted according to a small amount of mentioned above extracts the DNA of Region in Guanzhong Donkey;

To extract the DNA that obtains as template, increase according to following Amplification in following pcr amplification reaction system, obtain amplified production;

Pcr amplification reaction system can comprise:

Described pcr amplification parameter is:

Wherein, upstream primer as shown in SEQ ID No.3, shown in downstream primer SEQ ID No.4;

After pcr amplification terminates, electrophoresis is carried out to the pcr amplification product obtained, identify described amplified production, concrete grammar is as follows: prepare 1% concentration agarose gel, gets 8 μ L amplified productions, carries out electrophoresis, electrophoresis terminates, EB dyes, and observe under ultraviolet, result is see Fig. 4, Fig. 4 is the electrophoresis result of the amplified production that the embodiment of the present invention 4 obtains, wherein, 1 is the electrophoresis result of the amplified production that embodiment 4 obtains, and M is DL2000.As shown in Figure 4, method provided by the invention can increase and obtain goal gene, therefore, it is possible to for the detection of donkey derived components.

Embodiment 5

Detect donkey meat according to the method identical with embodiment 4, difference is, extracts the DNA of Dezhou donkey, result is the electrophoresis result of the amplified production that the embodiment of the present invention 4 obtains see Fig. 4, Fig. 4, wherein, the electrophoresis result of 2 amplified productions obtained for embodiment 5, M is DL2000.As shown in Figure 4, method provided by the invention can realize the detection of the donkey derived components to different varieties.

Comparative example 9 ~ 17

Respectively rabbit meat, goose, duck, chicken, mutton, horseflesh, beef and pork are detected according to the method identical with embodiment 4, result is see Fig. 5, Fig. 5 is the electrophoresis result of the amplified production that the embodiment of the present invention 4 and comparative example 9 ~ 17 obtain, and wherein M is DL2000.As shown in Figure 5, method provided by the invention has good specificity.

Embodiment 6

After obtaining amplified production according to the method identical with embodiment 1, adopt restriction enzyme xhoI to carry out enzyme to it and cut, result is see Fig. 3, and wherein M is the electrophoresis result of the endonuclease bamhi that DL2000, Fig. 3 provide for the embodiment of the present invention

Embodiment 7 ~ 16

According to the method identical with embodiment 1, horseflesh is detected, difference is, the concentration of DNA profiling is respectively 0.01,0.1,1.0,10,50,100,200,300,400 and 500ng/ μ L, result is see Fig. 6, Fig. 6 is the electrophoresis result of the amplified production that the embodiment of the present invention 7 ~ 16 obtains, wherein M be DL2000. as shown in Figure 6, method provided by the invention is 0.01ng/ μ L to the Monitoring lower-cut of horse derived components.

Embodiment 17 ~ 26

According to the method identical with embodiment 4, donkey meat is detected, difference is, the concentration of DNA profiling is respectively 0.01,0.1,1.0,10,50,100,200,300,400 and 500ng/ μ L, result is see Fig. 7, Fig. 7 is the electrophoresis result of the amplified production that the embodiment of the present invention 17 ~ 26 obtains, wherein M be DL2000. as shown in Figure 7, method provided by the invention is 1.0ng/ μ L to the Monitoring lower-cut of horse derived components.

Embodiment 27

Detect according to the method identical with embodiment 1, difference is, detected object is the mixture of horseflesh and donkey meat, and detect and all can detect that it contains horseflesh composition for 20 times, the accuracy rate of method provided by the invention can reach 100%.

Embodiment 28

Detect according to the method identical with embodiment 1, difference is, detected object is donkey meat, and detect and all cannot detect that it contains horseflesh composition for 20 times, the accuracy rate of method provided by the invention can reach 100%.

Embodiment 29

Detect according to the method identical with embodiment 4, difference is, detected object is the mixture of horseflesh and donkey meat, and detect and all can detect that it contains donkey meat composition for 20 times, the accuracy rate of method provided by the invention can reach 100%.

Embodiment 30

Detect according to the method identical with embodiment 4, difference is, detected object is horseflesh, and detect and all cannot detect that it contains donkey meat composition for 20 times, the accuracy rate of method provided by the invention can reach 100%.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (10)

1. for detecting a test kit for horse derived components in meat quail, it is characterized in that, comprising: the upstream primer as shown in SEQ ID No.1 and the downstream primer as shown in SEQ ID No.2.

2. test kit according to claim 1, is characterized in that, the concentration of described upstream primer is 0.1 μM ~ 0.5 μM, and the concentration of described downstream primer is 0.1 μM ~ 0.5 μM.

3. the detection method of horse derived components in meat quail, comprises the following steps:

Extract the DNA of meat sample or meat product sample;

With described DNA for template carries out pcr amplification, the upstream primer in described pcr amplification reaction system is as shown in SEQ ID No.1, and downstream primer is as shown in SEQ ID No.2;

Electrophoresis is carried out to described pcr amplification product, identifies described amplified production.

4. detection method according to claim 3, is characterized in that, also comprises:

Carry out enzyme to described amplified production to cut.

5. the detection method according to claim 3 or 4, is characterized in that, in described pcr amplification reaction system, the concentration of described upstream primer is 0.1 μM ~ 0.5 μM, and the concentration of described downstream primer is 0.1 μM ~ 0.5 μM.

6. detection method according to claim 5, is characterized in that, in described pcr amplification reaction system, the concentration of described upstream primer is 0.5 μM, and the concentration of described downstream primer is 0.5 μM.

7. for detecting a test kit for donkey derived components in meat quail, it is characterized in that, comprising: the upstream primer as shown in SEQ ID No.3 and the downstream primer as shown in SEQ ID No.4.

8. test kit according to claim 7, is characterized in that, the concentration of described upstream primer is 0.1 μM ~ 0.5 μM, and the concentration of described downstream primer is 0.1 μM ~ 0.5 μM.

9. the detection method of donkey derived components in meat quail, comprises the following steps:

Extract the DNA of meat sample or meat product sample;

With described DNA for template carries out pcr amplification, the upstream primer in described pcr amplification reaction system as shown in SEQ ID No.3, shown in downstream primer SEQ ID No.4;

Electrophoresis is carried out to described pcr amplification product, identifies described amplified production.

10. detection method according to claim 9, is characterized in that, in described pcr amplification reaction system, the concentration of described upstream primer is 0.1 μM ~ 0.5 μM, and the concentration of described downstream primer is 0.1 μM ~ 0.5 μM.