KR900001577B1 - Quantity analysis of gene by using polyethylene glycol - Google Patents

Abstract

A qutntitative virus gene is prepared using polyethylene glycol. Thus, HCMV AD 169 (ATCC) is cultured in human lung fibroblasts to isolate HCMV, which is cut by EcoRI, cloned in PBR 325, and inserted into recombinant vehicle by nick translation of 32P-dCTP to give a probe. Virus DNA is obtained from a treatment of 1ml of patient's urine with 300 ul or 40% polyethylene glycol. 50 ul of the virus DNA is placed in 96- well plate, treated with 50 ul of IN-NaOH, glued to nitro cellulose membrane, and packed in a polyvinyl bag. Cross- reaction solution preheated at 40oC is added into the above bag, reacted with probe, washed, and autoradiographed to quantify the virus gene.

Description

[Name of invention]

Method for quantifying viral genes present in a sample using polyethylene glycol and kits thereof

Detailed description of the invention

The present invention relates to a method for quantifying a viral gene present in a sample using polyethylene glycol and a kit thereof.

In more detail, a method of quantitatively analyzing a kit by which serum, urine, or saliva is used as a sample and reacted by adding polyethylene glycol or a salt solution thereof, followed by centrifugation, dissolving and separating the precipitate, and hybridizing it will be.

Conventionally, serological methods that have been commonly used for assaying viruses causing human or animal diseases have been assayed using antigen and antibody binding reactions. However, these inventions have many problems and many problems in the sensitivity and manufacturing process of kits. The disadvantage is that it takes time.

These problems have been proposed in accordance with the development of molecular biology since the mid-1970s. Among them, DNA Probe technology is a method of transferring a specific gene to a carrier and mass-repairing a recombinant carrier from a microorganism to attach radioisotopes or non-radioactive substances to a recombinant carrier to find the same gene present in the sample. . See Rigby et.at. J. Mol. Biol. 113, 237-251 (77); Ward et.al. PNAS 80, 4045-4049 (83)]

This probe technology has been used for research purposes, but has been applied in many fields since the 1980s. Probe technology has various problems to be solved in order to be applied to clinical field. Current research focuses on how to simplify hybridization, how to easily isolate sample genes, how to use non-radioactive isotopes, and the development of more sensitive probes.

For example, the sandwich method developed by Iri Palba et al. [Gene 21, 77-85 (83), J. of Clinical Microbiology 18 (1), 98-100 (83)], The part is attached to the stationary phase and the other part is attached to the radioisotope, and then reacted with the sample gene using a probe, and then the unreacted gene is removed to easily test.

At this time, when using I ¹²⁵ as a radioisotope, the amount of the sample gene is immediately measured in the r-counter.

In 1980, Dr. Ward's team at Yale University added biotin attached to [PNAS 80,4045-449 (83)] d UTP to specific genes to make probes, and then hybridized with avidin enzymes ( We developed a method to determine the result by color reaction by reacting with conjugates such as Avidin-Enzyme and Aviin-fluorescent dye.

Since 1982, research has been conducted on how to make probes more sensitive. Meshing, etc. [N.A.R. 9, 309-321 (81); Gene 19, 269-276 (82)] produced single stranded DNA probes using M13 phage, and Bulter et al. [J. of Biol. chem.257, 5772-5778 (82) developed single stranded RNA probes using the SP 6 transcription promoter. However, despite many of the developments described above, there are still problems in expanding clinical applications. That is, the development of the method of separating a sample gene from a sample is calculated | required.

Method [Hepatology 1 (5), 386 (81)] such as F. bonino [Mepatology 3 (3), 279 (83)] such as J. Skoto, method such as Emberninger [J.of Medical Virology 9 , 57 (82)] C. M. Chu [Hepatology 5 (3), 431 (85)], Sun Chow [The New England Journal of Medicine 308 (16), 921 (83)], Schuster et al. [Journal of Medical The separation methods used by Virology 19, 277 (86)] S. Specter (Clinical Chemistry 31 (9), 1514 (85)) are not available clinically.

In other words, the paper disclosed in the previous article describes the precipitation of virus particles using ultra-high-speed centrifuge when the virus is to be separated from serum and urine or saliva, and then the precipitates are treated with sodium dodecyl sulfate and proteinase K. After extraction with phenol / chloroform, virus DNA in aqueous solution was isolated. Another method was to add sodium dodecyl sulfate and proteinase K directly in serum to disrupt the virus in serum for 1-2 hours, and then to phenol / chloroform. Viral genes were isolated by extraction.

The conventional virus particle separation method is as follows.

1) Virus Gene Isolation Method Using High Speed Centrifuge

1-a) Separation method of viral genes present in serum using an ultrafast centrifuge.

600 μl serum was added to 400 μl of 30% sucrose cushion and centrifuged at 20,000 rpm for 20 hours at 4 ° C. to dissolve the additives in 35 μl of 1 mA EDTA, 10 mM Tris-HCL (pH 8.0), followed by 1% sodium After 5 μl of dodecyl sulfate solution was added and reacted at 37 ° C. for 2 hours, 45 μl of phenol saturated with 10 mm Tris-HCl (PH8.0) was added thereto, followed by shaking slightly, followed by centrifugation at room temperature for 12 minutes at 12.000 rpm. Was separated and used as a sample.

50 µl each of the 96-well plate was added, 50 µl of 1N NaOH was added thereto, followed by shaking for 10 minutes, followed by addition of 200 µl of 1M Tris-HCL (pH7.5) and 2.5M NaCL, followed by 6 × SSC ( SSC: After adding to a 96-Well Suction Apparatus equipped with a nitrocellulose membrane saturated with 0.15M NaCL, 0.015M Na ₃ Citrate, pH7.6), the nitrocellulose membrane was allowed to stand in air for 15 minutes. After leaving in a vacuum oven at 80 ° C., a nitrocellulose membrane was added to a polyvinyl bag, a solution was added thereto, and four edges were attached with a heat sealer, followed by at least 8 hours at 42 ° C. to perform a hybridization reaction. Cut one part of the bag, add the probe, reattach it, and react at 42 ℃ for 20-24 hours.

After washing with high concentration solution and low concentration solution, air drying and autoradiography assay is performed.

1-b) Isolation of viral genes present in urine

After centrifugation of 10 ml of urine for 1 hour at 20,000 rpm with SW-28 rotor, the precipitate was dissolved in DNA buffer [0.1 M NaCl, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)] and SDS, RNase, proteinina. Treat with zero, extract with phenol / chloroform and precipitate with ethanol. This was dissolved in an appropriate amount of TE (10 mM Tris-HCl, 1 mM EDTA (pH 8.0)] and used as a sample.The autoradiography was performed by attaching to a stationary phase and performing a hybridization reaction as described in 1-a above.

1-c) Viral Gene Isolation in Saliva

200 hr ultracentrifuged solution with isotonic saline solution at 20,000 rpm (Sorvall SS24), and precipitate was dissolved in a buffer solution (3% sarcosy1 0.075M Tris-HCl, pH8.5, 0.025M EDTA, pH8.1) Dissolved in and reacted with 100 μg / ml proteinase K for 1 hour at 37 ° C., extracted with phenol / chloroform, and used as a sample. Autoradiography was performed by attaching to a stationary phase and performing a hybridization reaction as described above in 1-a).

2) Isolation of Virus Genes Using Proteinase K

To 300 µl of serum, 100 µl of SDS cocktail [25 mM NaAc (pH7.5), 2.5 mM Na ₂ EDTA, 25 µg / ml, yeast t-RNA] containing proteinase K (10 µg / ml) was added, After the reaction, the reaction mixture was shaken by adding 400 µl of saturated phenol with water, and then shaken again by adding 40 µl of chloroform / isoamyl alcohol (24/1). 40 μl was added and shaken, followed by centrifugation at 12,000 rpm for 5 minutes at room temperature. The supernatant is separated and attached to a stationary phase as described above in 1-a) and subjected to hybridization by autoradiography.

As described above, both methods require a lot of time and complicated processes, and require expensive equipment and reagents, and thus clinical application is almost impossible. In addition, since the SDS or proteinase K used in the above-described method reacts nonspecifically with the sample protein, the recovery rate of the viral gene is very low after the reaction, and the loss of the viral gene during organic solvent extraction results in accurate quantification of the virus. Can not.

When quantifying gene extracts by performing hybridization reactions, the extracts should be attached to the stationary phase. Nitrocellulose membrane, gene screen, m-aminobenzyloxymethyl paper, and DBM paper (diazobenzyloxymethyl) paper and Zeta Probe. These stationary phases have affinity for both proteins, single-stranded DNA, and single-stranded RNA, so that not only the gene is attached to the stationary phase but also the protein mixed in the gene extract is limited. Competition between genes and proteins in space causes loss of the genes present in the extract. This problem is also a reason why the sample gene cannot be quantified.

The present inventors have diligently studied to eliminate these various disadvantages, and as a result, the envelope of the virus particles is composed of proteins, polysaccharides, and the like. You can do it easily.

Hereinafter, the present invention will be described in detail by dividing step by step.

1. Determination of the concentration of polyethylene glycol

Polyethylene glycol solution or a salt solution thereof was added to the patient's serum, urine or saliva so that the final concentration was 1, 2, 3, 4, 5, 6, 7, 10%, and the mixture was simply vortexed and allowed to stand at room temperature for 5 minutes. Then centrifuge for 5 min at 12,000 rpm at room temperature. Take out the supernatant, store it in a new tube, dissolve the precipitate in the solution, denature the solution, add neutralization solution, and add it to a 96-well suction device equipped with a nitrocellulose membrane. After confirming that the sample is completely sucked, take out the nitro cellulose membrane. Air dry and dry for 1 hour in a vacuum oven at 80 ℃.

The hybridization solution heated to 42 ° C. was added to a polyvinyl bag containing nitrocellulose membrane so as to be .01 ml / cm 2 + 1 ml, and then reacted at 42 ° C. for 1 hour. The bag was removed, a probe was added, and 42 ° C. was separated and quantified. I developed a method.

It is an object of the present invention to provide a method and kit for quantifying a sample gene by simply separating a virus gene from a sample and obtaining an adhesion rate of the isolated gene.

Hereinafter, the present invention will be described in detail.

In the present invention, when the virus particles are precipitated with a salt solution of polyethylene glycol or polyethylene glycol, other substances present in the sample are also precipitated, so that the adhesion rate of the viral gene should be obtained. The adhesion rate was only 25% of the extracted DNA when using the radioisotope attached DNA. Therefore, when analyzing the result after the hybridization reaction, the sample value should be multiplied by 4 and compared with the positive control.

The salt of the polyethylene glycol salt solution is reacted for 20 hours in use as an aqueous solution of sodium chloride, magnesium chloride, ammonium sulfate and potassium phosphate.

The reaction solution was washed 4 times at room temperature with high concentration solution (2 × SSC, 0.1% SDS) for 10 minutes and twice with low concentration solution (1 × SSC, 0.1% SDS) at 65 ° C for 45 minutes, followed by air drying and autoradiography. It was. As a result, as shown in Table 1, from 5% or more, the filtrate was completely precipitated without visible virus particles.

TABLE 1

2. Determination of reaction time after adding polyethylene glycol

After fixing the polyethylene glycol concentration to the final 5%, add 100 µl of polyethylene glycol salt solution to the sample, shake it slightly, and store the centrifuge in a new tube for 5 minutes while changing the reaction time to 3 minutes, 5 minutes, 10 minutes, and 20 minutes. The precipitates dissolved in the solution were attached to nitrocellulose membranes, and hybridization reactions and autoradiography were carried out according to the above 1 method. After 10 minutes and 15 minutes of reaction, some viral genes were observed in the supernatant. This is because the virus particles are damaged by polyethylene glycol and remain in the supernatant without precipitation when virus genes come out and centrifuge. The results are shown in Table 2 below.

TABLE 2

3. Recovery rate according to centrifugation time after adding polyethylene glycol and reacting

Polyethylene glycol salt solution was added to the sample so that the final concentration of polyethylene glycol was 5%, and the mixture was shaken slightly, and then reacted for 5 minutes at room temperature. The precipitate was centrifuged at 12,000 rpm for 1, 2, 3, 5, 7, and 15 minutes. The hybridization reaction and autoradiography were performed in the same manner as described above to obtain the same result from 5 minutes or more as shown in Table 3 below.

TABLE 3

4. Experiment on adhesion rate

Using 32 samples, virus particles extracted by polyethylene glycol method were attached to a 96-well suction well by nitrocellulose membrane saturated with 6-fold SSC, and two sheets were attached to each other in the same manner as described above. In the second nitrocellulose membrane, the viral genes present in the sample were measured. However, the modal control group did not adhere to the second nitrocellulose membrane. From this fact, the positive control group does not compete with other substances because it exists only in the state of DNA without contamination of other substances.

Therefore, the positive control group completely adheres to the first nitrocellulose membrane, whereas in the case of the sample, the viral gene demonstrates that it is not completely attached to the first nitrocellulose membrane and escapes due to competition with other substances.

For this reason, viral genes present in serum cannot be fully quantified. Therefore, if the adhesion rate of the sample is obtained for the quantification of the viral genes present in the sample, the quantification can be performed.

In the present invention, such an adhesion rate was obtained by performing as follows.

- 카운팅하여 전체 방사능양과 비교하여 부착율을 구하였다. 시료에 따라 약간의 차이는 있으나, 평균 25%의 바이러스 입자가 제1니트로셀루로즈막에 부착됨을 알 수 있었으며 100개의 시료를 사용하여 관찰한 결과 최대 부착율은 50%이었으며 최소부착율은 17%임을 알 수 있었다. 위의 사실로부터 시료내에 존재하는 바이러스 유전자의 양을 최대치, 최소치, 평균치 값으로 정량할 수 있다. 즉, 교잡반응된 니트로셀루로즈막을 칼로자르고,

-카운팅하여 그 값에 인자 4, 2, 6를 곱하여 양성대조군과 비교하여 시료의 평균값, 최소값, 최대값을 구할 수 있다. 이를 하기 표 4에 나타냈다.Viral gene was mixed with polymerase I 2U and 100 μM dATP, dGRP, dTTP, ³² P-CTP 100 μci, and allowed to react for 30 minutes at room temperature. 5 μl each was added to the stationary phase in the same manner as described above.

-Counting resulted in adhesion rate compared to total radioactivity. Although there were some differences depending on the samples, it was found that an average of 25% of the virus particles adhered to the first nitrocellulose membrane. The maximum adhesion rate was 50% and the minimum adhesion rate was 17% using 100 samples. I could see that. From the above facts, the amount of viral genes present in a sample can be quantified by the maximum, minimum and average values. That is, by cutting the hybridized nitro cellulose film with a knife,

Count and multiply the values by the factors 4, 2, and 6 to compare them with the positive control to find the average, minimum and maximum values of the sample. This is shown in Table 4 below.

TABLE 4

5. Comparison of Polyethylene Glycol Method, Proteinase K Method, and Ultracentrifugation Method

The results of comparative experiments in three ways using the same sample are shown in Table 5 below. In this table, it can be seen that the polyethylene glycol method has a 1.2 times higher recovery rate than the ultracentrifugation method and a 4 times higher recovery rate than the proteinase K method.

TABLE 5

Positive Control: 1pg: 109cpm Negative Control: 47cpm

50: 142 45

100: 210 42

200: 345 43

400: 478 52

600: 694 38

800: 974 47

1000: 14 120 44

In order to carry out the method of the present invention more simply and effectively, a DNA probe kit was made. The kit of the present invention can assay 88 samples at the same time. The kit consists of:

1.2×10⁸cpm/㎍1) DNA Probe (HBV)

1.2 × 10 ⁸ cpm / μg

2) Extract: Polyethylene glycol eye salt 20-40% solution 10-40ml

3) Solution: 15-60ml of TE (pH 8.0)

4) Denatured solution: 1N NaOH 5ml

5) Neutralization solution: 1M Tris-HCl (pH7.5) + 2.5M NaCl 20ml

6) Hybridization solution 15ml

50% formamide

5 × SSC

50 mM Sodium Phosphate

0.2% polyvinyl pyrrolidine

0.2% BSA

0.2% ficoll

1 mg / ml sheared salmon sperm DNA

(sheared salmon sperm DNA)

7) 20 × SSC 200ml

8) 10% SDS 20ml

9) 7 positive controls

10) 1 negative control

11) 1 polyvinyl bag

12) 1 nitrocellulose membrane

13) 4 sheets of Whatman 3M M Paper

14) 96-well plate

[Use of the present invention kit]

Place in a small centrifuge tube, add 300 µl of serum, add 100 µl of extract and shake slightly. After standing at room temperature for 5 minutes, centrifuging at 12,000 rpm for 5 minutes, discarding the supernatant, dissolving the precipitate with lysis solution, 50 μl was added to a 96-well plate, 50 μl of 1N NaOH was added to each well, and 10 minutes at room temperature. Leave it. 200 μl of neutralization solution was added to each well, and each sample was added to a 96-well suction device equipped with a nitrocellulose membrane saturated with 6 × SSC. The sample was then sucked with an aspirator for 10 minutes and washed twice with 6 × SSC. Take out the cellulose film, air-dry for 10 minutes, dry for 30 minutes in a vacuum oven at 80 ℃, transfer to polyvinyl bag, add 10ml of the hybridization reaction solution preheated in 42 ℃ water bath, and then stand still in 42 ℃ water bath for 1 hour. Add. After standing at 42 ° C for 20 hours, transfer the nitrocellulose membrane to a plastic box containing 2 × SSC, 01% SDS 200ml, wash it by shaking for 10 minutes at room temperature, and repeat this operation three more times. Transfer the nitrocellulose membrane to a plastic box containing 1xSSC, 01% 500ml preheated to 65 ℃, and let stand for 45 minutes in 65 ℃ water bath. Repeat this operation once more. After analyzing the results of absorbance at 405 nm, the absorbance values of the samples were multiplied by 2, 4, and 6 to obtain the minimum, average, and maximum values, respectively.

Alternatively, the nitrocellulose membrane was cut into each well and placed in a vial containing a 5 ml scintillation cocktail, and counted for 5 minutes, and the measured value of the positive control and the amount of DNA were log values. After converting and drawing a standard curve, multiply the measured values of the sample by 2, 4, and 6, respectively, to obtain the minimum, average, and maximum values, respectively.

Example 1

Hepatitis virus

1) Preparation of Probes

로타로 40000rpm으로 4시간 동안 원심 분리한다. 침전물을 상기 용액에 다시 현탁시킨 후 동일하게 조작한다. 이를 소량의 완충용액(10mM tris-HCl, pH 7.6, 0.1M NaCl, 10.5% Noniet-P

40, 0.1%

-mercaptoethanol)에 녹인 즉시 엔도제니우스 DNA 폴리머라제 액티비티(Endogeneous DNA Polymerase activity)를 이용하여 HBV DNA를 합성했다. 이때 조건은 40mM tris-HCl, pH 7.6, 16mM MgCl₂, 46mM NH₄Cl, 0.2mM dNTPs로 구성된 반응용액을 사용하였으며 37℃에서 3-5시간 반응시켰다. 반응이 끝난 것에서 DNA를 분리해 내기 위해 용액에 0.5% SDS, 5mM EDTA, 프로티나제 K(1mg/ml)를 각각 가하고 2시간 반응시킨 후 페놀/클로로포름(1:1)을 사용하여 불순물을 제거하고 알코올로 DNA를 침전시켜 얻었다.Centrifugation of HBsAg-positive plasma at 9000 rpm for 15 minutes to remove suspended solids and ultrafast centrifugation at 100,000 g for 3-4 hours to precipitate dan particles and other cellular components. This was again carefully suspended in buffer (10 nM Tris-HCl, pH 7.6, 0.1 M NaCl, 1 mg / ml Bovine Serum Albumine), and then placed on 30% sucrose cushion made of electric buffer and spinco SW 41

Centrifuge for 4 hours at 40000 rpm with Rota. The precipitate is resuspended in the solution and operated in the same manner. This was followed by a small amount of buffer (10 mM tris-HCl, pH 7.6, 0.1M NaCl, 10.5% Noniet-P

40, 0.1%

HBV DNA was synthesized using Endogeneous DNA Polymerase activity immediately after dissolution in -mercaptoethanol. At this time, the reaction solution using 40mM tris-HCl, pH 7.6, 16mM MgCl ₂ , 46mM NH ₄ Cl, 0.2mM dNTPs was used and reacted at 37 ℃ 3-5 hours. 0.5% SDS, 5mM EDTA, proteinase K (1mg / ml) was added to the solution to react with DNA for 2 hours, and then phenol / chloroform (1: 1) was used to remove impurities. It was obtained by precipitating DNA with alcohol.

HBV DNA was cleaved with Bam Hl and T ₄ DNA ligase was linked to the Bam Hl site of pBR 322 to transform HB101, thereby culturing the recombinant strain formed by mass culturing the recombinant strain. 1 μg of the isolated recombinant DNA was taken to perform a nick translation. 5 μl of 10 × nick translation buffer, 100 μl of ³² P-dCTP 100 μCi DNA polymerase I 2U, and 7 pg of DNase I. HBV DNA were reacted at 16 ° C. for 1 hour. The specific activity of the HBV probe thus obtained was 1.8 × 10 ⁸ cpm / μg.

2) HBV DNA Isolation from Serum

300 µl of serum was added to the centrifuge tube (1.5 ml), and 100 µl of 20% polyethylene glycol salt solution was added thereto, and vortexed for 10 seconds. After standing at room temperature for 5 minutes, the precipitate obtained by centrifugation at 12,000 rpm was distilled water and TE (pH 8.0). It was dissolved in 1% SDS or 1% NP40, etc. and used as a sample.

3) Attachment of sample DNA to nitrocellulose membrane

50 μl of the separated sample was added to a 96-well plate, and then 50 μl of 1N NaOH was added to the shaker for 10 minutes, followed by shaking on a shaker. Then, 200 μl of 1M Tris-HCl (pH 7.5) + 2.5M NaCl was added to the nitrocellulose membrane. It is added to a 96-well suction device, which is attached by suction for 10 minutes. The nitrocellulose film was taken out and dried for 10 minutes, dried in a vacuum oven at 80 ° C. for 30 minutes, sealed in a polyvinyl bag, and stored at room temperature until use.

4) hybridization reaction

The probe after the hybridization reaction solution is preheated to 42 ℃ polyvinyl 101 hours after the reaction in a water bath at 42 ℃ was added to 0.1ml / ㎠ + 1ml was added 1.4 × 10 ⁸ cpm / ㎍ was reacted for 20 hours.

5) washing

The nitrocellulose membrane was removed from the polyvinyl bag, transferred to a plastic box containing 2 × SSC and 200ml of 0.1% SDS, shaken at room temperature for 10 minutes, and the above operation was repeated three times. The nitrocellulose membrane was transferred to a plastic box containing 500 ml of 1 × SSC and 0.1% SDS preheated at 65 ° C. and allowed to stand for 45 minutes in a 65 ° C. water bath. The above operation was repeated once. The nitro cellulose membrane was taken out and analyzed in two ways.

-카운팅 분석방법6)

Counting method

Cut the sample area of the nitrocellulose membrane into a vial containing 5 ml scintillation cocktail and measure for 5 minutes. The average value was obtained by multiplying the measured value by 4, the maximum value was multiplied by 6, the minimum value was multiplied by 2, and then quantified by comparing with the measured value of the positive control group. The number of particle | grains of a sample can be calculated | required by the following formula.

X ÷ 2.7 × 10 ^-6 × 13 = particle count / ml

(Where X is the amount of DNA obtained by comparing the measured value of the sample with the positive control)

7) Autoradiography

(SARAN WRAP)으로 싼후 필터에 붙이고 강화스크린(intensifying screen)이 있는 카세트에 다시 붙이고 X- 레이 필름을 넣어 - 70℃에서 20시간 방치한 후 현상하여 96-웰 플레이트에 맞게 자른 후 멀티 타이터 택(multi titer tek)에서 405nm으로 흡광도를 측정하여측정된 흡광도 값에 각각 4, 6, 2를 곱해 평균값, 최대값, 최소값을 구하고 양성대조군의 흡광도 값과 비교하여 정량한다.I wrap up a nitrocellulose film

Wrap with SARAN WRAP, attach to filter, reattach to cassette with intensifying screen, add X-ray film-stand at 70 ℃ for 20 hours, develop and cut to 96-well plate Measure the absorbance at 405 nm in (multi titer tek) and multiply the measured absorbance values by 4, 6, and 2, respectively, to find the average, maximum, and minimum values, and quantify them by comparing them with the absorbance values of the positive control.

The number of particle | grains of a sample can be calculated | required by the following formula.

X ÷ 2.7 × 10 ^-6 × 13 = particle count / ml

(X is as described above)

Example 2

Cytomegalovirus

1) Preparation of Probes

Culturing the HCMV AD 169 (ATCC) in human lung fibroblast cells (human lung fibroblasts) to remove the HCMV was cut with EcoRI and the cleaning (Cloning) in pBR 325 match the cross-reactivity between HCMV strains key is selecting a recombinant strain ³² P-dCTP was inserted into the recombinant vehicle by nick translation and used as a probe.

2) Viral DNA Extraction

300 ml of 40% polyethylene glycol salt solution was added to 1 ml of the urine of the patient, briefly vortexed, and allowed to stand at room temperature for 5 minutes, followed by centrifugation at 12,000 rpm for 100 µl of water or TE (pH 8.0), 1% SDS, 10%. The precipitate was dissolved in NP-4- or the like and used as a sample.

3) Attached to the stationary phase

50 μl of the sample was added to a 96-well plate, and then 50 μl 1N NaOH was added thereto, followed by standing for 10 minutes, followed by 200 μl neutralization solution, which was immediately added to a 96-well suction machine. The sample was added to the nitrocellulose membrane for 10 minutes using an aspirator. Attach to The nitrocellulose membrane is taken out and left to stand in air for 10 minutes, dried in a vacuum oven at 80 ° C. for 30 minutes, and stored in a polyvinyl bag at room temperature.

4) hybridization reaction

The hybridization solution preheated to 40 ° C. was placed in a bag and left in a water bath for 1 hour, followed by the addition of probes and left for 20 hours.

5) washing

-카운팅하여 결과를 얻을 수 있다.Transfer the nitrocellulose membrane to a plastic box containing 200 ml of 2 × SSC, 0.1% SDS and shake for 10 minutes at room temperature. Repeat this operation three more times. Transfer the nitrocellulose membrane to a plastic box containing 500 ml of 1 × SSC, 0.1% SDS, preheated to 65 ° C and leave for 45 minutes at 65 ° C. Repeat this operation once more. Nitrocellulose film or autoradiography or

-You can count the results.

Example 3

HBV DNA Probe Kit

1.2×10⁸cpm/㎍1) DNA Probe (HBV)

1.2 × 10 ⁸ cpm / μg

2) Extract solution: Polyethylene glycol salt solution 10ml

3) Solution: 20ml

4) Denatured solution: 1N NaOH 5ml

5) Neutralization solution: 1M Tris-HCl (pH7.5) 20ml + 2.5M NaCl

6) Hybridization reaction solution 15 ml, 50% formamide, 5 x SSC, 50 mM sodium phosphate, 0.2% polyvinyl pyrrolidine, 0.2% BSA, 0.2% ficoll, 1 mg / ml shear salmon sperm DNA

7) 20 × SSC 200ml

8) 10% SDS 20ml

9) 7 positive controls

10) 1 negative control

11) 1 polyvinyl bag

12) One piece of nitrocellulose membrane

13) 4 sheets of Whatman 3MM Paper

14) 96-well plate

30 µl of serum was added to the centrifuge tube, 100 µl of the extract was added thereto, and the mixture was briefly vortexed and allowed to stand at room temperature for 5 minutes. After centrifugation at 12,000 rpm for 5 minutes, the supernatant was discarded and the precipitate was dissolved in 300 µl of the solution and assayed according to the above description. The above kit can simultaneously test 88 samples.

Example 4

CMV DNA Probe Kit

1) DNA probe (CMV) <1.2 × 10 ⁸ cpm / μg

2) Extract: 40% polyethylene glycol salt solution 40ml

3) Dissolution solution: 20ml 10mM TE (pH 8.0)

4) Denatured solution: 1N NaOH 5ml

5) Neutralization solution: 1M Tris-HCl (pH7.5) 20ml + 2.5M NaCl

6) Hybridization reaction solution 15 ml, 50% formamide, 5 x SSC, 50 mM sodium phosphate, 0.2% polyvinyl pyrrolidine, 0.2% BSA, 0.2% ficoll, 1 mg / ml shear salmon sperm DNA

7) 20 × SSC 200ml

8) 10% SDS 20ml

9) Positive Control

10) Negative Control

11) 1 polyvinyl bag

12) One piece of nitrocellulose membrane

13) 4 sheets of Whatman 3MM Paper

14) 96-well plate

In the method of use, 1 ml of urine is added to a centrifuge tube, 300 µl of extract is added thereto, briefly shaken, and allowed to stand at room temperature for 5 minutes. Centrifuge for 15 minutes at 12,000 ppm, discard the supernatant and dissolve the precipitate in the solution and assay according to the method described above.

Claims (3)

In the method for quantifying a viral gene present in a sample, a polyethylene glycol solution or a salt solution of polyethylene glycol is added to the sample, briefly vortexed, and allowed to stand at room temperature, followed by centrifugation to dissolve the precipitate as a solution and hybridization reaction. A method for quantifying a viral gene, characterized in that the result is multiplied by the adhesion rate. The method of claim 1 wherein the sample is serum, urine or saliva. Known as a kit for quantifying viral genes present in plasma, saliva or urine 1) DNA Probe

1.2 × 10 ⁸ cpm / μg 2) Denatured solution: 1N NaOH 5ml 3) Neutral Solution: 1M Tris-HCl (pH7.5) 20ml + 2.5M NaCl 4) Hybridization reaction 15, 50% formamide, 5 × SSC, 50 mM sodium phosphate, 0.2% polyvinyl pyrrolidine, 0.2% BSA, 0.2% Ficoll, 1 mg / ml shear salmon sperm DNA 5) 20 × SSC 200ml 6) 10% SDS 20ml 7) Positive Control 8) Negative Control 9) 1 polyvinyl bag 10) One piece of nitrocellulose membrane 11) 4 sheets of Whatman 3MM Paper 12) 96-well plate and new 13) Extract: 4-40% polyethylene glycol or its salt solution 10-40ml 14) Lysate: A kit for quantitating a viral gene consisting of 15-60 ml of one species selected from the group consisting of TE (pH 8.0), distilled water, SDS, and NP-4-.