KR100647474B1 - Method and test kit for detecting pathogenic rna virus - Google Patents

Abstract

The present invention performs three reverse transcriptase polymerase chain reaction (multiplex-RT PCR) to cause infectious diarrheal diseases in Korea, three representative RNA viruses, astrovirus (astrovirus), rotavirus (rotavirus), norovirus (norovirus genogroup) The present invention relates to a pathogenic RNA detection method for quickly and accurately detecting I and II) and a detection kit.

RNA virus, astrovirus, rotavirus, norovirus genogroup I, II, multiplex-RT PCR, detection method, detection kit

Description

METHOD AND TEST KIT FOR DETECTING PATHOGENIC RNA VIRUS

Figure 1 shows the result of electrophoresis of the amplified product after the multi-reverse transcription polymerase chain reaction of the sample mixed with three viruses.

The present invention relates to a method for detecting a pathogenic RNA virus, and a detection kit. More specifically, the present invention relates to three representative RNA viruses that cause infectious diarrheal diseases in Korea by performing multiplex reverse transcription polymerase chain reaction (multiplex-RT PCR). The present invention relates to a pathogenic RNA detection method for quickly and accurately detecting an astrovirus, a rotavirus, and a norovirus genogroup I, II, and a detection kit.

Despite the development of science and technology, it is still difficult to completely prevent food poisoning. Food poisoning includes food poisoning due to natural poisoning (blowfish poison, mushroom poisoning, etc.), food poisoning due to food decay based on the cause; And food poisoning by incorporation or reproduction of microorganisms in food. Among these food poisonings, food poisoning caused by natural poisoning can be easily prevented by avoiding the ingestion of the foods that cause the food poisoning. Food corruption is easily detected by changes in the appearance, smell, etc. of food poisonings. Relatively easy. However, in the case of food poisoning by microorganisms, it is the most difficult to prevent food poisoning because the incorporation or reproduction of microorganisms into foods is generally not observed with the naked eye and it is very difficult to detect them by professional methods.

Each year, the number of bacterial and viral food-borne gastrointestinal tract infections and food poisoning patients continues to increase, and despite the high socioeconomic costs, there is a lack of clear epidemiological studies of causative viruses or causative foods. I am having difficulty managing the disease. In particular, in the case of infectious diseases that occur collectively, promptness and accuracy are required for diagnosis and treatment to prevent the spread and damage of diseases.

While causative analysis and cultivation of bacterial food poisoning are relatively easy, viral food poisoning is very small and difficult to observe under microscopy, and culture is so difficult that rapid diagnosis cannot be performed. I can't catch it. In 2001 and 2002, the main causes of neonatal death and mass diarrhea in the postpartum care center in Ilsan and the obstetrics and gynecology department in Jeonnam were infections and enteritis caused by Astrovirus and Rotavirus. There is an increasing number of reports of infection with Norovirus. In particular, if two or more viruses are mixed (17 cases in 2000, reported by the National Institutes of Infections in March 2001), accurate confirmation is required. Therefore, rapid and specific development is possible through the development of genetic diagnosis based on molecular biology. Development of diagnostic systems should be established.

Recently, a test method for detecting such pathogenic viruses using polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (Reverse Transcription-PCR, RT-PCR) has been developed and used.

RT-PCR (Reverse Transcription-PCR) is a technique of synthesizing a corresponding cDNA (complementary DNA) by using RNA of a specific site as a template and then performing PCR amplification using it. transcriptase) is used to prepare cDNA from RNA and 2) amplify a specific region using cDNA. 2) process is the same as amplifying a specific gene region from genomic DNA.

Prior art related to enterovirus detection using reverse transcription polymerase chain reaction (RT-PCR) is a method of hybridizing with DNAchip after reverse transcription polymerase chain reaction, but there are few kinds of viruses that can be detected. In addition, there is a disadvantage of using DNAchip again after preceding RT-PCR, and there is a disadvantage of high cost. In addition, there is a rapid strip method as a method for detecting Rotavirus, Adenovirus, Astrovirus, etc., but this protein-targeted method cannot detect various mutations of the virus. It is less sensitive than PCR. On the other hand, the conventional detection method using PCR is a system for detecting each virus single species, there is a problem that the detection experiment for each virus is required for the detection of multiple viruses.

In order to solve the problems of the conventional pathogenic virus detection method described above, an object of the present invention can be identified by a single test of three viruses, rotavirus, astrovirus, norovirus, the conventional 4 to 7 days It is to provide a rapid and accurate pathogenic RNA virus detection method capable of shortening the diagnostic time to 4 to 5 hours.

Another object of the present invention is to provide a pathogenic RNA virus detection kit capable of detecting pathogenic RNA viruses quickly and accurately in a short time.

In order to achieve the above object, the present invention in the pathogenic RNA virus detection method using reverse transcriptase polymerase chain reaction (RT-PCR), Rotavirus specific reverse transcription primer, Astrovirus specific reverse transcription Provided is a method for detecting a pathogenic RNA virus, characterized in that the reverse transcription reaction is carried out using a primer and a Norovirus genogroup I, II specific reverse transcription primer.

In addition, the present invention uses a reverse transcription polymerase chain reaction (RT-PCR) to detect pathogenic RNA viruses, including a reverse transcription primer, primer for PCR, reaction buffer, reverse transcriptase, reverse transcriptase buffer, Taq DNA polymerase including pathogenic In the microbial detection kit, the reverse transcription primer includes a Rotavirus specific reverse transcription primer, an Astrovirus specific reverse transcription primer, and a Norovirus genogroup I and II specific reverse transcription primer. It provides a pathogenic RNA virus detection kit, characterized in that.

Hereinafter, the present invention will be described in detail.

The present inventors have studied the detection method and detection kit that can quickly and easily diagnose multiple pathogenic RNA viruses simultaneously using multiple reverse transcriptase polymerase chain reaction (multiplex RT-PCR). Synthesis of cDNA using specific reverse transfer primers simultaneously, and polymerase chain reaction using specific PCR primers for each virus using synthesized cDNA as a template to perform rotavirus, astrovirus, norovirus It was confirmed that the infection can be quickly detected without cross-reaction to three viruses of the present invention was completed based on this.

The detection method according to the present invention is a multiplex reverse transcriptase polymerase chain reaction (multiplex) for the infection of three viruses of the pathogenic RNA viruses Rotavirus, Astrovirus and Norovirus genogroup I, II. Simultaneous diagnosis with RT-PCR).

Rotavirus is a dsDNA virus, an important pathogen that causes acute diarrheal disease in up to 40% of infants worldwide. Rotavirus belongs to the family of Reoviridae, and the genome of the rotavirus consists of 11 dsRNA fragments and is surrounded by three-layer proteins of the nuclear, inner and outer layers. The inner layer is composed of Viral Protein 6 (VP6), which exhibits group specific antigenicity. The outer layer consists of VP4 and VP7 and is the main antigen that induces the production of virus neutralizing antibodies.

The astrovirus according to the present invention is a virus belonging to the newly established astroviridae, which is a virus that causes diarrhea, and is composed of a single layer capsid which does not form an envelope. In addition, the gene of the astrovirus is a single-stranded, positive-sense RNA of about 6.8 to 7.3kb. Astroviruses are named as Astroviruses because they form five to six stars on an electron microscope.

Norovirus genogroup I and II related to the present invention is a representative cause virus of infectious diarrheal disease, and has been derived from the name of the region since the onset of disease in Norwalk, Ohio, USA. Noroviruses of high diversity and variation can be classified as genogroups I and II.

Looking at the detection method according to the invention in detail as follows.

First, RNA is extracted from fecal specimens of people suspected of being contaminated with pathogenic RNA viruses.

Reverse transcription was performed using a Rotavirus specific reverse transcription primer, an Astrovirus specific reverse transcription primer, and a Norovirus genogroup I, II specific reverse transcription primer. A first strand cDNA is synthesized from the extracted RNA. The detection method according to the present invention performs a reverse transcription reaction using a specific reverse transcription primer for three kinds of viruses, and performs multiplex PCR (multiplex RT-PCR). Simultaneous diagnosis is possible. Therefore, there is a more convenient advantage compared to the conventional method of testing only a single species virus using one specific primer.

As a specific reverse transcription primer for performing the reverse transcription reaction, a rotavirus specific reverse transcription primer having a nucleotide sequence of SEQ ID NO: 1, an astrovirus specific reverse transcription primer having a nucleotide sequence of SEQ ID NO: 2, and a sequence It is preferable to use a norovirus specific reverse transcription primer having the nucleotide sequence of No. 3 simultaneously.

A cDNA was synthesized by performing a reverse transcription reaction using the specific reverse transcription primer together, using a synthesized cDNA as a template, a pair of primers for rotavirus-specific PCR, a pair of primers for astrovirus-specific PCR, and norovirus specific. Multiplex PCR is performed using primer pairs for PCR simultaneously. The detection method according to the present invention enables simultaneous diagnosis of three viruses in one experiment by carrying out a polymerase chain reaction (multiplex RT-PCR) using primer pairs specific for PCR for three kinds of viruses together. . Therefore, there is a more convenient advantage compared to the conventional method of testing only a single species virus using one specific primer.

As a specific PCR primer pair for performing the multiple polymerase chain reaction, a primer pair for rotavirus specific PCR having a nucleotide sequence described in SEQ ID NO: 1 and SEQ ID NO: 4; Primer pairs for an astrovirus specific PCR having the nucleotide sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 5; And primer pairs for norovirus-specific PCR having the nucleotide sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 together.

The primer pairs having the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 4 were designed to specifically detect Rotavirus, and the size of the amplification product was 877 bp.

The primer pairs having the nucleotide sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 5 were designed to specifically detect Astrovirus, and the size of the amplification product was 289 bp.

The primer pairs having the nucleotide sequences set forth in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 were designed to specifically detect noroviruses (Norovirus genogroup I, II), and amplified The size of the product is 123bp.

After the polymerase chain reaction, the amplified gene product is electrophoresed on agarose gel to determine whether the sample is infected according to the presence or absence of specific gene amplification.

According to the detection method of the present invention, three pathogenic viruses can be detected quickly and accurately in one test without non-specific amplification or interference.

In addition, the pathogenic RNA detection kit according to the present invention is a reverse transcription primer, PCR primer, reaction buffer, reverse transcriptase, reverse transcriptase buffer, Taq DNA to detect pathogenic RNA virus using reverse transcription polymerase chain reaction (RT-PCR) In a pathogenic microorganism detection kit including a polymerase, as a reverse transcription primer, Rotavirus specific reverse transcription primer, Astrovirus specific reverse transcription primer, and Norovirus genogroup I, II specificity Includes reverse transcription primers. The detection kit includes the specific reverse transcription primer and general components of the microbial detection kit using RT-PCR (reaction buffer, reverse transcriptase buffer, etc.).

The reverse transcription primer has a rotavirus specific reverse transcription primer having a nucleotide sequence of SEQ ID NO: 1, an astrovirus specific reverse transcription primer having a nucleotide sequence of SEQ ID NO: 2, and a base sequence of SEQ ID NO: 3 It is preferred to be a norovirus specific reverse transcription primer.

The primer for PCR of the detection kit according to the present invention preferably comprises a primer pair for rotavirus-specific PCR, a primer pair for astrovirus-specific PCR, and a primer pair for norovirus-specific PCR. Primer pairs for rotavirus specific PCR having the nucleotide sequence set forth in SEQ ID NO: 4; Primer pairs for an astrovirus specific PCR having the nucleotide sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 5; And a primer pair for norovirus specific PCR having the nucleotide sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

The detection kit of the present invention has an equivalent effect in terms of sensitivity and specificity compared to the conventional detection kit using a single primer, and can quickly detect three viruses in one kit.

 Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

(Example)

(Example 1)

(Design and Synthesis of Three Pathogenic RNA Virus Specific Primers)

Primers were designed and synthesized as follows to detect three pathogenic RNA viruses of rotavirus, astrovirus, and norovirus simultaneously without interference.

Rotavirus

A primer described in SEQ ID NO: 1 was designed as a reverse transcription primer to identify the VP4 region of rotavirus, and primer pairs described in SEQ ID NO: 1 and SEQ ID NO: 4 were prepared as primers for PCR.

2. Astrovirus

In order to detect an astrovirus, a primer described in SEQ ID NO: 2 was designed as a reverse transcription primer, and a primer pair described in SEQ ID NOs: 2 and 5 was prepared as a primer for PCR.

3. Norovirus

Norovirus produced primers that can distinguish genogroup I, II. Specific group I has a large number of serotypes, resulting in a wide variety of sequences. Therefore, based on ORF1 RNA polymerase gene region (M87661) among the most basically reported Norwalk virus nucleotide sequences registered on the genebank, a primer was developed to detect the entire group I. In the case of group II, Primers were prepared based on the nucleotide sequence of Lordsdalevirus (X86557).

The primers described in SEQ ID NO: 3 were designed such that group I and II were the same as the reverse transcription primers for reverse transcription, and were prepared to distinguish group I and II in the PCR step. For the detection of group I, primers described in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 were designed, and in the case of group II, primers described in SEQ ID NO: 6 were designed.

virus name  Sequence SEQ ID NO: Rotavirus RoR 5 'TGG CTT CGC CAT TTT ATA GAC A 3' One RoF 5 'ATT TCG GAC CAT TTA TAA CC 3' 4 Astrovirus AsR 5 'ACA TGT GCT GCT GTT ACT ATG 3' 2 AsF 5 'CGT CAT TAT TTG TTG TCA TAC T 3' 5 Norovirus NrR 5 'TGT CAC GAT CTC ATC ATC ACC 3' 3 NrllF 5 'TGG AAT TCC ATC GCG CAC TGG 3' 6 NrlF1 5 'GTG AAC AGC ATA AAT CAC TGG 3' 7 NrlF2 5 'GTG AAC AGT ATA AAC CAC TGG 3' 8 NrlF3 5 'GTG AAC AGT ATA AAC CAT TGG 3' 9

(Example 2)

Detection of Pathogenic RNA Viruses Using Multiple Reverse Transcriptase Chain Reactions

   1. RNA extraction

First, 560 μl of Buffer AVL was added to a 1.5 ml tube. 140 μl of the mixed samples of three viruses (in case of solids, dissolved in PBS, v / v 10%) was added and vortexed for 15 seconds. After standing at room temperature (15-25 ° C.) for 10 minutes, spin-down was performed, 560 μl of ethanol (96-100%) was added, vortexed for 15 seconds, mixed well, and spin-down. The solution was transferred to a column of 630 µL, centrifuge 8,000 rpm for 1 minute, and the collection tube was replaced twice. 500 μl of wash Buffer was added and centrifuged for 1 minute at 8,000 rpm. 500 μl of Buffer AW2 was added, centrifuged at 12,000 rpm for 3 minutes, and the collection tube containing the eluate was removed. Transfer the column to a new 1.5 ml tube, add 60 μl of elution Buffer and leave for 1 minute. Elution was performed by centrifugation at 8,000 rpm for 1 minute.

2. Reverse Transcription

 2.5 μl of DMSO and 5 μl of RNA extracted from the sample were added to the PCR-tube, mixed well, and spin-down. Immediately after 5 min at 70 ° C., ice was denatured. At this time, the temperature was kept constant by using a PCR machine. 2 μl 10X RT buffer (FINZYME), 4 μl dNTP (10 mM), 2.5 μl multi-primer mixture (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3) 10 pmole, 0.2 μl M-MuLV reverse trascriptase (FINZYME), 11.8μl DEPC treated dw was mixed well to make 25μl reverse ranscription reaction mixture, and cDNA was synthesized for 1 hour at 42 ℃ using PE 9700 PCR machine.

   3. Multiplex PCR

Using 5.0 μl of cDNA as a template, 5.0 μl 5X Taq polymerase buffer, 0.3 μl of 10 mM dNTP mix, 4.0 μl of 5 pmole multi primer mixture (SEQ ID NOS: 1 to 9), 1.0 μl of Taq polymerase, and 10 μl of sterilization. PCR reaction solution containing the distillation was prepared. The reaction was repeated 40 times at 95 ° C. for 5 minutes, at 94 ° C. for 45 seconds, at 50 ° C. for 45 seconds, and at 72 ° C. for 1 minute 15 seconds, followed by 72 ° C. for 7 minutes to amplify the virus.

4. Electrophoresis

The PCR product obtained above was electrophoresed on 2% agarose gel to confirm amplification of the virus. Figure 1 shows the results of electrophoresis of the amplification product.

Lane M: 100bp ladder

Lane 1: voice control

Lanes 2 to 7: rotavirus

Lanes 8 to 11: astrovirus

Lanes 12 and 13: norovirus

Lane P: internal RNA control

(Example 3)

(Configuration of Pathogenic RNA Virus Detection Kit)

A multiplex primer mixture was prepared containing the nine pairs of primers (SEQ ID NOS: 1 to 9) of Example 1 to construct a detection kit as follows.

* Kit Components

Reverse transcriptase

Reverse transcription buffer

5 × reaction buffer

Multiplex Primer Mixture (SEQ ID NOS: 1-9)

Taq DNA Polymerase

100bp DNA ladder

DNA loading dye

As described above, according to the present invention, it is possible to quickly and accurately check whether three major viruses causing gastrointestinal infections in Korea are infected with rotavirus, astrovirus, and norovirus by a single test, such as enteritis or diarrhea disease. It can be effectively used for the diagnosis and treatment of diseases, it is possible to significantly reduce the time and expense by reducing the conventional diagnostic time of 4 to 7 days to 4 to 5 hours.

Attach sequence list electronic file  

Claims (8)

In the pathogenic RNA virus detection method using reverse transcription polymerase chain reaction (RT-PCR), Reverse transcription reaction using Rotavirus specific reverse transcription primer having the nucleotide sequence of SEQ ID NO: 1 and Norovirus genogroup I, II specific reverse transcription primer having the nucleotide sequence of SEQ ID NO: 3 Then, A primer pair for rotavirus specific PCR having the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4 after the reverse transcription reaction; And simultaneously performing a polymerase chain reaction using primer pairs for norovirus-specific PCR having the nucleotide sequences set forth in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 9 Method for detecting pathogenic RNA virus. delete delete delete In a pathogenic microorganism detection kit comprising a reverse transcription primer, a primer for PCR, a reaction buffer, a reverse transcriptase, a reverse transcriptase buffer, and a Taq DNA polymerase to detect a pathogenic RNA virus using reverse transcriptase chain reaction (RT-PCR). , The reverse transfer primer is a Rotavirus specific reverse transfer primer having a nucleotide sequence as shown in SEQ ID NO: 1, and a Norovirus genogroup I, II specific reverse transfer primer having a nucleotide sequence as shown in SEQ ID NO: 3; , The primer for PCR is a rotavirus-specific PCR primer pair having a nucleotide sequence described in SEQ ID NO: 1 and SEQ ID NO: 4; And a pair of primers for norovirus-specific PCR having the nucleotide sequences shown in SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 9. delete delete delete

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