KR20030031243A - Primers used in nested RT-PCR for diagnosing various cancers including stomach cancer and liver cancer, gastritis, hepatocirrhosis and hepatitis at the same time - Google Patents

Abstract

PURPOSE: Primers used in nested RT-PCR for diagnosis of various cancers including stomach cancer and liver cancer, gastritis, hepatocirrhosis and hepatitis at the same time are provided, thereby rapidly diagnosing various diseases. CONSTITUTION: The primers for detecting 16s rRNA, vacA(vacuoloating cytotoxin A) and cagA(cytotoxin-associated gene A) of Helicobacter pylori, cea and afp genes as makers of cancers, and 5'-UTR(untranslated region) of HCV under the same condition of nested RT-PCR(reverse transcription-polymerase chain reaction) have the nucleotide sequences of SEQ ID NOs: 1 to 28, wherein the RT-PCR condition is characterized by the annealing at 50 to 53 deg. C. A diagnostic kit contains the primers.

Description

Primers used in nested RT-PCR for diagnosing various cancers including stomach cancer and liver cancer, gastritis, hepatocirrhosis and hepatitis at at the same time to diagnose various cancers including gastric cancer and liver cancer, gastritis, cirrhosis and hepatitis the same time}

The present invention diagnoses all cancer types including gastritis and gastric cancer, hepatitis, liver cirrhosis and liver cancer, and colon cancer, prostate cancer, breast cancer, uterine cancer and lung cancer in one nested reverse transcription-polymerase chain reaction (RT-PCR) reaction. The present invention relates to a nested primer for RT-PCR and a kit for cancer diagnosis comprising the same.

After a certain period of time, normal cells die by the apoptosis system that the cells themselves possess. However, due to mutation of genes involved in apoptosis and cell proliferation, normal cells become cancer cells that do not die. In addition, these cancer cells metastasize to other cells to destroy normal tissues nearby, and local recurrence or distant metastasis is easy, and cancer is recognized as a chronic wasting disease.

Meanwhile, in Korea, liver cancer occupies the first place in the rankings of liver cancer, second place is the stomach cancer, and third place is lung cancer. In addition, since cancer has a high cure or treatment effect at the time of early diagnosis, research on the early diagnosis method of cancer has been actively conducted in the art.

On the other hand, conventional cancer diagnostic methods are generally made through surgical methods such as endoscopy and biopsy using biochemical methods such as enzyme test and antigen antibody test. However, in the case of endoscopy, cancer is diagnosed according to a subjective point of view of a doctor, and there is a problem that the cancer is not diagnosed unless the cancer tissue develops to a certain size. In addition, the biochemical method also has a problem that the diagnosis can take a long time because the detection of the protein or metabolites in the tissue can be generated, since the diagnosis is likely to occur, and the time required for tissue culture.

Recently, in order to solve this problem, researches on cancer diagnosis using molecular biology methods have been conducted. For example, studies have been conducted to diagnose cancer by detecting specific genes related to cancer or mutations within the genes using polymerase chain polymerization reaction (PCR).

However, the mechanism by which cancer develops is not caused by any one single gene, and since the genes causing cancer differ by cancer sites, it is important to detect several genes related to cancer.

On the other hand, the PCR reaction is performed by a cycle of denaturation-annealing-extension, and the optimum annealing temperature and time of the primers depend on the template DNA binding site, GC base content ratio and length It is not possible to determine the exact annealing temperature by the primer sequence alone, but it is necessary to experimentally determine the optimum annealing temperature and time.

If the annealing reaction is not carried out under the correct annealing conditions, a nonspecific PCR product is formed, suspicious of the PCR results and, in the case of PCR to detect cancer markers, results in misdiagnosis of the cancer. Therefore, in order to detect several genes, the optimal annealing conditions should be measured for each primer, and if the optimal annealing conditions are different, the inconvenience of having to perform multiple PCRs with different annealing conditions occurs. Therefore, by making the annealing conditions of the primers for amplifying each gene in the same manner, enabling gene amplification at the same time under the same PCR conditions, it is possible to save a lot of time and money in cancer diagnosis through the detection of several genes.

On the other hand, in detecting a target gene for diagnosing cancer, another important factor to consider is sensitivity. PCR technology yields large amounts of DNA through multiple cycles of amplification, even with small amounts of DNA. However, in the early or latent period of infection with a virus causing cancer, the target gene or its expression is outside the detection range of the PCR technique, and thus, even if the patient is not diagnosed with cancer, even though the patient may develop and possibly develop cancer. Occurs.

To address this problem, nested RT-PCR techniques have been developed. Nested RT-PCR isolates RNA from a sample, synthesizes cDNA from reverse RNA by reverse transcription, and performs primary PCR using the synthesized cDNA as a template, followed by primary PCR product and primary By performing the secondary PCR using a primer that binds inside the PCR product, very good sensitivity is shown. That is, the mRNA detectable range of the nested RT-PCR is detectable in an amount of 0.000001 ng or less. However, since the nested RT-PCR requires one pair of primers for the first RT-PCR and one pair of primers for the secondary PCR, there is a problem in that a combination of four primers having the same annealing temperature must be found.

Therefore, under the same nested RT-PCR condition, if several genes can be detected simultaneously, not only the diagnosis of cancer but also the prognosis of cancer development will be a very effective technique. For this purpose, it is most important that all primers used in the first and second PCR have the same annealing temperature.

Therefore, the present inventors also conducted a number of studies to develop the primers for nested RT-PCR for the simultaneous diagnosis of several types of cancer, 16s rRNA, vacA of the Helicobacter pylori strain isolated from most gastric cancer or gastritis patients And cagA gene; Afp and cea genes as markers of colorectal cancer, prostate cancer, liver cancer, breast cancer, gastric cancer, uterine cancer and lung cancer cells; And when the 5'-untranslated region (5'-UTR) of HCV virus involved in hepatitis, cirrhosis and liver cancer is detected simultaneously under the same nested RT-PCR conditions, the sequence of SEQ ID NO: 1 to SEQ ID NO: 28 Using, it was confirmed that each gene can be amplified under the same nested RT-PCR conditions, and came to complete the present invention.

Accordingly, it is an object of the present invention to provide primers for nested RT-PCR for diagnosing several types of cancer using a single nested RT-PCR.

In addition, another object of the present invention is to provide various cancer diagnostic kits comprising the primers.

Figure 1 is from the result of synchronous detection using a 16s rRNA, vacA (vacuoloating cytotoxin A) and cagA (cytotoxin-associated gene A) gene nested RT-PCR from the DNA of pylori H. pylori isolates (Figure 1, Figure 1a Gel electrophoresis of the PCR product obtained by performing the primary PCR, Figure 1b shows the result of electrophoresis of the PCR product obtained by performing the secondary PCR; Lane M is the size marker, lanes 1 to 4 shows amplification of 16s rRNA using 100, 1, 0.01 and 0.0001ng of template DNA, respectively, and lanes 5 to 8 show vacA gene with 100, 1, 0.01 and 0.0001ng of template DNA, respectively. The results of the amplification, lanes 9 to 12 show the results of amplifying the cagA gene with the amount of template DNA 100, 1, 0.01, and 0.0001 ng, respectively.).

Figure 2 shows the cea gene as a template using 9 types of cancer cell lines distributed from the National Institutes of Health, DNA isolated from serum of hepatitis B patients, and cDNA synthesized from total RNA isolated from tissues of gastric cancer patients obtained at the clinical site. The result of amplification using Steed RT-PCR (in FIG. 2, lane M is size marker, lanes 1 to 9 are colorectal cancer cell line ACT116, prostate cancer cell line PC3, liver cancer cell line SK-Hep1, and liver cancer Korean cell line SNU449 , Breast cancer cell line MCF7, gastric cancer cell line AGS, uterine cancer cell line Hela, lung cancer cell line A 549, and Korean gastric cancer cell line SNU628. Results for gastric tissue obtained).

Figure 3 shows the afp gene as a template of the nine cancer cell lines distributed from the National Institutes of Health, DNA isolated from the serum of hepatitis B patients, and cDNA synthesized from total RNA isolated from tissues of gastric cancer patients obtained at the clinical site. The result of amplification using Steed RT-PCR (in FIG. 3, lane M is size marker, lanes 1 to 9 are colorectal cancer cell line ACT116, prostate cancer cell line PC3, liver cancer cell line SK-Hep1, liver cancer Korean cell line SNU449 , Breast cancer cell line MCF7, gastric cancer cell line AGS, uterine cancer cell line Hela, lung cancer cell line A 549, Korean gastric cancer cell line SNU628, lanes 10 and 11 show the results for DNA isolated from hepatitis B patients, lanes 12 to 15 Results for gastric tissue obtained).

Figure 4 is a view showing the results of the nested RT-PCR for the plasmid DNA containing the HCV gene (Fig. 4A PCR using the group A primer for HCV 5'-UTR amplification of SEQ ID NO: 21 to 24) The results of the amplification are shown in lanes M to 100, 0.01, 0.0001 and 0.000001 for the gene markers, and lanes 1 to 4, respectively, as template DNA; FIG. 4B shows HCV 5 ′ of SEQ ID NOs: 25 to 28; PCR amplification was performed using the B-group primer for UTR amplification. Lane M shows the results of using the gene markers and lanes 1 to 4 using the amounts of template DNA as 100, 0.01, 0.0001, and 0.000001, respectively. ).

Figure 5 shows the results of performing nested RT-PCR on serum from patients infected with HCV (in Figure 5, lane M is the size marker, lanes 1 to 4 are group A primer for HCV 5'-UTR amplification Lanes 5 to 8 show amplification results using HCV 5′-UTR amplification group B primers. Lanes 1, 3, 5 and 7 show the results of performing only the first PCR. 2, 4, 6, and 8 show the results of amplifying the 5'-UTR of HCV by performing the second PCR.).

In order to achieve the above object, the present invention is characterized by performing nested RT-PCR using the primers described in Table 1 below. Here, '1st' means a primer used for the primary PCR in performing the nested RT-PCR, '2nd' is used for secondary PCR using the amplification product obtained by performing the primary RT-PCR as a template On the other hand, the primer for secondary PCR is bound to the inner sequence region of the primary PCR product.

Target genes PCR step Primer Name order SEQ ID NO: Amplification 16s rRNA 1st 16-1D 5'-ctggaacatt actgacgctg-3 ' SEQ ID NO: 1 320 bp 16-1U 5'-ctagcgattc cagcttcatg-3 ' SEQ ID NO: 2 2nd 16-2D 5'-cttgcagtaa tgcagctaac gc-3 ' SEQ ID NO: 3 16-2U 5'-cgaaggcagt ctccttagag-3 ' SEQ ID NO: 4 cagA 1st cag1D 5'-ctctgatgat aaagaaaaag c-3 ' SEQ ID NO: 5 170 bp cag1U 5'-caggttctat gctgctatgt a-3 ' SEQ ID NO: 6 2nd cag2D 5'-caatcttccg atctcaaaga aac-3 ' SEQ ID NO: 7 cag2U 5'-catccttcat ttccatatca cc-3 ' SEQ ID NO: 8 vacA 1st vac1D 5'-gatacgggta atggtggttt c-3 ' SEQ ID NO: 9 180 bp vac1U 5'-ggctataatc catgactgca ttg-3 ' SEQ ID NO: 10 2nd vac2D 5'-caatcaatcg cgcatcaaca c-3 ' SEQ ID NO: 11 vac2U 5'-gcaagtttgt tggtgatttc-3 ' SEQ ID NO: 12 cea 1st cea 1D 5'-c cagA atgac acaggattct ac-3 ' SEQ ID NO: 13 230 bp cea 1U 5'-gactgtgatc gtcgtgactg-3 ' SEQ ID NO: 14 2nd cea 2D 5'-gacacagcaa gctacaaatg-3 ' SEQ ID NO: 15 cea 2U 5'-gagctcttgg gtggattgct gg-3 ' SEQ ID NO: 16 afp 1st afp 1D 5'-gctacctgcc tttctggaag-3 ' SEQ ID NO: 17 210 bp afp 1U 5'-gcacatctcc tctgcaacag-3 ' SEQ ID NO: 18 2nd afp 2D 5'-gacattcatg aacaaattc-3 ' SEQ ID NO: 18 afp 2U 5'-cacatgcatg ttgatttaac-3 ' SEQ ID NO: 20 5'UTR A of HCV 1st hcvA1D 5'-ccctgtgagg aactactg-3 ' SEQ ID NO: 21 140 bp hcvA1U 5'-tgatgcacgg tctacgagac-3 ' SEQ ID NO: 22 2nd hcvA2D 5'-cgttagtatg agtgttgtgc ag-3 ' SEQ ID NO: 23 hcvA2U 5'-gcccaaatct ccaggcattg a-3 ' SEQ ID NO: 24 HCV 5'UTR B 1st hcvB1D 5'-gacactccac catagatc-3 ' SEQ ID NO: 25 270 bp hcvB1U 5'-caacaggtaa actccaccaa-3 ' SEQ ID NO: 26 2nd hcvB2D 5'-catagtggtc tgcggaacc-3 ' SEQ ID NO: 27 hcvB2U 5'-gggaacttaa cgtcctgtg-3 ' SEQ ID NO: 28 Control group (GAPDH) gapdhAD 5'-cgtcttcacc accatggaga ag-3 ' SEQ ID NO: 29 260 bp gapdhAU 5'-gtcttctggg tggcagtgat g-3 ' SEQ ID NO: 30 gapdhBD 5'-ctcatgacca cagtccatgc-3 ' SEQ ID NO: 31 340 bp gapdhBU 5'-cgctgttgaa gt cagA ggag-3 ' SEQ ID NO: 32

Since the primer sequences described in Table 1 specifically bind only to the complementary sequences of the corresponding target genes at the same annealing temperature of 50 to 53 ° C, only a corresponding gene region can be specifically amplified in a PCR reaction. In other words, the primers described in Table 1 were prepared in consideration of the GC content ratio, length, and expected PCR amplification product size in each oligonucleotide sequence, and specific for each gene of interest under the same nested RT-PCR condition. It is designed to be amplified by.

These primers were prepared from conventionally known sequences, and the 16s rRNA, vacA, and cagA genes of the Helicobacter pylori strains used the sequences of AE000547, AE000511, and AF030402 of the International Gene Bank (Am. J. Gastroenterol. 92 (8). ), 1316-1321 (1997); J. Clin. Microbiol. 38 (7), 2740-2742 (2000)). In addition, for the cea and afp genes, the primers were prepared using the known documents of the conventional literatures and the International Bank of Japan's Accession Nos. AC008999 and NM_001134 (Mol. Cell. Biol. 7 (9), 3221-3230 ( 1987); Genomics 3 (1), 59-66 (1988); Gene 20 (3), 415-422 (1982). In addition, the primer sequence for amplifying the 5'-UTR region of HCV was prepared based on the sequence of the pCV-H77C vector prepared by inserting the HCV gene.

The method for detecting each gene by performing nested RT-PCR using the primers,

1) extracting total RNA from cancer tissue or serum of the patient and synthesizing cDNA therefrom or obtaining genomic DNA from cancer tissue or serum of the patient;

2) cDNA or genomic DNA obtained in step 1) as a template, and 5 minutes of heat denaturation at 94 ° C using a primer for primary PCR in SEQ ID NOS: 1 to 28; 25-30 cycles of 1 minute thermal denaturation at 94 ° C, 1 minute annealing at 50-53 ° C, and 1 minute polymerization at 72 ° C; Performing a primary PCR at 72 ° C. under a PCR reaction condition of 7 minutes;

3) Using the PCR product obtained in step 2) as a template, and performing a second PCR under the same conditions as in the step 2) using a secondary PCR primer among the sequences described in SEQ ID NOs: 1 to 28 step; And,

4) by electrophoresis of the secondary PCR product obtained in step 3), characterized in that made by a method comprising the step of confirming whether the PCR product is generated.

On the other hand, the present invention provides a kit comprising a primer of SEQ ID NO: 1 to 28 as the primer sequence for nested RT-PCR described in Table 1. In addition, the kit according to the present invention may be, for example, a kit for separating mRNA from a sample or a part of a component thereof, a kit for synthesizing cDNA from an mRNA or a part of a component thereof, a buffer for performing a PCR reaction. After performing dNTP, Taq polymerase, and nested RT-PCR, DNA size markers for measuring the size of PCR amplification products or a user's manual containing an optimal reaction performance condition may be further included. Herein, DNA size markers can use nested RT-PCR products containing products previously amplified using primers according to the present invention, and these size markers are the same gels as the results of the nested RT-PCR. By electrophoresis at, the nested RT-PCR results can be analyzed more accurately.

In addition, the kit according to the present invention does not necessarily include all of the primers described in SEQ ID NO: 1 to SEQ ID NO: 28, 16s rRNA, cagA and vacA genes of Helicobacter pylori strains; cea and afp genes; And a kit containing appropriate primers for simultaneously amplifying and detecting one or more genes selected from the group consisting of the 5'-UTR region of HCV virus, and appropriately performing nested RT-PCR. The control primer for judging whether or not may further comprise the GAPDH primers described in SEQ ID NOs: 29 to 32.

Hereinafter, the present invention will be described in more detail.

The kit containing the primer according to the present invention can be used as a kit for detecting Helicobacter , an optional kit for detecting cancer, and a kit for detecting HCV virus.

I. Helicobacter Detection kit

Kit according to the present invention is to amplify the primers of SEQ ID NO: 1 to 4 to amplify 16s rRNA of Helicobacter strain, primers of SEQ ID NO: 5 to 8 to amplify the vacA gene of the same strain, cagA gene of the same strain To primers SEQ ID NOs: 9-12. Such primers specifically amplify each gene of interest to enable identification and detection of Helicobacter pylori strains, and the identification and detection of Helicobacter strains may affect the development or development of various gastric diseases such as gastric cancer, gastritis and gastric ulcers in patients. May suggest.

Helicobacter is a 2-7 μm long spiral bacterium that is parasitic on the gastrointestinal mucosa and is treated with special staining to be observed under a microscope. In addition, Helicobacter has three or four flagella, 20 times stronger than E. coli, and has the property of strongly penetrating the gastric mucus layer.

Due to the characteristics of Helicobacter alone, Helicobacter bacteria can cause acute gastritis, chronic active gastritis, liver, chronic atrophic gastritis, non-ulcer dyspepsia, gastric ulcer, duodenal ulcer, gastric adenocarcinoma, lymphoma, and duodenal ulcer. have. Clinical studies show that Helicobacter spp. Is found in 90-95% of duodenal ulcers, 60-80% of gastric ulcers, and 92-100% of lymphomas in the stomach, and eradication of Helicobacter significantly reduces recurrence rates. Have been reported. Helicobacter infection also has a four to six-fold increase in the risk of developing stomach cancer decades later than non-infected people. Therefore, due to the carcinogenicity of Helicobacter , in 1994, the International Cancer Research Organization (IARC) under the World Health Organization officially recognized Helicobacter as 'Group I carcinogenic factor' (benzine and asbestos). did.

On the other hand, the path of Helicobacter infection is not yet known, but there are reports that the infected person ingested food, water or foods in the stool, or someone else's mouth ingested, or flies coming and going from the food and stool. . There are also reports of direct infections from mouth to mouth. Koreans ought, more than half of infections and Helicobacter pylori (Helicobacteria), with age as a according to different regions and professions adults 50-60%, 1.1% and less than 5 years old, 5-9 years 12.8% and 20.4% aged 10-14 infected There is a report that the rate increases rapidly.

Infection with Helicobacter spp. Can result in various upper gastrointestinal lesions, depending on the variety of strains and susceptibility of the infected people, and the majority of infected people continue to have asymptomatic infections and develop symptoms.

Therefore, early diagnosis of Helicobacter carriers is important in the prevention and treatment of various gastric diseases, and for this purpose, a small amount of the gene product produced by Helicobacter in the early stage of infection should be detected quickly and reproducibly.

Meanwhile, the kit according to the present invention can be used in a nested RT-PCR using the primer sequences of SEQ ID NOs. 1 to 12 to be used in a method that can more efficiently and rapidly diagnose Helicobacter infection and bacteriostatic status in a patient. Can be.

II. As tumor markers of liver cancer, lung cancer, breast cancer, stomach cancer, gastritis and hepatitis cea And afp Gene Detection Kit

Detection kit according to the present invention is to detect the cea and afp genes used as markers of various cancers, such as liver cancer, lung cancer, breast cancer, gastric cancer, prostate cancer, and the like through SEQ ID NOS: 13-16 and It may include primers for nested RT-PCR for detection of the corresponding gene of SEQ ID NO: 17 to 20.

Tumor marker refers to a gene capable of diagnosing the presence of cancer cells and the progression of cancer cells by quantitatively and qualitatively analyzing gene expression. These tumor markers are not only genes but also substances produced by cancer cells or substances that normal cells in the body react with cancer cells, which are useful as indicators for diagnosing or treating cancer. Also included. Currently known tumor markers include many types of glycoproteins, hormones, enzymes, and blood coagulation-related substances, and about 30 of them are used in hospitals for clinical tests using blood and urine.

The kit according to the present invention detects fetal cancer antigens (Human carcinoembryonic antigen; cea ) and alpha-fetoprotein ( afp ) genes as tumor markers.

cea and afp are a type of fetal protein (alpha) that all fetuses have, and they are rapidly lost at birth and disappear as adults. However, tumor markers such as cea and afp are detected again when various cancers occur, and their numbers increase when the cancer momentum is active. In addition, when the cancer tissue becomes smaller or cured through treatment, the level of the tumor marker also decreases or disappears, and thus may be useful for detecting cancer tissue, diagnosing cancer progression, and diagnosing cancer recurrence.

However, although the kit according to the present invention can provide strong evidence of the possibility of having cancerous tissue in the body, it is impossible to distinguish and diagnose various cancers such as liver cancer, lung cancer, breast cancer, stomach cancer and prostate cancer. However, in the early diagnosis of cancer, it is economical and timely for the patient to determine the presence of cancer tissue by detecting genes commonly expressed in various cancers before performing each test separately for each cancer. It can reduce the burden on you.

In addition, since the kit according to the present invention can detect a very small amount of tumor markers expressed in very early cancers, it can lead to a positive diagnosis even in early cancer patients who can be judged negative by current test accuracy. Diagnostic sensitivity, which may suggest the possibility of cancer from patient to cancer.

On the other hand, the tumor markers cea and afp genes to be detected in the present invention are reported to be expressed in a very small amount, but also in patients such as hepatitis, cirrhosis. In other words, if alpha fetal protein is detected to be less than 10 ng of alpha fetal protein per 1 ml of blood in healthy adults, it is judged to be negative, and it is reported that it rises to about 500 ng in hepatitis and cirrhosis. However, alpha-fetoprotein values of 10-500 ng alone cannot accurately diagnose the presence of liver cancer. However, in most all cancers, cea and afp genes are expressed, and cirrhosis, infection, or gastritis are likely to develop into cancer, so the kit according to the present invention detects very small amounts of cea and afp as tumor markers. Not only the diagnosis but also the prognosis of the possibility of developing cancer can be diagnosed.

III. HCV Hepatitis Virus Detection Kit

Kit according to the present invention, HCV virus causing the hepatitis C virus by amplifying the 5'-untranslated region (5'-UTR) of HCV virus using the primers for nested RT-PCR of SEQ ID NO: 21 to 28 Can be detected.

In Korea, most of the causes of hepatocellular carcinoma are known as hepatitis virus infection. There are types of hepatitis virus A, B, C, D, E and F, two of which are related to liver cancer, type B and C. In addition, 85% of liver cancer patients are infected with hepatitis B or C virus.

In addition, HCV serotypes detected among patients infected with hepatitis C virus have been reported to have 6 serotypes of 1-6 so far, and each serotype is classified into subgroups of a and b. Among Koreans infected with HCV, most of them are serotypes 2a and 1b.

Hepatitis virus infects normal hepatocytes, causing mutations in normal hepatocyte genes, thereby turning normal hepatocytes into cancerous tissues, causing liver cancer. Therefore, a person infected with hepatitis virus is treated as a 'high risk group of liver cancer' that is susceptible to liver cancer.

Infection with hepatitis virus causes most cases of hepatitis. Symptoms include anorexia, general malaise, bloating, constipation and diarrhea. , Cold sweats, dyspnea, tachycardia, etc.). However, hepatocarcinoma has few characteristic symptoms, and the same symptoms as hepatic disorder caused by hepatitis and cirrhosis occur.

Therefore, liver cancer is often misdiagnosed as hepatitis and cirrhosis. Symptoms specific to liver cancer include hepatic swellings that occur only when liver cancer ruptures and bleeding into the abdomen (abdominal cavity), or rupture of liver cancer such as sudden abdominal pain or anemia. As there is no treatment, there is a problem in diagnosing liver cancer based only on symptoms.

The HCV virus, on the other hand, is a single chain (+) RNA virus of about 9.4 kb, in which protein precursors are formed from one long ORF, from which the unique HCV protein is produced by the action of proteases of cells and viruses. Is generated. On the other hand, the 5'-UTR is composed of up to 344 bp base, the sequence is well conserved between HCV virus strains.

Therefore, the kit according to the present invention amplifies the 5'-UTR of HCV virus with nested RT-PCR, so that a very small amount of genes can be detected at the beginning of HCV virus hepatitis, thereby prematurely developing the onset or possibility of developing hepatitis. You can diagnose it. In addition, such diagnosis can be detected by classifying serotypes of HCV 2a and 1b which are frequently detected in Koreans. That is, the primers represented by the group A in Table 1 can detect the 2a serotype of HCV, and the primers represented by the group B can specifically detect the 1b serotype of HCV.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto, and modifications that can be commonly recognized and performed by those skilled in the art are included in the scope of the present invention.

Reference Example 1 Blood of a Patient Helicobacter Preparation of Genomic DNA from Bacteria

To extract genomic DNA from the patient's blood, a wizard prep kit (promega) was used and a WPA S2000 Lightwave UV / VIS diode spectrophotometer was used. The isolated DNA was quantified, of which 10 ng DNA was used for subsequent nested RT-PCR reactions.

Detailed methods for extracting genomic DNA from patient blood are as follows.

First, 900 μl of cell lysis buffer (Tris-HCl, pH7.5, 1 mM EDTA, 10 ug / ml RNAse) was added to the microcentrifuge tube, 200 μl of patient blood was added and mixed well. Lyse the cells. Then, it is left at room temperature for 10 minutes, and then centrifuged for 20 seconds at 13,000 ~ 15,000g. Only 10-20 μl of supernatant is obtained and vigorously vortexed to mix thoroughly. To this, 300 μl of nuclear lysis buffer (0.2N NaOH, 1% SDS) was added, followed by pipetting 5-6 times and incubating at 37 ° C. for 1 hour to dissolve the nuclei. However, if nuclei are not completely lysed and agglomerates remain, add 100 μl of cytolysis buffer and further incubate to completely lyse the cells. Then, 100 μl of protein precipitating solution was added to the cell lysate and vigorously vortexed, followed by centrifugation at 13,000 to 16,000 g for 3 minutes to generate a dark brown protein precipitate. The supernatant is then transferred to a new tube, 300 μl of isopropanol is added, followed by gentle stirring. Then, after standing at room temperature for 10 minutes, centrifuged again for 1 minute at 13,000 ~ 16,000g and then remove the supernatant. The precipitate is washed with 70% EtOH, then the supernatant is removed and dried in air for 15 minutes. Then, add 100 µl of tertiary distilled water and dissolve at 65 ° C for 1 hour.

On the other hand, genomic DNA was extracted by using the wizard prep kit (promega, USA) from Helicobacter pylori strains (clinical isolates isolated from ATCC 43526, 43504 and Korean gastric cancer patients) by the same method as described above. Then, after quantifying the isolated genomic DNA, a small amount of 10-50ng was used in subsequent experiments. In addition, after diluting the genomic DNA to 0.000001 ng to determine whether a small amount of Helicobacter attached to the gastric mucosa can be detected, detection was attempted using the nested RT-PCR method described below.

Reference Example 2 RNA Extraction and cDNA Synthesis

Total RNA derived from various cancer tissues, experimental cancer cell models and serum of hepatitis patients was isolated using RNA extraction kit (promega). 5 μg of RNA was obtained from the total RNA isolated, which was used for the synthesis of cDNA. First, the isolated total RNA was denatured by incubation at 70 ° C. for 10 minutes, and then rapidly cooled on ice. Then, 5 μl of random primer, 10 × PCR buffer, 10 mM dNTP (promega), AMV RTase 10 unit, RNasin 5 unit, and 44 μl of H ₂ O were added to the reaction tube. After the solution was prepared, the mixture was incubated at 42 ° C for 1 hour to synthesize cDNA, boiled at 95 ° C for 5 minutes to stop the activity of each enzyme, and then cooled at room temperature. The synthesized cDNA was then stored at -20 ° C for later experiments.

Reference Example 3 PCR Performance Conditions

In the first PCR reaction, 10-100ng / μl of cDNA synthesized from genomic DNA or total RNA prepared in Reference Example 1 or Reference Example 2, 5 μl of 10X buffer (promega), 10 mM dNTP, and each gene specific 1 50 pmol of primary primer, 5 units of Taq polymerase (promega), and 50 μl of final volume were added to distilled water to prepare a PCR reaction solution, and the PCR reaction solution was heat-modified at 94 ° C. for 5 minutes; 30 cycles of heat denaturation at 94 ° C. for 1 minute, annealing at 53 ° C. for 1 minute and polymerization at 72 ° C. for 1 minute; As a final polymerization reaction, the reaction was carried out at 72 ° C. for 7 minutes.

The second PCR reaction was the same as the first PCR reaction solution except that 5 µl of the PCR product obtained by the primary PCR reaction was obtained as the template DNA, and a specific secondary primer of each gene was used as the primer. After preparing a reaction solution having a composition, it was carried out under the same conditions as the first PCR reaction.

Reference Example 4 Control Genes

As a control gene for confirming that the cDNA synthesis and PCR reaction were properly performed from the total RNA, a GAPDH gene, which is a house keeping gene, was used, and a primer for performing PCR was SEQ ID NO. 29 to 32 gene primers were used.

Reference Example 5 Electrophoresis

5 μl of the PCR product amplified by performing the first and second PCR were subjected to electrophoresis for 30 minutes on 2% agarose gel (FMC Co.) containing ethidium bromide (EtBr), followed by ultraviolet irradiation. UV transilluminator was used to confirm the amplification of specific genes and photography.

On the other hand, as the DNA size marker for identifying the DNA size of the amplified PCR product was developed by the present inventors, the size of the DNA represented by each band is 100bp, 200bp, 400bp, 600bp, 800bp, 1kb and 1.5 kb.

Example 1 Helicobacter Pylori 16s rRNA of strain, vacA And cagA Simultaneous Detection of Genes

Genomic DNA was extracted from the sample containing the Helicobacter pylori strain by the method of Reference Example 1, and primary and secondary PCR reactions were performed by the method of Reference Example 3. Here, in amplifying the 16s rRNA, the primers used for the primary PCR were SEQ ID NOs: 1 and 2, and the secondary PCR primers SEQ ID NOs: 3 and 4; In amplifying the vacA gene, primers used for the first PCR were SEQ ID NOs: 5 and 6, and for the second PCR, primers of SEQ ID NOs: 7 and 8 were used; In amplifying the cagA gene, the primers used for the primary PCR were SEQ ID NOs: 9 and 10, and for the secondary PCR, the primers of SEQ ID NOs: 11 and 12 were used. For each gene, PCR reaction was performed by diluting the amount of template DNA to 100, 1, 0.01, and 0.0001 ng, and each reaction tube was the result of simultaneous PCR in the same PCR instrument.

After terminating the primary PCR, the electrophoresis of the generated PCR products by the method of Reference Example 5 is shown in FIG. 1A, and FIG. 1B for the PCR products after the completion of the secondary PCR. Shown in

As shown in Figures 1A and 1B, amplification of 16s rRNA, vacA and cagA genes using nested RT-PCR using primers according to the present invention, resulting in 320bp, 180bp and 170bp products as single bands, respectively It can be seen that the primers according to the present invention do not form a nonspecific band and specifically amplify only the corresponding gene. In addition, the nested RT-PCR method according to the present invention can be amplified accurately and reproducibly, even if a small amount of 16s rRNA, vacA and cagA genes, by performing two PCRs, many gastric diseases such as gastric cancer It is possible to accurately identify Helicobacter pylori related to. In addition, since the primers according to the present invention can specifically amplify each corresponding gene under the same PCR conditions, it is possible to simultaneously identify 16s rRNA, vacA and cagA genes through one PCR reaction, and thus any one gene The detection of Helicobacter pylori bacteria can be more accurately compared to the method of identification only.

Example 2 From Various Cancer Cells, Hepatitis B Patients, and Gastric Cancer Patients cea Gene detection

Nine cancer cell lines (colon cancer cell line ACT116, prostate cancer cell line PC3, liver cancer cell line SK-Hep1, liver cancer Korean cell line SNU 449, breast cancer cell line MCF7, gastric cancer cell line AGS, uterine cancer cell line Hela, lung cancer cell line A 549) DNA was isolated from the blood of the Korean gastric cancer cell line SNU628) and hepatitis B patients by the method of Reference Example 1 above, and 10 ng of each DNA was isolated as a template for the following PCR reaction. In addition, from the gastric cancer cells isolated from gastric cancer patients in the clinical field, total RNA was isolated by the method of Reference Example 2, and cDNA was synthesized from each total RNA. Then, 50 ng of each synthesized cDNA was used as a template DNA in the following PCR reaction.

As the primers, the nested RT-PCR was performed by using PCR primers of SEQ ID NOS: 13 to 16 under the same conditions as those of the reference examples 3 and 1 above. . The results are shown in FIG.

As shown in Figure 2, as a result of performing nested RT-PCR in all kinds of cancer cells, a 230bp size cea gene amplification product was confirmed, these results are cancer markers in which the cea gene is expressed in all kinds of cancer cells, the present invention Primer according to specifically amplify the cea gene. In addition, since the results of FIG. 2 are performed under the same PCR conditions as in Example 1 and Example 2, multiple PCR reaction solutions can be amplified at the same time under the same PCR instrument and PCR execution conditions. That is, when using the primers according to the present invention, the cea gene can be detected simultaneously with the 16s rRNA, vacA and cagA genes of the Helicobacter pylori strain.

Example 3 From Various Cancer Cells, Hepatitis B Patients and Gastric Cancer Patients afp Gene detection

Nine cancer cell lines (colon cancer cell line ACT116, prostate cancer cell line PC3, liver cancer cell line SK-Hep1, liver cancer Korean cell line SNU 449, breast cancer cell line MCF7, gastric cancer cell line AGS, uterine cancer cell line Hela, lung cancer cell line A 549) DNA was isolated from the blood of the Korean gastric cancer cell line SNU628) and hepatitis B patients by the method of Reference Example 1 above, and 10 ng of each DNA was isolated as a template for the following PCR reaction. In addition, from the gastric cancer cells isolated from gastric cancer patients in the clinical field, total RNA was isolated by the method of Reference Example 2, and cDNA was synthesized from each total RNA. Then, 50 ng of each synthesized cDNA was used as a template DNA in the following PCR reaction.

Then, each synthesized cDNA 50ng as a template, using the primers for nested RT-PCR of SEQ ID NOS: 17 to 20 as primers, under the same conditions as the PCR conditions of Reference Examples 3 and 1 above. PCR was performed to perform nested RT-PCR. The results are shown in FIG.

As shown in Figure 3, as a result of performing the nested RT-PCR in all kinds of cancer cells, a 230bp size cea gene amplification product was confirmed, these results are a cancer marker that the cea gene is expressed in all kinds of cancer cells, the present invention Primer according to show that specifically amplify the afp gene.

[Example 4] 5'-UTR amplification and sensitivity

A plasmid pCV-H77C (US) containing the HCV gene as a template, a primer for nested RT-PCR of SEQ ID NOS: 21 to 24, and a primer of SEQ ID NOs: 25 to 28 for amplifying 5'-UTR of HCV Using, the PCR reaction was carried out according to the PCR conditions of Reference Example 3. At this time, in order to measure the sensitivity of the template DNA, PCR reaction solutions were prepared in amounts of 100, 1, 0.01, 0.0001 and 0.000001 ng.

On the other hand, gene amplification in the present embodiment was performed by a method similar to the multiplex PCR technique that performs a single PCR for a reaction solution containing both pairs of PCR primers.

The results obtained using the sequences of SEQ ID NOs: 21 to 24 as primers (Group A primers) are shown in FIG. 4A, and the results obtained using the sequences of SEQ ID NOs: 25 to 28 (Group B primers) as primers are shown in FIG. 4B. It was.

As shown in Figures 4A and 4B, each primer specifically amplified each corresponding HCV 5'-UTR region to form two bands, and a very small amount of 0.000001 ng of DNA could be amplified and detected. Confirmed. That is, it was confirmed that the primers according to the present invention can simultaneously detect HCV serotypes of 2a and 1b under the same PCR conditions.

Meanwhile, in the present invention, the multiplex PCR is performed instead of the nested RT-PCR, in order to confirm whether each of the primary and secondary primers amplify the corresponding region correctly under the same annealing conditions.

Example 5 5'-UTR Detection from Serum of HCV Patient

Total RNA was isolated from the sera of hepatitis C patients by the method of Reference Example 2 above, and cDNA was synthesized from the isolated RNA. Synthesized cDNA as a template, and the reaction was carried out using primers for nested RT-PCR of SEQ ID NOS: 21 to 24 and primers of SEQ ID NOs: 25 to 28 as primers, and PCR reaction conditions were described in Reference Example 3 above. It was carried out by the same method as. The results are shown in FIG.

As shown in FIG. 5, when PCR was performed using the primers of SEQ ID NOs: 21 to 24, no amplification products were shown, but when PCR was performed using SEQ ID NOs: 25 to 28, 5 'at a position of 270 bp size -UTR was amplified and detected. In addition, although only 1st PCR was performed to detect HCV infection, amplification products were reproducibly detected in nested RT-PCR results up to 2nd PCR. Therefore, from the results of this example, it can be diagnosed that the HCV infected patient is a patient with the HCV 1b serotype.

As described above, the kits according to the present invention and the primers for nested RT-PCR, both can specifically amplify the respective genes under the same PCR conditions, to diagnose various gastric cancer, gastritis, various cancers or hepatitis In this case, since only one PCR is performed under the same conditions, the presence or absence of various cancers and hepatitis, gastritis, and the like can be diagnosed at the same time, so that a lot of time and money can be saved in a patient who is trying to diagnose cancer.

In addition, since the primer according to the present invention can be detected by using nested RT-PCR for DNA of 0.000001 ng or less, the possibility of occurrence of various cancers can be diagnosed even in patients at the very beginning of the development of various cancers.

Claims (7)

16s rRNA, vacA and cagA genes of Helicobacter pylori strains, cea and afp genes as cancer markers, and 5'-UTR of HCV virus were used for simultaneous detection by the same nested reverse transcription-polymerase chain reaction A primer for nested RT-PCR according to SEQ ID NOs: 1 to 28, characterized by the above. The primer for nested RT-PCR according to claim 1, wherein the method having the same nested RT-PCR condition includes annealing conditions at 50 to 53 ° C. The method of claim 1, wherein the method has the same nested RT-PCR condition, 1) extracting total RNA from cancer tissue or serum of the patient to obtain synthesized cDNA, or obtaining genomic DNA from cancer tissue or serum of the patient; 2) cDNA or genomic DNA obtained in step 1) as a template, and 5 minutes of heat denaturation at 94 ° C using a primer for primary PCR in SEQ ID NOS: 1 to 28; 25-30 cycles of 1 minute thermal denaturation at 94 ° C, 1 minute annealing at 50-53 ° C, and 1 minute polymerization at 72 ° C; Performing a primary PCR at 72 ° C. under a PCR reaction condition of 7 minutes; 3) Using the PCR product obtained in step 2) as a template, and performing a second PCR under the same conditions as in the step 2) using a secondary PCR primer among the sequences described in SEQ ID NOs: 1 to 28 step; And, 4) Primed nested RT-PCR characterized in that the conditions comprising the step of electrophoresis the secondary PCR product obtained in step 3) to determine whether the PCR product generation. A kit for the simultaneous diagnosis of cancer, gastritis and hepatitis, comprising the primer according to claim 1. [Claim 5] The kit for simultaneously diagnosing cancer, gastritis, hepatitis and cirrhosis of claim 4, further comprising a DNA size marker in which the amplification products obtained by amplifying the respective genes with the primers of claim 1 are mixed. [Claim 6] The kit for simultaneously diagnosing cancer, gastritis, hepatitis and cirrhosis of claim 4 or 5, further comprising GAPDH primers for amplifying control genes. The cancer, gastritis, hepatitis and liver cirrhosis simultaneous diagnosis kit, characterized in that the cancer is selected from the group consisting of colon cancer, prostate cancer, liver cancer, breast cancer, stomach cancer, uterine cancer and lung cancer.