CN1314492A - Method for simultaneously extracting pathogen RNA and DNA in body fluid and special extracting liquid thereof - Google Patents

Abstract

The simultaneous extraction process includes the steps of: taking at least 5 micronliter for body fluid and adding 1-100 micronliter extracting liquid at 25-45 deg.c for 3-15 min; builing 5-15 min; maintaining at 50-80 deg.c for 10-30 min before uncovering; adding 2-10 micronliter of superpure water for suspension and resting for 5-20 min; centrifugalizing at 12,000-15,000 rpm for 5-15 min; and final preversing supernatant liquor. The special extracting liquid contains diethyl pyrocarbonate (DEPC) and NP40. The said process is simple, fast, high in yield and stable.

Description

The method of pathogenic agent RNA and DNA and special-purpose extract thereof in a kind of while extracting body fluid

The invention belongs to the gene engineering field, relate to the diagnostic gene chip, relate in particular to a kind of while extracting body fluid especially in the blood in the method for pathogenic agent RNA and DNA.

Gene chip is a forward position biotechnology that grows up nineteen nineties.With a large amount of target fragments in an orderly manner, to high-density (distance between points is generally less than 500 μ m) be arranged on the carriers such as glass, silicon, be referred to as gene chip.

U.S. affymetrix company in later 1980s to the beginning of the nineties, taken the lead in carrying out this respect research (Fodor, S.P.A.et al. (1991) Science 251,767-773.).1992, the said firm's utilization semi-conductor photograph plate technique was at 1cm ²About slide on the synthetic oligonucleotide fragment of original position, first gene chip (Southern, E., Maskos, U.﹠amp in the world have been born; Elder, R. (1992) Genomics 13,1008-1017.).Simultaneously, the fluorescent mark of probe, laser confocal scanning (U.S Patent 5981965) and Computer Analysis technology such as (U.S Patent 5974164) be development thereupon also.Nineteen ninety-five, first is that the gene chip (micromatrix) of carrier is at (the Schena that is born of U.S. Stanford university with glass, (1995) Science.270. (20) such as M.: 467-480.), this indicates that biochip technology stepped into the period of broad research and application.

The synthetic oligonucleotide chip of original position has the dense degree height, can synthesize the advantages such as oligonucleotide of arbitrary sequence, is applicable to determined dna sequence, snp analysis etc.But its shortcoming is synthetic oligonucleotide limited length, thereby gene specific is poor, and increases with the increase resultant fault rate of length thereupon, and research can not show a candle to the cDNA chip as gene expression profile.The cDNA chip be with micro-cDNA fragment on carriers such as glass by the matrix dense arrangement and solidify, also cry micromatrix (Microarray) (DeRisi, J. etc. (1997) Science278,680-686.).Though gene point sample density is more much higher as the point sample density of cellulose mixture filter membrane or nylon membrane than using conventional carriers not as good as the synthetic oligonucleotide chip height of original position, can reach 40,000 genes of every slide glass.And cDNA chip biggest advantage is that the target gene detection specificity is very good, and is reliable as the express spectra result of study.Present many National Laboratories and big drugmaker all use this type of chip (Baldwin, D etc. (1999) Curr Opin Plant Biol 2 (2): 96-103.).

Gene chip is that the demand owing to high-throughput, broad scale research gene function produces the earliest, but along with the maturation day by day of biochip technology, people find that pleasantly surprisedly gene chip has unique application prospect and huge commercial market in the diagnosis of communicable disease and inherited disease.The infectious pathogen diagnosing chip is exactly that characterizing gene fragment (target gene) with pathogenic agent to be measured is fixed in and makes chip on the slide, will be from the patients serum extracting DNA or the RNA that go out pathogenic agent behind amplification label fluorescence, hybridize with chip, hybridization signal is by scanner scanning, yin and yang attribute is judged in machine analysis as calculated again.The superiority that diagnosing chip is different from other detection meanss is:

(1) test sample is all kinds of Disease-causing gene fragments, has improved detection efficiency;

(2) because of need not organism immune response, can diagnose early, and the testing sample consumption is less;

(3) the diagnosing chip technology is the molecular diagnosis method that DNA hybridization technique and fluorescent mark technology combine, and high sensitivity, specificity and reliability are arranged;

(4) the level of automation height is beneficial to large-scale promotion application.Aspect medical diagnosis on disease, more existing single disease gene diagnosing chip launch, for example the Affymetrix company of the U.S. has utilized gene chip to carry out the research work of HIV (human immunodeficiency virus) (HIV), and business-like GeneChip HIV PRT diagnosing chip listing is arranged, be used for the early diagnosis of AIDS.But the application of chip technology in medical diagnosis on disease still is in the starting stage, also big area is not promoted, reason mainly contains: (1) biochip technology is a brand-new technology, requirement to the talent, technology, fund is very high, this technology of having had only a fraction of scientific research institution and high-tech company at present on top of; (2) theoretically, diagnosing chip can detect all already present pathogenic agent in the body fluid, thereby accomplishes the early prevention and the treatment of disease.In order to detect the DNA or the RNA pathogenic agent that may exist in the body fluid, the dna profiling of the corresponding pathogenic agent of requirement acquisition or acquisition RNA template are carried out reverse transcription and are obtained its DNA, carry out the pcr amplification fluorescent mark then, and amplified production is used for the DNA hybridization detection of diagnosing chip.Therefore, the DNA, the RNA template that obtain the various pathogenic agent that may exist in the body fluid with easier method have become a technical barrier in this field, still do not have good method at present.

The object of the present invention is to provide the method for middle pathogenic agent RNA of a kind of while extracting body fluid (including but not limited to blood, urine, saliva) and DNA, by corresponding extraction agent and method for extracting, obtain DNA, the RNA template of the various pathogenic agent that body fluid may exist with easier method, be used for pcr amplification, help to improve the accuracy and the specificity of diagnostic gene chip technology.

The present invention also is achieved in that

Disclosed in this invention is pathogenic agent RNA and DNA method in a kind of method for extracting body fluid, specific as follows:

(1) get body fluid 50ul, add extract 1-10 μ l, 25-45 ℃, 3-15 minute.(2) boil 5-15 minute.(3) 50-80 ℃, 10-30 minute, uncap.(4) add 2-10 μ l ultrapure water again and suspend static 5-20 minute.(5) 12000-15000rpm, centrifugal 5-15 minute.(6) get supernatant 8 μ l and be RT-PCR.Extract (1000 μ l) is formulated as follows: the chemical reagent name that 100 μ lDEPC (diethylpyrocarbonate)+5 μ lNP40+895 μ l water NP40 produce for AMRESCO company, molecular weight is 603.

Below will relevant details of the present invention be further elaborated by embodiment; but embodiment does not limit protection scope of the present invention; relevant technician fully can be according to design of the present invention and disclosed technical scheme; be applied to pathogenic agent RNA and DNA in the body fluid of extracting simultaneously with drawing inferences about other cases from one instance;, this is also within protection scope of the present invention.

Embodiment 1

(1) extract one really the infection of disease the blood samples of patients of hepatitis B virus (dna virus) and hepatitis C virus (RNA viruses) is arranged, get serum 50ul, adding extract 5 μ l, 37 ℃, 5 minutes.

(2) boil 10 minutes.

(3) 70 ℃, 20 minutes, uncap.

(4) add 5 μ l ultrapure waters again and suspend, left standstill 10 minutes.

(5) 13000rpm, centrifugal 10 minutes.

(6) get supernatant 8 μ l and be RT-PCR.

(7) probe and the target gene on the diagnosing chip with preparation carries out DNA hybridization;

(8) with diagnosing chip ScanArray 3000 scanner scanning, and use Imagene software analysis results of hybridization, find that this serum sample and hepatitis B virus target gene and hepatitis C virus target gene are positive.

Show that by disclosed technical scheme of specification sheets and embodiment the method for pathogenic agent RNA and DNA has the following advantages in the while extracting body fluid of the present invention: (1) method is easy, and speed is fast; (2) because operation steps is less, yield height, good stability.

Claims (3)

1, the method for pathogenic agent RNA and DNA in a kind of while extracting body fluid is characterized in that extractive step is:

(1) get body fluid 5ul at least, add extract 1-100 μ l, 25-45 ℃, 3-15 minute.

(2) boil 5-15 minute.

(3) 50-80 ℃, 10-30 minute, uncap.

(4) add 2-10 μ l ultrapure water again and suspend static 5-20 minute.

(5) 12000-15000rpm, centrifugal 5-15 minute.

(6) it is standby to leave and take supernatant.

2, pathogenic agent RNA and DNA while method for extracting in a kind of body fluid as claimed in claim 1 is characterized in that optimum condition is:

(1) get serum 50ul, add extract 5 μ l, 37 ℃, 5 minutes.

(2) boil 10 minutes.

(3) 70 ℃, 20 minutes, uncap.

(4) add 5 μ l ultrapure waters again and suspend, left standstill 10 minutes.

(5) 13000rpm, centrifugal 10 minutes.

(6) it is standby to leave and take supernatant.

3. a special-purpose extract that is used for the described method of claim 1 is characterized in that, extract (1000 μ l) is formulated as follows:

100 μ lDEPC (diethylpyrocarbonate)+5 μ lNP40+895 μ l water.