CN110157788A - Primer, probe and kit for quantitatively detecting SENP2 mRNA level - Google Patents
Primer, probe and kit for quantitatively detecting SENP2 mRNA level Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, and particularly relates to a primer, a probe and a kit for quantitatively detecting SENP2 mRNA level, wherein the primer comprises a primer F1 and a primer R1 for amplifying SENP2 gene, and a primer F2 and a primer R2 for amplifying control gene GADPH. The probes include a probe P1 for detecting SENP2 gene and a probe P2 for detecting a control gene GAPDH. The kit comprises the primer and the probe. The primer and the probe have good specificity and sensitive and accurate detection. The kit of the invention can accurately and quantitatively analyze the expression of SENP2 mRNA by using a dual-channel FAM and VIC, and has the advantages of rapidness, simplicity, convenience, high sensitivity and the like.
Description
Technical field
The present invention relates to technical field of biological, and in particular to a kind of drawing for quantitative detection SENP2 mRNA level in-site
Object, probe and kit.
Background technique
Vital myocarditis (viral myocarditis, VMC) is myocardium limitation or diffused caused by virus
Acute or chronic inflammatory disorders.Morbidity of the vital myocarditis in China in recent years is in rising trend, it has also become children and blueness are few
One of the main reason for year sudden death.Some patientss are in self-limited course, and severe one may occur in which heart failure, cardiogenic shock and sudden
Extremely, some patientss can develop into dilated cardiomyopathy, and lethality is up to 50%.Vital myocarditis has become China's urgent need to resolve
Great public health problem.Release is in serum after being concentrated mainly on myocardial damage to the clinical detection of vital myocarditis at present
In myocardium enzyme CK and CK-MB change and electrocardiogram dynamic monitoring.And clinical discovery, myocardium enzyme detection often lack
With the positive correlation of vital myocarditis, it is unable to the actual conditions of reflecting myocardium inflammation patient sometimes.Therefore, effective virus is found
Property myocarditis distinguishing mark object, be the key that reduce the disease death rate.SENP2 is cysteine proteinase family member, by
589 amino acid residues are constituted, and are the major protein enzymes of protein s UMOization modification.The discovery of our early-stage studies, SENP2 condition
Property knock-out mice there is the defect of embryonic heart development, result of study shows that SENP2 has important work to the development of cardiac muscle cell
With.The Coxsackie virus A group of enterovirus genus, coxsackie virus group B, Chinese mugwort can virus, poliovirus etc. be common
Myocarditis virus is caused, wherein 3 type of coxsackie B group (CVB3) is most important virus.We have discovered that causing through CVB3 infection
Mice with viral myocarditis in, the mRNA level in-site of SENP2 and protein level all significantly increase that (experimental result is as schemed in heart
Shown in 4A, B).Meanwhile there are a large amount of inflammatory cells in pathological picture of myocarditis slice HE dyeing and Showed by immune group result, heart
Infiltration, and occur the high expression of a large amount of SENP2 albumen in inflammatory cell (experimental result is as shown in Fig. 5 A, B).Therefore,
SENP2 is positively correlated in vital myocarditis with degree of inflammation, can be used as the molecule mark of vital myocarditis identifying and diagnosing
Will object.The present invention will use fluorescent quantitative PCR technique, detect SENP2 expression in mRNA level in-site, be vital myocarditis
Detection method accurate, quickly, efficiently, sensitive that diagnosis provides.
Summary of the invention
In order to overcome shortcoming and defect existing in the prior art, the purpose of the present invention is to provide one kind for quantitatively examining
The PCR primer of SENP2 mRNA level in-site is surveyed, the primer specificity is good, detects sensitive accurate.
It is another object of the present invention to provide a kind of PCR probes for quantitative detection SENP2 mRNA level in-site, should
Probe specificity is good, detects sensitive accurate.
Another purpose of the invention is that a kind of PCR kit for quantitative detection SENP2 mRNA level in-site is provided,
The kit can carry out accurate quantitative analysis to the expression of SENP2 mRNA using FAM and VIC binary channels, have quick, letter
Just, sensitivity height etc. detects advantage.
The purpose of the invention is achieved by the following technical solution: a kind of PCR for quantitative detection SENP2 mRNA level in-site
Primer, the primer include primers F 1 for expanding SENP2 gene and primer R1 and for expanding crt gene GADPH
Primers F 2 and primer R2, the nucleotide sequence of 4 primers be respectively as follows:
F1:5 '-GGACACTGATGAAATACCAG-3'
R1:5 '-CCGTGTTCCATTACAAGCAG-3'
F2:5 '-CCTGCCAAGTATGATGACATCAAGA-3'
R2:5 '-GTAGCCCAGGATGCCCTTTAGT-3'.
Another object of the present invention is achieved through the following technical solutions: one kind being used for quantitative detection SENP2 mRNA level in-site
PCR probe, the probe includes the probe P1 for detecting SENP2 gene and the probe for detecting crt gene GAPDH
The nucleotide sequence of P2, the probe are respectively as follows:
P1:5 '-TCTCTACAATGCTGCCAGCT-3'
P2:5 '-TGGTGAAGCAGGCGGCCGAG-3'；
Wherein, 5 ' the equal mark fluorescent groups in end of the probe P1 and probe P2,3 ' ends mark quenching group.
Preferably, 5 ' the end mark fluorescent group FAM of the probe P1,3 ' end label quenching group TAMRA.
Preferably, 5 ' the end mark fluorescent group VIC of the probe P2,3 ' end label quenching group TAMRA.
An also purpose of the invention is achieved through the following technical solutions: one kind is for quantitative detection SENP2 mRNA level in-site
PCR kit, the kit includes primer described above and probe described above.
Preferably, the kit is by 2 × NASBA reaction solution, 5 × NASBA reaction enzyme mixation, standard items, positive matter
Control product and negative quality-control product composition.
Preferably, 2 × RT-PCR reaction solution includes following specific ingredient: 50mM Tris-HCl(pH8.3), 100mM
KCl、10mM MgCl2, 1.0% Triton X-100,0.5mM dNTP, primers F 1 and each 1 μm of ol/L of primer R1,2 and of primers F
Each 0.5 μm of 1 μm of ol/L, P1 probe, 0.4 μm of ol/L and P2 probe ol/L of primer R2.
Preferably, 5 × RT-PCR reaction enzyme mixation include 4U/mL Taq enzyme, 1.6U/ μ L MMLV reverse transcriptase,
3U/ μ L RNase inhibitor and 30mg/mL BSA.
Preferably, the standard items are the recombination pUC57 plasmid containing standard items sequence, the standard items sequence are as follows:
GGACACTGATGAAATACCAGCCAAAAGACCAAGATTAGATTGCTTTATTCACCAAGTGAAAAACAGTCTCTA
CAATGCTGCCAGCTTATTTGGATTCCCATTCCAGCTGACCACAAAGCCCATGGTAACTTCTGCTTGTAATGGAACA
CGG。
Preferably, the positive quality control product is the cell pyrolysis liquid sample containing SENP2 mRNA, and negative quality-control product is nothing
The DEPC-H of RNA enzyme2O。
The beneficial effects of the present invention are: primer and probe specificity of the invention is good, detects sensitive accurate.Of the invention
Kit can carry out accurate quantitative analysis to the expression of SENP2 mRNA using FAM and VIC binary channels, have quickly, it is easy,
Sensitivity height etc. detects advantage.
Detailed description of the invention
Fig. 1 is the fluorescence quantitative PCR detection result of standard items.
Fig. 2 is the standard curve of the fluorescence quantitative PCR detection of standard items.
Fig. 3 is 42 clinical case SENP2 gene magnification curves.
Fig. 4 is the detection of CVB3 virus load and SENP2 mRNA level in-site in CVB3 infection C57BL/6J mouse after a week heart
Detection.Wherein, A:CVB3 infects the CVB3 virus load detection in heart after a week of C57BL/6J mouse；B:CVB3 infection
The SENP2 mRNA level in-site detection in heart after a week of C57BL/6J mouse.RpL35A is mouse cDNA loading internal reference.* P <
0.05；* P < 0.01；* * P < 0.001, vs Control (n=5).
Fig. 5 is that CVB3 infects C57BL/6J mouse heart tissue HE coloration result and immunohistochemistry detection SENP2 after a week
Albumen distribution and expression.Wherein, A:CVB3 infects C57BL/6J mouse heart tissue HE coloration result after a week；B:CVB3
Infect C57BL/6J the mouse distribution of heart tissue immunohistochemistry detection SENP2 albumen and expression after a week.Close quarters generation
The expression of table SENP2, color more deeply feel show SENP2 expression it is more.
Specific embodiment
For the ease of the understanding of those skilled in the art, the present invention is made further below with reference to examples and drawings
Bright, the content that embodiment refers to not is limitation of the invention.
The design and composition of 1 kit of embodiment
Kit of the invention is by 2 × NASBA reaction solution, 5 × NASBA reaction enzyme mixation, standard items, positive quality control product and yin
Property quality-control product composition.
1, the design and synthesis of primer and probe:
The conserved genetic sequences that SENP2 and GAPDH are found out from GenBank are distinguished using 5.0 software of Primer Primier
Design amplimer and probe sequence, it is ensured that every pair of primers can specific amplification go out SENP2 and GAPDH.
For expanding the primers F 1 and primer R1 of SENP2 gene, and 2 He of primers F for expanding crt gene GADPH
The nucleotide sequence of primer R2 is respectively as follows:
F1:5 '-GGACACTGATGAAATACCAG-3'
R1:5 '-CCGTGTTCCATTACAAGCAG-3'
F2:5 '-CCTGCCAAGTATGATGACATCAAGA-3'
R2:5 '-GTAGCCCAGGATGCCCTTTAGT-3'.
For detecting the nucleotide sequence of the probe P1 and the probe P2 for detecting crt gene GAPDH of SENP2 gene
It is respectively as follows:
P1:5 '-TCTCTACAATGCTGCCAGCT-3'
P2:5 '-TGGTGAAGCAGGCGGCCGAG-3'.
Wherein, 5 ' the end mark fluorescent group FAM of probe P1,3 ' end label quenching group TAMRA；The 5 ' of the probe P2
Hold mark fluorescent group VIC, 3 ' end label quenching group TAMRA.
2, the preparation of reaction solution and reaction enzyme mixation:
2 × NASBA reaction solution and 5 × NASBA reaction enzyme mixation are determined as specifically optimal as follows match according to Optimum Experiment
Just: 50mM Tris-HCl(pH8.3), 100mM KCl, 10mM MgCl2, 1.0% Triton X-100,0.5mM dNTP, draw
Each 1 μm of ol/L of object F1 and primer R1, primers F 2 and each 0.5 μm of 1 μm of ol/L, P1 probe, 0.4 μm of ol/L and P2 probe ol/ of primer R2
L；It includes 4U/mL Taq enzyme, 1.6U/ μ L MMLV reverse transcriptase, 3U/ μ L RNase inhibitor that 5 × RT-PCR, which reacts enzyme mixation,
With 30mg/mL BSA.
3, standard items
Standard items are final concentration of 1 × 108 The recombination pUC57 plasmid containing standard items sequence of copies/mL, TE solution are molten
Solution (group of TE solution becomes 10mmol/L tri- (methylol) aminomethane hydrochloride, 1mmol/L ethylenediamine tetra-acetic acid and water).
Recombinate the standard items sequence of pUC57 plasmid insertion are as follows:
GGACACTGATGAAATACCAGCCAAAAGACCAAGATTAGATTGCTTTATTCACCAAGTGAAAAACAGTCTCTA
CAATGCTGCCAGCTTATTTGGATTCCCATTCCAGCTGACCACAAAGCCCATGGTAACTTCTGCTTGTAATGGAACA
CGG。
4, positive quality control product and negative quality-control product
Positive quality control product is the cell pyrolysis liquid sample containing SENP2 mRNA, and negative quality-control product is the DEPC-H without RNA enzyme2O。
The preparation of 2 standard curve of embodiment
Reaction solution is prepared: 2 × NASBA reaction solution is 10 μ L × n, and it is 4 μ L × n, DEPC-H that 5 × RT-PCR, which reacts enzyme mixation,2O
For 2 μ L × n.Each component is mixed, every 16 μ L of pipe is dispensed into eight connecting legs (n is reaction tube number).
The gradient dilution of standard items: the method that standard solution is used into 10 times of gradient dilutions, successively it is diluted to 1 ×
107copies/mL、1×106copies/mL、1×105Copies/mL and 1 × 104copies/mL。
The production of standard curve: taking the standard dilutions of 4 each concentration of μ L, is added into the reaction solution for having been loaded with 16 μ L,
PCR amplification is carried out on the LightCycler480 quantitative PCR apparatus of Roche Holding Ag.PCR reaction condition are as follows: 50 DEG C, 15min → 95
DEG C, 3min → (95 DEG C, 15s → 55 DEG C, 30s, 72 DEG C, 30s).PCR amplification step carries out 40 circulations altogether.
Standard curve is drawn according to the fluorescence quantitative PCR detection result (Fig. 1) of standard items, standard curve is as shown in Figure 2.It is horizontal
Coordinate is X, represents the logarithm (Log10) of standard items starting copy number, and ordinate Y represents Ct value.The equation of standard curve
For Y=- 3.74X+52.37, related coefficient 0.9994.
Kit of the invention can carry out accurately quantitative point using FAM and VIC binary channels to the expression of SENP2 mRNA
Analysis has the advantages that the detection such as quick, simplicity, sensitivity height.
Above-described embodiment is the preferable implementation of the present invention, and in addition to this, the present invention can be realized with other way,
Do not depart under the premise of present inventive concept it is any obviously replace it is within the scope of the present invention.
Claims (10)
1. a kind of PCR primer for quantitative detection SENP2 mRNA level in-site, it is characterised in that: the primer includes for expanding
The primers F 1 and primer R1 of SENP2 gene and the primers F 2 for expanding crt gene GADPH and primer R2, described 4 are drawn
The nucleotide sequence of object is respectively as follows:
F1:5 '-GGACACTGATGAAATACCAG-3'
R1:5 '-CCGTGTTCCATTACAAGCAG-3'
F2:5 '-CCTGCCAAGTATGATGACATCAAGA-3'
R2:5 '-GTAGCCCAGGATGCCCTTTAGT-3'.
2. a kind of PCR probe for quantitative detection SENP2 mRNA level in-site, it is characterised in that: the probe includes for detecting
The probe P1 of SENP2 gene and probe P2 for detecting crt gene GAPDH, the nucleotide sequence of the probe are respectively as follows:
P1:5 '-TCTCTACAATGCTGCCAGCT-3'
P2:5 '-TGGTGAAGCAGGCGGCCGAG-3'；
Wherein, 5 ' the equal mark fluorescent groups in end of the probe P1 and probe P2,3 ' ends mark quenching group.
3. a kind of PCR probe for quantitative detection SENP2 mRNA level in-site according to claim 2, it is characterised in that:
5 ' the end mark fluorescent group FAM of the probe P1,3 ' end label quenching group TAMRA.
4. a kind of PCR probe for quantitative detection SENP2 mRNA level in-site according to claim 2, it is characterised in that:
5 ' the end mark fluorescent group VIC of the probe P2,3 ' end label quenching group TAMRA.
5. a kind of PCR kit for quantitative detection SENP2 mRNA level in-site, it is characterised in that: the kit includes right
It is required that primer described in 1 and the described in any item probes of claim 2-4.
6. a kind of PCR kit for quantitative detection SENP2 mRNA level in-site according to claim 5, feature exist
In: the kit is by 2 × NASBA reaction solution, 5 × NASBA reaction enzyme mixation, standard items, positive quality control product and negative matter
Control product composition.
7. a kind of PCR kit for quantitative detection SENP2 mRNA level in-site according to claim 6, feature exist
In: 2 × RT-PCR reaction solution include following specific ingredient: 50mM Tris-HCl(pH8.3), 100mM KCl, 10mM
MgCl2, 1.0% Triton X-100,0.5mM dNTP, primers F 1 and each 1 μm of ol/L of primer R1, primers F 2 and each 1 μ of primer R2
0.5 μm of mol/L, P1 probe, 0.4 μm of ol/L and P2 probe ol/L.
8. a kind of PCR kit for quantitative detection SENP2 mRNA level in-site according to claim 6, feature exist
In: 5 × RT-PCR reaction enzyme mixation includes 4U/mL Taq enzyme, 1.6U/ μ L MMLV reverse transcriptase, 3U/ μ L RNA enzyme
Inhibitor and 30mg/mL BSA.
9. a kind of PCR kit for quantitative detection SENP2 mRNA level in-site according to claim 6, feature exist
In: the standard items are the recombination pUC57 plasmid containing standard items sequence, the standard items sequence are as follows:
GGACACTGATGAAATACCAGCCAAAAGACCAAGATTAGATTGCTTTATTCACCAAGTGAAAAACAGTCTCTA
CAATGCTGCCAGCTTATTTGGATTCCCATTCCAGCTGACCACAAAGCCCATGGTAACTTCTGCTTGTAATGGAACA
CGG。
10. a kind of PCR kit for quantitative detection SENP2 mRNA level in-site according to claim 6, feature exist
In: the positive quality control product is the cell pyrolysis liquid sample containing SENP2 mRNA, and negative quality-control product is the DEPC- without RNA enzyme
H2O。
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