CN204079950U - Fowl Sex Rapid identification test kit - Google Patents

Abstract

The utility model discloses a kind of Fowl Sex Rapid identification test kit, belong to biological technical field, comprise the liner in box body and box body, described liner is provided with vestibule, is respectively equipped with Auele Specific Primer mixture JYp, amplification buffer, DNAMarker and agar Icing Sugar in described vestibule.The utility model is according to the CHD1 gene order appropriate design PCR primer be present on poultry sex chromosome W karyomit(e) and Z chromosome simultaneously, utilize PCR-agarose electrophoresis technology and methods of genotyping, determine individual sex, have simple to operation, qualification fast, the advantage such as result is accurate and cheap, avoid and adopt that traditional method is consuming time, effort and the shortcoming of easily makeing mistakes.

Description

Fowl Sex Rapid identification test kit

Technical field

The utility model belongs to biological technical field, utilizes PCR-agarose electrophoresis technology, provides a kind of Fowl Sex qualification Rapid identification test kit.

Background technology

For the bird of large-scale cultivation, the Forepart identification of its sex will contribute to saving feeding cost, improve cultivation efficiency.Female raising is generally tended in the cultivation of egg fowl, correctly identifies sex during hatching, can eliminate male early, saves cultured space and feeding cost; In poultry cultivation, its nutritional needs of the individuality of different sexes is different, and distinguishing sex early can raise individual differentiation; For kind of a fowl cultivation, different plant species needs different rational sex ratios, and early stage sex identification will contribute to realizing this process.In current production, the sex appraisal method of bird mainly still turns over anus method, especially for all the more so chicken, duck.Although it has certain irritability to individuality, cost of labor cheap, has the advantage that it is certain.Turn over the application of anus method on goose, dove unsatisfactory.In production application, rely on the experience of people to judge the sex of squab, its accuracy only has about 70%; For the sex determination of goose, mainly by pharynx bag and the observation of other outward appearances, but effect bad.In recent years, the progress of molecular biotechnology, the solution for this difficult problem provides another kind of thinking, also for we bring the brand-new means of the another kind of sex identification.

The sex chromosome of bird has two kinds---Z karyomit(e) and W karyomit(e).Female bird genotype is ZW type, and public fowl genotype is ZZ type.Z karyomit(e) and W karyomit(e) differ greatly in size, and the most gene be therefore positioned on Z karyomit(e) does not exist on W karyomit(e).But also find that there is minority gene at present all to exist on bird Z and W karyomit(e).Some intron of these genes is different with the sequence size in Z karyomit(e) at W karyomit(e), this just makes us can by this regional sequence of over-designed primer amplification, products therefrom, through agarose gel electrophoresis, finally judges individual sex according to the band difference of the PCR product of electrophoresis formation.

Utility model content

The purpose of this utility model is the deficiency for existing Fowl Sex authenticate technology, provides a kind of Fowl Sex Rapid identification test kit, can carry out Fowl Sex qualification accurately and rapidly, cheap.

The utility model is achieved through the following technical solutions, a kind of Fowl Sex Rapid identification test kit, comprise the liner in box body and box body, liner is provided with vestibule, is respectively equipped with Auele Specific Primer mixture JYp, amplification buffer, DNA Marker and agar Icing Sugar in described vestibule.

Further, described box body is provided with lid.

Further, the shape of described box body is square.

Further, described box portion left right side wall top is respectively equipped with inner cap.

Wherein JYp details are as follows:

Primer mixture JYp represents the upstream and downstream primer mixture for the amplification of CHD1 gene PCR, and primer information is as follows:

Upstream primer 5'-TGCAGAAGCAATATTACAAGT-3',

Downstream primer 5'-AATTCATTATCATCTGGTGG-3'.

The using method of the utility model Fowl Sex Rapid identification test kit: with the STb gene of poultry to be measured for template, adopts Fowl Sex qualification PCR primer to carry out PCR amplification, then adopts agarose gel electrophoresis to detect PCR amplified production.Be judged to be when a specific amplification band only appears in amplified production male (ZZ type, a large PCR product fragment); Be judged to be when two specific amplification bands appear in amplified production simultaneously female (ZW type, small one and large one two PCR product fragments).

1, PCR amplification object fragment

1) PCR amplification reaction system prepares

In the PCR thin-walled tube of 200 μ L, add 2 μ L template DNAs (about 50 ng), 12.5 μ L amplification buffers, 2 μ L primer JYp (10 μm of ol/L) and 8.5 μ L water, the reaction system of PCR is 25 μ L, fully of short duration centrifugal after mixing.

2) PCR amplified reaction programming

The reaction conditions that PCR instrument arranges PCR is: after 94 DEG C of denaturation 4 min, 94 DEG C of sex change 30 s, 51 DEG C of renaturation 30 s, 72 DEG C extend 30 s totally 35 circulations, last 72 DEG C extend 6 min also 4 DEG C save backup.After programming, PCR reaction tubes is put into PCR instrument and carry out reaction amplification.

2, sex is identified

2% sepharose is placed in 1 × TBE electrophoresis liquid.Get 4-6 μ L reaction product point sample in gel pore.Under room temperature, suitable voltage (according to electrophoresis chamber width × 6-8 V) electrophoresis 30 minutes.According to PCR amplified production electrophoretogram qualification sex.

Compared with prior art, the utility model has following beneficial effect:

First, upstream and downstream primer mixture JYp, amplification buffer and agar Icing Sugar that the utility model is increased by CHD1 gene PCR form, the utility model is by design Auele Specific Primer, PCR amplified production has more specificity, ensure that the accuracy of being carried out Fowl Sex gene type by specific PCR product.The utility model is reasonable in design, easy to use, reliable results and with low cost.

Second, the utility model is according to the CHD1 gene order appropriate design PCR primer be present on poultry sex chromosome W karyomit(e) and Z karyomit(e) simultaneously, utilize PCR-agarose electrophoresis technology and methods of genotyping, determine individual sex, have simple to operation, differentiate fast, the advantage such as result is accurate, avoid and adopt that traditional method is consuming time, effort and the shortcoming of easily makeing mistakes.

Accompanying drawing explanation

Fig. 1 is the structural representation of the utility model Fowl Sex Rapid identification test kit;

In figure: 1-box body, 2-liner, 3-Auele Specific Primer mixture JYp, 4-amplification buffer, 5-DNA marker, 6-agar Icing Sugar, 7-lid, 8-inner cap.

Embodiment

Be described further the utility model with accompanying drawing in conjunction with the embodiments.Should be understood that these embodiments are only for illustration of object, and be not used in restriction protection domain of the present utility model.

As shown in Figure 1, a kind of Fowl Sex Rapid identification test kit, comprises Auele Specific Primer mixture JYp3, amplification buffer 4, DNA marker 5 and the agar Icing Sugar 6 of inserting respectively in each vestibule of the liner 2 in box body 1, box body, liner.

Box body is provided with lid 7.

The shape of box body is square.

Box portion left right side wall top is respectively equipped with can the inner cap 8 of open and close.

Upstream and downstream primer mixture JYp, amplification buffer and agar Icing Sugar that the utility model is increased by CHD1 gene PCR form, reasonable in design, cheap;

The utility model is according to the CHD1 gene order appropriate design PCR primer be present on poultry sex chromosome W karyomit(e) and Z karyomit(e) simultaneously, utilize PCR-agarose electrophoresis technology and methods of genotyping, determine individual sex, have simple to operation, differentiate fast, the advantage such as result is accurate, avoid and adopt that traditional method is consuming time, effort and the shortcoming of easily makeing mistakes.

Embodiment 1

This test kit is used to carry out sex Molecular Identification to 8 of known sex Beijing Fatty Chickens, 8 large bone chicken, 8 Youxian County's sheldrakes, 8 Beijing ducks, 8 Anser anser, 8 wild goose geese, 8 stone qi doves, 8 silver-colored plumage Wang Ge.

1, sample

Gather the blood of poultry individuality, apply traditional phenol/chloroform method and extract DNA, and by DNA concentration dilution to 50 ng/ μ L.

2, PCR amplified reaction and result

1) PCR amplification reaction system prepares

In the PCR thin-walled tube of 200 μ L, add 2 μ L template DNAs (about 50 ng), 12.5 μ L amplification buffers, 2 μ L primers JYp(10 μm of ol/L) and 8.5 μ L water, the reaction system of PCR is 25 μ L, fully of short duration centrifugal after mixing.

2) PCR amplified reaction programming

The reaction conditions that Eppendorf PCR instrument arranges PCR is: after 94 DEG C of denaturation 4 min, 94 DEG C of sex change 30 s, 51 DEG C of renaturation 30 s, 72 DEG C extend 30 s totally 35 circulations, last 72 DEG C extend 10 min also 4 DEG C save backup.After programming, PCR reaction tubes is put into PCR instrument and carry out reaction amplification.

3, the agarose electrophoresis qualification of PCR amplified production

2% sepharose is placed in 1 × TBE electrophoresis liquid.Get 4-10 μ L reaction product point sample in gel pore.Under room temperature, suitable voltage (according to electrophoresis chamber width × 6-8 V) electrophoresis 30 min.According to PCR amplified production electrophoretogram qualification sex.

According to electrophoretogram, identified gene type.The PCR primer locating to occur under corresponding DNA marker 500 bp omits is Z band, and the PCR product occurred at corresponding DNA marker 250 about bp is W band.Therefore, if only occur what Z was with, be male ZZ type; Occurring Z, W two band, is female ZW type.Sample individual molecular sex identification result is respectively: No. 1-4, Beijing Fatty Chicken for male, No. 5-8 for female, No. 9-12, large bone chicken be male, No. 13-16 be mother; No. 1-4, Youxian County's sheldrake be male, No. 5-8 for female, No. 9-13, Beijing duck be female, and No. 14-16 is public affairs; No. 1-4, Anser anser be male, No. 5-8 for female, wild goose goose No. 9-14 be male, 15, No. 16 be mother; Stone qi dove No. 1-4 be male, No. 5-8 for female, No. 10,12, silver-colored plumage Wang Ge be male, 9,11, No. 13-16 be mother.By the sex result of Molecular Identification with there is no difference by the result of anatomic observation sexual gland.

Claims (4)

1. a Fowl Sex Rapid identification test kit, comprise the liner in box body and box body, it is characterized in that, described liner is provided with vestibule, is respectively equipped with the Auele Specific Primer mixture JYp, amplification buffer, DNA Marker and the agar Icing Sugar that are made up of upstream primer 5'-TGCAGAAGCAATATTACAAGT-3' and downstream primer 5'-AATTCATTATCATCTGGTGG-3' in described vestibule.

2. Fowl Sex Rapid identification test kit according to claim 1, it is characterized in that, described box body is provided with lid.

3. Fowl Sex Rapid identification test kit according to claim 2, is characterized in that, the shape of described box body is square.

4. Fowl Sex Rapid identification test kit according to claim 3, is characterized in that, described box portion left right side wall top is respectively equipped with inner cap.