CN101575641B - Method for detecting fluorescence quantitative PCR genotypes of predisposing genes of hepatic cell carcinoma and kit thereof - Google Patents

Abstract

The invention discloses a method for detecting fluorescence quantitative PCR genotypes of predisposing genes of hepatic cell carcinoma (HCC), which comprises the following steps: aiming at the polymorphism of a fifth intron rs430397 site of a GRP78 gene, designing a PCR reaction primer and a fluorescent probe; adding the PCR reaction primer, the fluorescent probe and PCR reaction solution to a reaction system; and placing the mixture on a fluorescence quantitative PCR instrument for reaction. The invention also discloses a kit for the method for detecting the fluorescence quantitative PCR genotypes of the predisposing genes of the hepatic cell carcinoma. The kit comprises the PCR reaction solution, the PCR reaction primer and the fluorescent probe. The method for detecting fluorescent probe genotypes of TaqMan and the kit have the advantages of simplicity, quickness, high flux and strong specificity, are applicable to large-scale screening of HCC susceptible population, provide a potential drone for gene therapy, and are advantageous for guiding the prevention and treatment of the HCC.

Description

A kind of quantitative fluorescent PCR methods of genotyping and test kit thereof that detects the tumor susceptibility gene of primary hepatocellular carcinoma

Technical field

The present invention relates to a kind of quantitative fluorescent PCR methods of genotyping and test kit thereof, particularly a kind of quantitative fluorescent PCR methods of genotyping and test kit thereof that detects the tumor susceptibility gene of primary hepatocellular carcinoma.

Background technology

(hepatocellular carcinoma is one of the malignant tumour of high incidence in the world wide HCC) to primary hepatocellular carcinoma, and its sickness rate occupies the 5th in kinds of tumor; HCC year death toll and the same year the neopathy number roughly similar, in the relevant mortality ratio of tumour, account for the 3rd, only be lower than lung cancer and cancer of the stomach.China also is liver cancer country occurred frequently, and HCC accounts for 90% of China's primary hepatocarcinoma, and HCC is one of modal malignant tumour of China, and the serious threat people's is healthy, also is one of problem of China's primary study therefore.

Know that at present it is a polygene and the rapid process of multistep that liver cancer forms, and relates to karyomit(e) 1p, 4q, 8p; 8q, 10q, 11p, 13q; 16p, 17p, chromosome aberrations such as 19p and 22q (chromosomalaberrations), some gene functions that cause controlling cell growth or inhibition change.

Glucose regulated protein 78 (glucose-regulated protein 78; GRP78); Have another name called 70 kilodalton heat shock protein(HSP)s 5 (heat shock 70kDa protein 5, HSPA5), with heat shock protein 70 (heat shockprotein 70; HSP70) family has homology, is considered to one of member of HSP70 family.The human GRP78 assignment of genes gene mapping is on karyomit(e) 9q33.The GRP78 gene contains 8 exon 7 introns.In endoplasmic reticulum, found the gene product of GRP78, in endoplasmic reticulum, (immunoglobulin, Ig) heavy chain mortise stop self combining of prematurity heavy chain, combine until heavy chain and light chain for it and Tegeline.This proof GRP78 albumen is the resident albumen of endoplasmic reticulum.

As a kind of stress (stress) relevant important molecule companion (chaperone), GRP78 relates to the kinds of tumors evolution of (comprising HCC).The proteic expression of GRP78 raises in the HCC tissue, is usually indicating the bad of prognosis.But polymorphum and the risk of HCC and the relation of prognosis of present GRP78 gene; The research that the gene diagnosis affinity tag of seeking HCC at gene level and prognostic factor etc. are relevant is fewer, does not particularly also have forecast China people primary hepatocellular carcinoma susceptibility test kit and detection method at present.

Summary of the invention

Main purpose of the present invention is to provide a kind of quantitative fluorescent PCR methods of genotyping that detects the tumor susceptibility gene of primary hepatocellular carcinoma.

The quantitative fluorescent PCR methods of genotyping of the tumor susceptibility gene of this detection primary hepatocellular carcinoma comprises the steps:

(1) to the polymorphum in the 5th intron rs430397 site of GRP78 gene, design PCR reacts primer and fluorescent probe;

(2) PCR that in reaction system, adds in the said step (1) reacts primer, fluorescent probe and PCR reaction solution;

(3) mixing solutions that described step (2) is obtained places on the quantitative real time PCR Instrument and reacts.

Obtain GRP78 gene reference sequences and rs430397 site through ncbi database and dbSNP DB, design amplimer and the fluorescent probe of this SNP.GRP78 Gene Partial reference sequences is following:

GTATGTTCCT?TGTTTTCTGC?TTTGCTAATG?AGATCTCCTT?AGACTCTGAA

TTCAGGACAT TGCATCTAGA ?TACTTAGATA ?ACAGACATCA

CAGTAACCAT

TCTTTTTTC TAGGATCTGT TCCGGTCTAC

TATGAAGCCC GTCCAGAAAG ?TGTTGGAAGA ?TTCTGATTTG

AAGAAGTCTG?ATATTGA

The site that top square frame marks is polymorphic site.

The sequence of described PCR reaction primer is following:

Forward primer: 5 '-CCT TGT TTT CTG CTT TGC TAA TGA-3 ';

Reverse primer: 5 '-CAT AGT AGA CCG GAA CAG ATC CTA GA-3 '.

The sequence of described fluorescent probe is following:

5 '-FAM-CAT CACAGTAAC CATATC-3 ' and

5’-VIC-CATCAC?AGT?AAC?CAT?GTC-3’。

5 ' end mark fluorescent probe of probe, an allelotrope mark VIC, another allelotrope mark 6-carboxyfluorescein (FAM).

The final concentration of described PCR reaction primer is 500nM.

The final concentration of described fluorescent probe is 200nM.

The reaction conditions of said step (3) be 92 ℃ 15 seconds, 60 ℃ 1 minute, react 40 circulations.

Second purpose of the present invention is to provide a kind of test kit of quantitative fluorescent PCR methods of genotyping of the tumor susceptibility gene that detects primary hepatocellular carcinoma.This test kit comprises the PCR reaction solution, also comprises:

Forward primer: 5 '-CCT TGT TTT CTG CTT TGC TAA TGA-3 ';

Reverse primer: 5 '-CAT AGT AGA CCG GAA CAG ATC CTA GA-3 ';

Fluorescent probe: 5 ' FAM-CAT CAC AGT AAC CAT ATC-3 ' and 5 ' VIC-CATCAC AGT AAC CAT GTC-3 '.

5 ' end mark fluorescent probe of probe, an allelotrope mark VIC, another allelotrope mark 6-carboxyfluorescein (FAM).3 ' end then the mark coupling the minor groove wedding agent (minorgroove binder, non-fluorescent quenching agent MGB) (non-fluorescent quencher, NFQ).

TaqMan fluorescent probe genotype tests method of the present invention and test kit be simple, fast, high-throughput, high responsive, high specificity; Be applicable to the extensive examination of HCC Susceptible population; And, help instructing prevention and the treatment of HCC for gene therapy provides the potential target.The closely-related GRP78 polymorphic site of announcement of the present invention and primary hepatocellular carcinoma to having established crucial basis based on the individualized treatment of genetic background, and will bring very considerable society and economic benefit.

Embodiment

For making the present invention be more prone to understand, will further set forth specific embodiment of the present invention below.

A preferred embodiment of the quantitative fluorescent PCR methods of genotyping of the tumor susceptibility gene of detection primary hepatocellular carcinoma of the present invention and detection kit is specific as follows:

1, the extraction of DNA

Blood specimen comes from tumour hospital of Zhongshan University and ophthalmologic hospital.Use the DNA blood specimen to extract test kit (QIAamp DNA blood mini kit) and extract the white corpuscle genomic dna.To the rs430397 site, we have detected 428 primary hepatocellular carcinoma patients, and 410 normal peoples' matching of age and sex genotype.

2, real-time fluorescence quantitative PCR reaction

Utilize the quantitative fluorescent PCR gene parting detecting reagent of the tumor susceptibility gene of detection primary hepatocellular carcinoma of the present invention to carry out:

In a preferred embodiment; This test kit contains the PCR reaction solution; PCR primer and fluorescent probe; The PCR reaction solution is not for containing the TaqMan gene type main reaction liquid (TaqMan GenotypingMaster Mix without UNG (Applied Biosystems)) of UNG, and the sequence of PCR primer is following:

Forward primer: 5 '-CCT TGT TTT CTG CTT TGC TAA TGA-3 ';

Reverse primer: 5 '-CAT AGT AGA CCG GAA CAG ATC CTA GA-3 '.

The sequence of fluorescent probe is following:

5 '-CAT CAC AGTAAC CATATC-3 ' and

5’-CAT?CAC?AGT?AAC?CAT?GTC-3’。

Comprise the genomic dna of 50ng in the 20 μ l PCR reaction systems and do not contain the TaqMan gene type main reaction liquid (TaqMan Genotyping Master Mix without UNG (AppliedBiosystems)) of UNG, contain the above-mentioned primer of 500nM and the above-mentioned probe of 200nM.The special-purpose Sptting plate of the ABI in 96 holes place 7900HT quick real-time PCR instrument system (Applied Biosystems, Foster City, CA, USA) in, with 92 ℃ 15 seconds, 60 ℃ 1 minute, react 40 circulations.

3, data statistics and analysis

(or between different case groups) allelotrope and genotypic distributional difference use the unconditional logistic regression model after age, sex are proofreaied and correct between case group and the control group; And calculate odds ratio (oddsratios simultaneously; ORs) and 95% credibility interval (confidence intervals, CIs).All checks all are two-tailed tests, with P=0.05 for having statistical significance.

In order to guarantee that selected control group and case group all come from the same crowd of Han nationality, have carried out the Hardy-Weinberg balance check earlier.The result sees table 1; Table 1 is normal control group and primary hepatocellular carcinoma group GRP78 gene rs430397 genotype frequency and HWE (Hardy-Weinberg equilibrium) examination table; The result shows that selected crowd meets the mendelian population genetics principle.

Table 1

The P value of pHWE:Hardy-Weinberg equilibrium check

Hardy-Weinberg law (Hardy-Weinberg Law) is meant in panmictic population; If a locus has the frequency of two kinds of allelotrope A and a to be respectively p and q, the frequency of frequency of genotypes AA, Aa and aa is respectively p2,2pq and q2; Then this colony is a genetic equilibrium colony, not with changing from generation to generation.Between the generation; The gene frequency of genetic equilibrium colony is (in the colony; The ratio of certain allelotrope number of one locus and the whole allelotrope numbers of this locus) with genotype frequency (in the colony; The ratio of certain genotype number of individuals and the whole number of individuals of this colony) this constant relationship; By Britain mathematician Ha Di (D.H.Hardy) and German doctor Weinberg (W.Weinberg) independent respectively discovery in 1908, be called hardy-Weinberg law, also claim the law of genetic equilibrium (genetic equilibrium law).The condition of keeping genetic equilibrium is: colony is infinitely great, does not have sudden change, selection, migration and genetic drift.The meaning of hardy-Weinberg law in theory, is the theoretical foundation stone of population genetic and quantitative inheritance, and the genetic model and the parameter estimation of these two subdisciplines of genetics derive out according to this law.In practice; It points out us when introducing a fine variety, reserve seed for planting, hiving off and setting up inbred lines, does not make colony too small, otherwise; Will cause the gene frequency of colony and the change of genotype frequency, thereby cause the forfeiture of former kind (strain) " kind property " or some good economic characters.

The full name of OR value is odd ratio, and the OR value is a relative risk, claims odds ratio, odds ratio again, and for the very low disease of sickness rate, the OR value promptly is the accurate estimated value of relative risk.When the situation occurred of our known disease, when comparing the situation difference of disease group and non-disease group Hazard Factor exposure (during retrospective study), be quantitatively described with OR.Whether OR meaningful its P value of also will seeing, the general 95%CI upper limit possibly be the protection factor less than explanation in 1 o'clock, if opposite lower limit is greater than 1 then explanation possibly be Hazard Factor.

During logistic returned, OR value=1 represented that this factor is inoperative to the generation of disease; The OR value representes that greater than 1 this factor is Hazard Factor; The OR value representes that less than 1 this factor is a protection factor.

Table 2

^aProofreaied and correct the logistic regression analysis of age and sex

Table 2 is the allelotrope of Rs430397 and the correlation results table of genotype and HCC onset risk, and table 2 shows that the allelotrope A of rs430397 and frequency of genotypes AA significantly increase ill risk (OR=1.42,95%CI=1.09-1.84, the P=0.009 of HCC; OR=2.26,95%CI=1.10-3.46, P=0.013).Show that rs430397 allelotrope A and frequency of genotypes AA are the susceptible marker sites of primary hepatocellular carcinoma, explain that also rs430397 is the susceptibility loci of HCC.

Last institute should be noted that; Above embodiment is only in order to technical scheme of the present invention to be described but not to the restriction of protection domain of the present invention; Although the present invention has been done detailed description with reference to preferred embodiment; Those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement technical scheme of the present invention, and do not break away from the essence and the scope of technical scheme of the present invention.

Sequence table

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ccttgttttc?tgctttgcta?atga 24

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catagtagac?cggaacagat?cctaga 26

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catcacagta?accatatc 18

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catcacagta?accatgtc 18

Claims (1)

1. a quantitative fluorescent PCR gene type test kit that detects the 5th the intron rs430397 site of GRP78 gene of primary hepatocellular carcinoma is characterized in that, comprises the PCR reaction solution, also comprises:

Forward primer: 5 '-CCT TGT TTT CTG CTT TGC TAA TGA-3 ';

Reverse primer: 5 '-CAT AGT AGA CCG GAA CAG ATC CTA GA-3 ';

Fluorescent probe: 5 '-FAM-CAT CAC AGT AAC CAT ATC-3 ' and

5’-VIC-CAT?CAC?AGT?AAC?CAT?GTC-3’。