CN106399469A - Dairy cow early-gestation diagnosis primer pairs, probes and method based on duplex quantitative PCR - Google Patents
Dairy cow early-gestation diagnosis primer pairs, probes and method based on duplex quantitative PCR Download PDFInfo
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Abstract
The invention discloses dairy cow early-gestation diagnosis primer pairs, dairy cow early-gestation diagnosis probes and a dairy cow early-gestation diagnosis method based on duplex quantitative PCR, relating to the technical field of molecular biology and nucleic acid detection. The invention discloses the primer pairs corresponding to the bovine ISG15 gene, the bovine RSAD2 gene and the reference gene beta-Actin during the dairy cow early-gestation diagnosis based on duplex quantitative PCR, and further discloses the probes corresponding to the three genes. The invention further discloses the dairy cow early-gestation diagnosis method based on the duplex quantitative PCR, the duplex fluorescent quantitative PCR reaction on one target gene and a reference gene is carried out simultaneously in a PCR tube by adopting a TaqMan probe method, meanwhile, the duplex fluorescent quantitative PCR reaction on two target genes is also carried out in one PCR reaction board, and the dairy cow early-gestation is judged according to the expression difference of the two target genes. With the adoption of the primer pairs, the probes and the method, the manufacture cost of a mold is saved, the cost of the reaction reagent also can be reduced, and meanwhile, the reaction time is shortened.
Description
Technical field
The present invention relates to molecular biology and nucleic acid detection technique field and in particular to a kind of based on dual quantitative PCR
Milk cow diagnosis of early gestation primer pair, probe and method.
Background technology
Cyesiognosis is a vital part in cow reproduction work, quickly, accurately and quickly and easily milk cow is entered
Row diagnosis of early gestation can investigate nonpregnant ox in advance, reduces the consumption of the aspects such as the nonpregnant feed leading to, the energy, manpower, with
When can also pregnant ox be implemented precisely raise, reduce the generation of early abortion event.Using Protocols in Molecular Biology to milk cow
Carry out cyesiognosis, due to this technology have sampling convenience, economic and reliable, can the various advantages such as batched operation and produced
Person receives and progressively widely uses, and the commercial kit of wherein comparative maturity is PAG (PAG) ELISA detection
Kit.But, the method could accurately judge whether milk cow becomes pregnant in 28 days after milk cow artificial insemination, and actual production
In, part milk cow without successfully being joined, occurs after a feelings phase (i.e. 21 days) and returns feelings after artificial insemination, therefore,
How using molecular biology technology 21 days after milk cow artificial insemination before carry out cyesiognosis and be increasingly becoming vast milk cow
Culturist and the emphasis of researcher concern.
At present, quantitative PCR technique is increasingly favored by people due to the features such as its reaction time is short, sensitivity is high.
The patent of invention of Patent No. 2014106572061 provides fluorescent quantificationally PCR detecting kit and the inspection of milk cow early pregnancy
Survey method, this invention detects the table of gene in 18 days PBLs after milk cow artificial insemination by fluorescent quantitative PCR technique
Reach level to carry out cyesiognosis, realize differentiating pregnant ox and nonpregnant ox in the feelings phase after milk cow artificial insemination.But, should
Invention is confined to substance fluorescent quantitative PCR technique, and that is, Single tube amplification once can only detect a kind of gene：Genes of interest or internal reference
Gene, same blood sample at least needs to detect the expression of two genes at twice, this undoubtedly increased workload, and
Need the sample size providing also can increase, the cost of detection also can increase.
Content of the invention
For defect present in prior art, it is an object of the invention to provide a kind of milk based on dual quantitative PCR
Ox diagnosis of early gestation primer pair, probe and method, it is possible to decrease reaction actual cost, save the reaction time.
For reaching object above, the present invention adopts the technical scheme that：A kind of milk cow early stage based on dual quantitative PCR is pregnant
It is pregnent the primer pair of diagnosis, including the specific primer pair of ox ISG15 gene, ox RSAD2 gene and reference gene β-Actin, its
In：The primer pair of described ox ISG15 gene is SEQ ID NO:1 and SEQ ID NO:Sequence shown in 2；Described ox RSAD2 gene
Primer pair be SEQ ID NO:4 and SEQ ID NO:Sequence shown in 5；The primer pair of described reference gene β-Actin is SEQ
ID NO:7 and SEQ ID NO:Sequence shown in 8.
The invention also discloses a kind of probe of the milk cow diagnosis of early gestation based on dual quantitative PCR, including ox ISG15
The specific probe of gene, ox RSAD2 gene and reference gene β-Actin, wherein：The probe of described ox ISG15 gene is SEQ
ID NO:Sequence shown in 3；The probe of described ox RSAD2 gene is SEQ ID NO:Sequence shown in 6；Described reference gene β-
The probe of Actin is SEQ ID NO:Sequence shown in 9.
The invention also discloses a kind of milk cow diagnosis of early gestation method based on dual quantitative PCR, visited using TaqMan
The skill of handling needles carries out double fluorescent quantitative PCR detection to two kinds of genes of interest and reference gene, carries out one kind in single PCR pipe simultaneously
Genes of interest is reacted with the PCR of reference gene, and to carry out the PCR of two kinds of genes of interest in same PCR reaction plate anti-simultaneously
Should, milk cow early pregnancy is judged according to the differential expression of two kinds of genes of interest.
On the basis of technique scheme, genes of interest described in one of which is ox ISG15 gene, another kind of described mesh
Gene be ox RSAD2 gene, described reference gene be β-Actin.
On the basis of technique scheme, comprise the following steps：
S1, collect blood sample, the blood sample of 18 days after 0 day and artificial insemination after artificial insemination after collection cow oestrus
1.5-2mL, anti-freezing, deepfreeze；
S2, extracts blood sample RNA, in 2 hours after Blood specimen collection, extracts blood with blood total RNA extraction reagent box
Total serum IgE in liquid sample, and analyze RNA concentration of specimens and purity, detect RNA integrality simultaneously, after the sample of quality inspection dispenses,
Cord blood is standby；
S3, reverse transcription reaction becomes cDNA, and carrying out reverse transcription reaction using Reverse Transcriptase kit will extract from blood sample
RNA reverse transcription become cDNA；
S4, design with synthetic primer to and probe, the primer pair of described ox ISG15 gene is SEQ ID NO:1 and SEQ
ID NO:Sequence shown in 2；The primer pair of described ox RSAD2 gene is SEQ ID NO:4 and SEQ ID NO:Sequence shown in 5；Institute
The primer pair stating reference gene β-Actin is SEQ ID NO:7 and SEQ ID NO:Sequence shown in 8；Described ox ISG15 gene
Probe is SEQ ID NO:Sequence shown in 3；The probe of described ox RSAD2 gene is SEQ ID NO:Sequence shown in 6；Described internal reference
The probe of gene β-Actin is SEQ ID NO:Sequence shown in 9；
S5, carries out ox ISG15 gene, ox RSAD2 gene and reference gene in double fluorescent quantitative PCR detection blood sample
β-Actin, detects ox ISG15 gene and reference gene β-Actin in single PCR pipe simultaneously, and simultaneously anti-in same PCR
Answer and in plate, detect ox RSAD2 gene and reference gene β-Actin simultaneously；
S6：After the completion of double fluorescent quantitative PCR, the cycle threshold (Ct) according to each gene calculates gene ISG15 and base
Because of the relative expression quantity of RSAD2, and milk cow diagnosis of early gestation is carried out according to relative expression quantity.
On the basis of technique scheme, the reaction system of gene ISG15 and gene β-Actin in step S5：2×
Premix Ex TaqMix 10 μ L, the upstream primer that each gene is 10 μm of ol/L using concentration and each 0.2uL of downstream primer, respectively
The each 0.4uL of probe that gene is 10 μm of ol/L using concentration, cDNA template 0.6 μ L, ddH2O 7.8 μ L, reaction system totally 20 μ L；
Gene RSAD2 and the reaction system of gene β-Actin：2 × Premix Ex TaqMix 10 μ L, it is 10 μ that each gene adopts concentration
The upstream primer of mol/L and each 0.2uL of downstream primer, each 0.4uL, cDNA of the probe that each gene is 10 μm of ol/L using concentration
Template 0.6 μ L, ddH2O 7.8 μ l, reaction system totally 20 μ l.
On the basis of technique scheme, the reaction condition in step S5 is：95 DEG C of denaturations 30 seconds；95 DEG C of denaturation 15
Second, anneal and extend 30 seconds for 61 DEG C, 40 circulations, the 61 DEG C of stages of annealing and extend circulated at each collect fluorescence signal.
On the basis of technique scheme, the concretely comprising the following steps of step S6：
S601：Calculate difference DELTA Ct of genes of interest Ct average and reference gene Ct average in detection blood sample；
S602：Calculate the relative expression quantity 2 of genes of interest in blood sample-△ΔCt, wherein Represent after artificial insemination 0 day or 18 days blood sample genes of interest Ct average,Represent this blood sample corresponding
The Ct average of reference gene；
S603：The relative expression quantity of 0 day each gene after milk cow artificial insemination is corrected to 1, the phase of 18 days after artificial insemination
The multiple of relative expression quantity when expression is corrected to 0 day after artificial insemination；Calculate 18 days cow blood samples after artificial insemination
In this, the relative expression quantity of ox ISG15 gene is X1, and the relative expression quantity of ox RSAD2 gene is X2；
S604：X1 and X2 is brought into formula P=1/ [1+e-(-6.53+2.830X1+0.866X2)] in, if P value is more than 0.680,
It is gestation.
Compared with prior art, it is an advantage of the current invention that：
1st, the primer pair such as SEQ ID NO of the ox ISG15 gene that the present invention adopts:1 and SEQ ID NO:Shown in 2, probe
As SEQ ID NO:Shown in 3；The primer pair of ox RSAD2 gene such as SEQ ID NO:4 and SEQ ID NO:Shown in 5, probe such as SEQ
ID NO:Shown in 6；The primer pair of reference gene β-Actin such as SEQ ID NO:7 and SEQ ID NO:Shown in 8, probe such as SEQ
ID NO:Shown in 9；When double fluorescent quantitative PCR being carried out using above-mentioned primer pair and probe reacting, genes of interest ox ISG15 gene
Or ox RSAD2 gene when reacting in same PCR pipe with reference gene β-Actin mutual interference minimum, and react bar
Part is consistent, so that genes of interest and reference gene are able to react in same PCR pipe and carry out two kinds of purpose bases simultaneously
The PCR reaction of cause.
2nd, present invention testing goal gene and reference gene in same PCR pipe, decreases the usage amount of template, saves
The cost of template construct, can also mitigate the damage that sampling causes to milk cow to a certain extent.Simultaneously as the contracting of reaction tube
Subtract, the cost of reaction reagent also can decrease.
3rd, the present invention can in same PCR reaction plate testing goal gene ISG15 and RSAD2 simultaneously expression, contracting
The short reaction time；Simultaneously as the detection time of dual quantitative PCR technique is traditional SYBO Green substance fluorescent quantitation
/ 2nd of the reaction time of round pcr, improve the efficiency of detection.
Brief description
Fig. 1 is the schematic flow sheet of the milk cow diagnosis of early gestation method in the embodiment of the present invention based on dual quantitative PCR；
Fig. 2 is primer Ago-Gel detection electrophoresis result figure, wherein M in the embodiment of the present invention：DNA relative molecular mass
Standard DL2000；Swimming lane 1-2:The pcr amplification product of reference gene β-Actin；Swimming lane 3-4：The PCR amplification of ox ISG15 gene
Product；Swimming lane 5-6：The pcr amplification product of ox RSAD2 gene；
Fig. 3 is that in the embodiment of the present invention, double fluorescent quantitative PCR and substance quantitative fluorescent PCR react amplification curve contrast
Figure；
Fig. 4 is pregnant ox double fluorescent quantitative PCR amplification curve in the embodiment of the present invention；
Fig. 5 is nonpregnant ox double fluorescent quantitative PCR amplification curve in the embodiment of the present invention.
Specific embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
The invention discloses a kind of primer pair of the milk cow diagnosis of early gestation based on dual quantitative PCR：Including ox ISG15
The specific primer pair of gene, ox RSAD2 gene and reference gene β-Actin, wherein：The primer pair of described ox ISG15 gene
It is SEQ ID NO:1 and SEQ ID NO:Sequence shown in 2；The primer pair of described ox RSAD2 gene is SEQ ID NO:4 and SEQ
ID NO:Sequence shown in 5；The primer pair of described reference gene β-Actin is SEQ ID NO:7 and SEQ ID NO:Sequence shown in 8
Row.
The invention also discloses a kind of probe of the milk cow diagnosis of early gestation based on dual quantitative PCR：Including ox ISG15
The specific probe of gene, ox RSAD2 gene and reference gene β-Actin, wherein：The probe of described ox ISG15 gene is SEQ
ID NO:Sequence shown in 3；The probe of described ox RSAD2 gene is SEQ ID NO:Sequence shown in 6；Described reference gene β-
The probe of Actin is SEQ ID NO:Sequence shown in 9.
When double fluorescent quantitative PCR being carried out using above-mentioned primer pair and probe reacting, genes of interest ox ISG15 gene or ox
When RSAD2 gene is reacted in same PCR pipe with reference gene β-Actin, mutual interference is minimum, and reaction condition one
Cause so that genes of interest and reference gene be able in same PCR pipe reaction and in same PCR reaction plate simultaneously
Carry out the PCR reaction of two kinds of genes of interest.
The invention also discloses a kind of milk cow diagnosis of early gestation method based on dual quantitative PCR：Visited using TaqMan
The skill of handling needles carries out double fluorescent quantitative PCR detection to two kinds of genes of interest and reference gene, carries out one kind in single PCR pipe simultaneously
Genes of interest is reacted with the PCR of reference gene, and to carry out the PCR of two kinds of genes of interest in same PCR reaction plate anti-simultaneously
Should, milk cow early pregnancy is judged according to the differential expression of two kinds of genes of interest.Genes of interest described in one of which is ox ISG15 base
Cause, another kind of described genes of interest is ox RSAD2 gene, and described reference gene is β-Actin.
【Embodiment 1】Milk cow diagnosis of early gestation method based on dual quantitative PCR
Shown in Figure 1, comprise the following steps：
S1, collect blood sample, the blood sample of 18 days after 0 day and artificial insemination after artificial insemination after collection cow oestrus
1.5-2mL, anti-freezing, deepfreeze；
S2, extracts blood sample RNA, in 2 hours after Blood specimen collection, extracts blood with blood total RNA extraction reagent box
Total serum IgE in liquid sample, and analyze RNA concentration of specimens and purity, detect RNA integrality simultaneously, after the sample of quality inspection dispenses,
Cord blood is standby；
S3, reverse transcription reaction becomes cDNA, and carrying out reverse transcription reaction using Reverse Transcriptase kit will extract from blood sample
RNA reverse transcription become cDNA；
S4, design with synthetic primer to and probe, the primer pair of described ox ISG15 gene is SEQ ID NO:1 and SEQ
ID NO:Sequence shown in 2；The primer pair of described ox RSAD2 gene is SEQ ID NO:4 and SEQ ID NO:Sequence shown in 5；Institute
The primer pair stating reference gene β-Actin is SEQ ID NO:7 and SEQ ID NO:Sequence shown in 8；Described ox ISG15 gene
Probe is SEQ ID NO:Sequence shown in 3；The probe of described ox RSAD2 gene is SEQ ID NO:Sequence shown in 6；Described internal reference
The probe of gene β-Actin is SEQ ID NO:Sequence shown in 9；
S5, carries out ox ISG15 gene, ox RSAD2 gene and reference gene in double fluorescent quantitative PCR detection blood sample
β-Actin, detects ox ISG15 gene and reference gene β-Actin in single PCR pipe simultaneously, and simultaneously anti-in same PCR
Answer and in plate, detect ox RSAD2 gene and reference gene β-Actin simultaneously；
S6：After the completion of double fluorescent quantitative PCR, the cycle threshold (Ct) according to each gene calculates gene ISG15 and base
Because of the relative expression quantity of RSAD2, and milk cow diagnosis of early gestation is carried out according to relative expression quantity.
The reaction system of gene ISG15 and gene β-Actin in step S5：2 × Premix Ex TaqMix 10 μ L, respectively
Upstream primer and each 0.2uL of downstream primer that gene is 10 μm of ol/L using concentration, each gene is 10 μm of ol/L using concentration
The each 0.4uL of probe, cDNA template 0.6 μ L, ddH2O 7.8 μ L, reaction system totally 20 μ L；
Gene RSAD2 and the reaction system of gene β-Actin：2 × Premix Ex TaqMix10 μ L, each gene adopts dense
Spend the upstream primer for 10 μm of ol/L and each 0.2uL of downstream primer, the probe that each gene is 10 μm of ol/L using concentration each
0.4uL, cDNA template 0.6 μ L, ddH2O 7.8 μ L, reaction system totally 20 μ L.
Reaction condition in step S5 is：95 DEG C of denaturations 30 seconds；95 DEG C of denaturation 15 seconds, anneal and extend 30 seconds for 61 DEG C,
40 circulations, the 61 DEG C of stages of annealing and extend circulated at each collect fluorescence signal.
The concretely comprising the following steps of step S6：
S601：Calculate difference DELTA Ct of genes of interest Ct average and reference gene Ct average in detection blood sample；
S602：Calculate the relative expression quantity 2 of genes of interest in blood sample-△ΔCt, wherein Represent after artificial insemination 0 day or 18 days blood samples in genes of interest Ct average,Represent this blood sample to correspond to
Reference gene Ct average；
S603：The relative expression quantity of 0 day each gene after milk cow artificial insemination is corrected to 1, the phase of 18 days after artificial insemination
The multiple of relative expression quantity when expression is corrected to 0 day after artificial insemination；Calculate 18 days cow blood samples after artificial insemination
In this, the relative expression quantity of ox ISG15 gene is X1, and the relative expression quantity of ox RSAD2 gene is X2；
S604：X1 and X2 is brought into formula P=1/ [1+e-(-6.53+2.830X1+0.866X2)] in, if P value is more than 0.680,
It is gestation.
The present invention testing goal gene and reference gene in same PCR pipe, decrease the usage amount of template, save mould
The cost that plate makes, can also mitigate the damage that sampling causes to milk cow to a certain extent.Simultaneously as the contracting of reaction tube
Subtract, the cost of reaction reagent also can decrease.The present invention can in same PCR reaction plate testing goal gene simultaneously
The expression of ISG15 and RSAD2, shortens the reaction time；Simultaneously as the detection time of double fluorescent quantitative PCR technology is to pass
/ 2nd of the reaction time of SYBO Green substance fluorescent quantitative PCR technique of system, improve the efficiency of detection.
【Embodiment 2】The accuracy testing of the milk cow diagnosis of early gestation method based on dual quantitative PCR
1st, primer checking
By the blood sample of the accuracy testing of the double fluorescent quantitative PCR of freezen protective with the primer pair in step S4 and
Probe enters performing PCR amplification, and its reaction system is：Premix TaqTM10uL, concentration is that the upstream and downstream primer of 10 μm of ol/L is each
0.2uL, cDNA template 0.6 μ L, finally uses ddH2O mends to 20 μ L.Its reaction condition is：94 DEG C of denaturations 3 minutes, 94 DEG C of denaturation
30 seconds, anneal 30 seconds for 61 DEG C；72 DEG C extend 25 seconds, totally 30 circulations；Last 72 DEG C 10 minutes.With Ago-Gel (1.2%)
Electrophoresis detection PCR primer.
Conclusion：Shown in Figure 2, the gel electrophoresis figure of quantitative pcr amplification is shown in during amplification no primer dimerization
Body, does not have miscellaneous band yet, can be used for the quantitative PCR detection of next step.
2nd, the accuracy that double fluorescent quantitative PCR reaction is reacted with substance quantitative fluorescent PCR compares
Detect ox ISG15 gene in each blood sample, ox RSAD2 gene, internal reference in same PCR reaction plate simultaneously
Gene β-Actin, ox ISG15 gene and reference gene β-Actin, ox RSAD2 gene and reference gene β-Actin.Wherein, single
Quantitative fluorescent PCR reaction system is again：2 × Premix Ex Taq Mix 10 μ L, concentration is the upstream and downstream primer of 10 μm of ol/L
Each 0.2uL, concentration is the probe 0.4uL of 10 μm of ol/L, cDNA template 0.6 μ L, finally uses ddH2O mends to 20 μ L.Double fluorescent
Quantitative PCR reaction system：Taking ox ISG15 gene and reference gene β-Actin as a example, 2 × Premix Ex Taq Mix 10 μ L,
Concentration is the ox ISG15 gene of 10 μm of ol/L and each 0.2uL of upstream and downstream primer (10 μm of ol/L) of reference gene β-Actin, dense
Spend the ox ISG15 gene for 10 μm of ol/L and reference gene β-Actin corresponding probe 0.4uL, cDNA template 0.6 μ L, finally
Use ddH2O mends to 20 μ L.In the amplification of CFX Connect quantitative real time PCR Instrument, reaction condition is：95 DEG C of denaturations 30 seconds；95℃
Denaturation 15 seconds, anneals and extends 30 seconds for 61 DEG C, 40 circulations, and the 61 DEG C of stages of annealing and extend circulated at each collect fluorescence letter
Number.Analysis software calculates the cycle threshold (Ct) of each blood sample in real time, shown in Figure 3, compares same blood sample list
△ Ct when weight fluorescence quantitative PCR detection is detected with double fluorescent quantitative PCR.According to the examination criteria in step S604：Will
The relative expression quantity of ISG15 (X1) and RSAD2 (X2) gene substitutes into formula P=1/ [1+e-(-6.53+2.830X1+0.866X2)], when P is big
It is gestation when 0.680.Whole blood sample is judged, and SYBO Green technology widely used with laboratory inspection
The expression surveying single ox ISG15 gene and single ox RSAD2 gene in blood sample carries out accuracy ratio carrying out cyesiognosis
Relatively.Wherein table 1 is substance fluorescence quantitative PCR detection ISG15 and the testing result of RSAD2 gene, and table 2 is double fluorescent quantitative
PCR detects the testing result of ISG15 and RSAD2 gene.
Table 1：The Ct value of substance fluorescence quantitative PCR detection ISG15 and RSAD2 gene
| Sample | ISG15 | RSAD2 | β-Actin | ΔCt(ISG15) | ΔCt(RSAD2) |
| 1 | 26.02 | 29.19 | 22.91 | 3.11 | 6.28 |
| 2 | 26.36 | 29.73 | 21.41 | 4.94 | 8.32 |
| 3 | 26.83 | 29.42 | 19.56 | 7.27 | 9.86 |
| 4 | 33.18 | 35.55 | 26.11 | 7.07 | 9.44 |
| 5 | 31.17 | 31.67 | 23.00 | 8.17 | 8.67 |
| 6 | 29.18 | 32.27 | 21.41 | 7.77 | 10.85 |
Table 2：Double fluorescent quantitative PCR detects the Ct value of ISG15 and RSAD2 gene
Conclusion：From Table 2, it can be seen that same sample is when carrying out double fluorescent quantitative PCR detection, in ox ISG15 base
Under cause and reference gene β-Actin and ox RSAD2 gene and the combination of reference gene β-Actin both, reference gene β-
The Ct of Actin is very stable, and difference is less than 0.6, illustrates that the repeatability of this test is relatively good.Relatively Tables 1 and 2, result shows,
The detection of same blood sample double fluorescent quantitative PCR is higher by 0～2.36 than the Ct value of substance fluorescence quantitative PCR detection, but, identical
Blood sample double fluorescent quantitative PCR detection differ with Δ Ct (the ox ISG15 gene) value of substance fluorescence quantitative PCR detection-
0.84～0.02, Δ Ct (ox RSAD2 gene) value difference -1.23～0.04, shows the process of double fluorescent quantitative PCR reaction
In, genes of interest is higher with the primer specificity of reference gene, between vie each other little；Genes of interest is glimmering with reference gene
Light probe cross jamming is smaller, and whole result shows, dual quantitative PCR detecting method is more stable.
3rd, carry out the accuracy detection of cyesiognosis using double fluorescent quantitative PCR
Per rectum inspection is defined as with pregnant ox (19) and the blood sample of nonpregnant ox (17) is expanded, amplification
Curve is as shown in Figure 4 and Figure 5.With 2-△ΔCtRepresent genes of interest mRNA relative expression quantity in blood sample, according to the morning of the present invention
Phase cyesiognosis method, is detected to the sample of 36 cow heads.Using the detection per rectum inspection of double fluorescent quantitative PCR method
Look into 19 oxen being defined as gestation, result is：18 is gestation, and 1 is nonpregnant, and the accuracy of pregnant ox detection is 94.73%；
It is defined as nonpregnant 17 ox using the detection per rectum inspection of double fluorescent quantitative PCR method, it is nonpregnant that result is 16,1
For gestation, the accuracy of nonpregnant ox is 94.12%.
Conclusion：The present invention is applied to the diagnosis of early gestation of milk cow, the sensitivity of detection and substance fluorescent quantitative PCR technique
Unanimously, the accuracy of detection is 94.12%, can meet the requirement for cyesiognosis in Cow product.
The present invention is not limited to above-mentioned embodiment, for those skilled in the art, without departing from
On the premise of the principle of the invention, some improvements and modifications can also be made, these improvements and modifications are also considered as the protection of the present invention
Within the scope of.The content not being described in detail in this specification belongs to prior art known to professional and technical personnel in the field.
Claims (8)
1. a kind of primer pair of the milk cow diagnosis of early gestation based on dual quantitative PCR it is characterised in that：Including ox ISG15 base
The specific primer pair of cause, ox RSAD2 gene and reference gene β-Actin, wherein：The primer pair of described ox ISG15 gene is
SEQ ID NO:1 and SEQ ID NO:Sequence shown in 2；The primer pair of described ox RSAD2 gene is SEQ ID NO:4 and SEQ ID
NO:Sequence shown in 5；The primer pair of described reference gene β-Actin is SEQ ID NO:7 and SEQ ID NO:Sequence shown in 8.
2. a kind of probe of the milk cow diagnosis of early gestation based on dual quantitative PCR it is characterised in that：Including ox ISG15 gene,
Ox RSAD2 gene and the specific probe of reference gene β-Actin, wherein：The probe of described ox ISG15 gene is SEQ ID
NO:Sequence shown in 3；The probe of described ox RSAD2 gene is SEQ ID NO:Sequence shown in 6；Described reference gene β-Actin's
Probe is SEQ ID NO:Sequence shown in 9.
3. a kind of milk cow diagnosis of early gestation method based on dual quantitative PCR it is characterised in that：Using TaqMan probe method pair
Two kinds of genes of interest and reference gene carry out double fluorescent quantitative PCR detection, carry out a kind of purpose base in single PCR pipe simultaneously
Because react with the PCR of reference gene, and carry out the PCR of two kinds of genes of interest in same PCR reaction plate simultaneously and react, according to
The differential expression of two kinds of genes of interest judges milk cow early pregnancy.
4. as claimed in claim 3 a kind of milk cow diagnosis of early gestation method based on dual quantitative PCR it is characterised in that：
Genes of interest described in one of which is ox ISG15 gene, and another kind of described genes of interest is ox RSAD2 gene, described internal reference base
Because β is-Actin.
5. as claimed in claim 4 a kind of milk cow diagnosis of early gestation method based on dual quantitative PCR it is characterised in that
Comprise the following steps：
S1, collect blood sample, the blood sample 1.5- of 18 days after 0 day and artificial insemination after artificial insemination after collection cow oestrus
2mL, anti-freezing, deepfreeze；
S2, extracts blood sample RNA, in 2 hours after Blood specimen collection, extracts blood sample with blood total RNA extraction reagent box
Total serum IgE in this, and analyze RNA concentration of specimens and purity, detect RNA integrality simultaneously, after the sample of quality inspection dispenses, low temperature
Save backup；
S3, reverse transcription reaction becomes cDNA, carries out, using Reverse Transcriptase kit, the RNA that reverse transcription reaction will extract from blood sample
Reverse transcription becomes cDNA；
S4, design with synthetic primer to and probe, the primer pair of described ox ISG15 gene is SEQ ID NO:1 and SEQ ID
NO:Sequence shown in 2；The primer pair of described ox RSAD2 gene is SEQ ID NO:4 and SEQ ID NO:Sequence shown in 5；Described interior
The primer pair of ginseng gene β-Actin is SEQ ID NO:7 and SEQ ID NO:Sequence shown in 8；The probe of described ox ISG15 gene
It is SEQ ID NO:Sequence shown in 3；The probe of described ox RSAD2 gene is SEQ ID NO:Sequence shown in 6；Described reference gene
The probe of β-Actin is SEQ ID NO:Sequence shown in 9；
S5, carry out ox ISG15 gene in double fluorescent quantitative PCR detection blood sample, ox RSAD2 gene and reference gene β-
Actin, detects ox ISG15 gene and reference gene β-Actin in single PCR pipe simultaneously, and reacts in same PCR simultaneously
Detect ox RSAD2 gene and reference gene β-Actin in plate simultaneously；
S6：After the completion of double fluorescent quantitative PCR, the cycle threshold (Ct) according to each gene calculates gene ISG15 and gene
The relative expression quantity of RSAD2, and milk cow diagnosis of early gestation is carried out according to relative expression quantity.
6. as claimed in claim 5 a kind of milk cow diagnosis of early gestation method based on dual quantitative PCR it is characterised in that：
The reaction system of gene ISG15 and gene β-Actin in step S5：2 × Premix Ex TaqMix 10 μ L, each gene
The upstream primer being 10 μm of ol/L using concentration and each 0.2uL of downstream primer, the probe that each gene is 10 μm of ol/L using concentration
Each 0.4uL, cDNA template 0.6 μ L, ddH2O 7.8 μ L, reaction system totally 20 μ L；
Gene RSAD2 and the reaction system of gene β-Actin：2 × Premix Ex TaqMix 10 μ L, each gene adopts concentration
Upstream primer for 10 μm of ol/L and each 0.2uL of downstream primer, the probe that each gene is 10 μm of ol/L using concentration each
0.4uL, cDNA template 0.6 μ L, ddH2O 7.8 μ L, reaction system totally 20 μ L.
7. as claimed in claim 5 a kind of milk cow diagnosis of early gestation method based on dual quantitative PCR it is characterised in that：
Reaction condition in step S5 is：95 DEG C of denaturations 30 seconds；95 DEG C of denaturation 15 seconds, anneal and extend 30 seconds for 61 DEG C, 40 circulations,
The 61 DEG C of stages of annealing and extend circulated at each collect fluorescence signal.
8. as claimed in claim 5 a kind of milk cow diagnosis of early gestation method based on dual quantitative PCR it is characterised in that：
The concretely comprising the following steps of step S6：
S601：Calculate the difference △ Ct of genes of interest Ct average and reference gene Ct average in detection blood sample；
S602：Calculate the relative expression quantity 2 of genes of interest in blood sample-△△Ct, wherein Represent after artificial insemination 0 day or 18 days blood sample genes of interest Ct average,Represent this blood sample corresponding
The Ct average of reference gene；
S603：The relative expression quantity of 0 day each gene after milk cow artificial insemination is corrected to 1, the relative table of 18 days after artificial insemination
The amount of reaching is corrected to the multiple of relative expression quantity when 0 day after artificial insemination；Calculate in 18 days cow blood samples after artificial insemination
The relative expression quantity of ox ISG15 gene is X1, and the relative expression quantity of ox RSAD2 gene is X2；
S604：X1 and X2 is brought into formula P=1/ [1+e-(-6.53+2.830X1+0.866X2)] in, if P value is more than 0.680, as
Gestation.
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