KR102278402B1 - Primers and probes for Measles virus and Measles virus vaccine strain and detection method using them - Google Patents

Abstract

The present invention relates to a primer and a probe for detecting measles virus and vaccine strain, and a method for detecting measles virus using the same. Specifically, the primer and probe consisting of a specific nucleotide sequence targeting the nucleoprotein gene (N gene) of the present invention can distinguish between measles virus and vaccine strain. In addition, rapid and accurate diagnosis is possible by using the optimized real-time PCR reagent composition including the primer and probe.

Description

Primers and probes for Measles virus and Measles virus vaccine strain and detection method using them}

The present invention relates to a primer set for detecting a measles virus and a measles virus vaccine strain, and a composition or method for detecting the measles virus using the same.

Measles is an acute febrile infectious infectious virus disease caused by Measles virus of the genus Morbillivirus belonging to the Paramyxoviridae family. The virus is a negative-stranded RNA template (SS(-)RNA) virus with a spherical shape of 100-250 nm in diameter. Atypical measles symptoms may appear in children vaccinated against measles, and diagnosis is difficult due to attenuated clinical symptoms. Gene analysis through self-diagnosis is the only way to distinguish measles with unknown symptoms.

In the 10 years before the development of a live measles vaccine in 1063, there were 549,000 cases of measles in the United States and an average of 495 deaths per year from measles. Almost all Americans have been affected by measles at some point in their lives. Since the development of the measles vaccine, the worldwide incidence of measles has decreased, and in 2000 the United States declared measles eradication.

In Korea, the number of cases has drastically decreased due to vaccination, and the number of cases of measles continues to decrease. However, despite the measles eradication certification in 2014, cases of measles outbreaks continue to be reported mainly from imported cases. It is necessary to establish a diagnostic system to enable continuous monitoring by expanding the area of measles testing that can only be performed in some institutions.

Although both serological tests and genetic tests are used as current diagnostic methods, the current test methods are less effective in managing diseases and monitoring patients due to their low sensitivity. The WHO, which is conducting a measles eradication project, also recommends a real-time RT-PCR method with high gene sensitivity. However, since there is no commercialized genetic diagnostic kit for measles, a legal group 2 infectious disease, it is necessary to develop a real-time genetic diagnostic kit that can be used in front-line hospitals with speed, sensitivity, and accuracy for rapid epidemic prevention and management of patients and contacts. .

Through the commercialization and general dissemination of a diagnostic method based on real-time reverse polymerase chain reaction rather than the current serological test-dependent diagnostic method, each test institution uses standardized diagnostic reagents centered on the central management institution and secures a certain level of test quality This is very important for effective management.

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In order to solve the above problems, an object of the present invention is SEQ ID NO: 1 and SEQ ID NO: 2 for the detection of an outdoor strain of measles virus; And to provide a primer set comprising SEQ ID NO: 4 and SEQ ID NO: 5 for detecting the measles virus strain.

Another object of the present invention is to provide a composition for detecting a measles virus comprising the primer set and a method for detecting a measles virus and a measles virus vaccine strain using the same.

In order to achieve the above object, measles virus (Measles virus) SEQ ID NO: 1 and SEQ ID NO: 2 for field strain detection; And it provides a primer set comprising SEQ ID NO: 4 and SEQ ID NO: 5 for detecting the measles virus strain.

In addition, the present invention provides a primer set further comprising a probe of SEQ ID NO: 3 or SEQ ID NO: 6.

The present invention also provides a composition for detecting measles virus comprising a primer set.

The present invention also provides a composition, wherein the sample used in the composition is isolated from blood, serum, sputum, urine or tissue in vivo.

In addition, the present invention provides a composition that performs the detection of a real-time reverse transcription polymerase chain reaction (RT-PCR).

The present invention also provides a method for detecting a measles virus and a measles virus vaccine strain in an isolated sample, comprising the following steps.

(a) preparing forward and reverse primer sets of SEQ ID NOs: 1, 2, 4 and 5;

(b) isolating RNA from the sample;

(c) performing a reverse transcription reaction using the isolated RNA;

(d) performing amplification by adding one or more primer sets to the sample on which the reverse transcription reaction has been performed; and

(e) analyzing the sequence of the amplified product;

The set for the detection of the measles virus and vaccine strain of the present invention and the detection method using the same are faster and more efficient because two targets can be simultaneously identified in one reaction compared to a system capable of single detection of the existing measles virus or vaccine strain. In addition, it was confirmed that the clinical usefulness of the present invention is high through a comparative test with the WHO recommended diagnostic method. Through the present invention, it will be possible to prevent the spread at an early stage through rapid diagnosis of suspected patients, and effective management will be possible when an exposed person occurs. In the case of a vaccine reaction by rapidly differentially diagnosing the measles symptoms and the reaction due to vaccination, By preventing unnecessary measures such as contact investigation, it can contribute to the preparation of effective quarantine measures.

1 is a result of optimizing primer and probe concentration conditions for simultaneous detection of Measles virus and vaccine strains of Measles virus.
Figure 2 shows the results of comparison of the detection efficiency of the WHO standard diagnostic method and the primer and probe set of the present invention.
3 shows the results for confirming the detection efficiency of Measles virus vaccine strains of the primer and probe set of the present invention (* FAM fluorescence Ct value: 10 ³ copies 31.6, 10 ² copies 33.6, 10copies 35.8; * JOE fluorescence Ct value : 10 ³ copies 30.4, 10 ² copies 33.6, 10 copies 36.3).

Real-time reverse transcription PCR can be performed using the primer and probe set for detecting the measles virus and vaccine strain of the present invention.

The base sequence of the measles virus was collected from the NCBI database and the common conserved sequence between strains was found from the multi-alignment result, and the target gene (N gene) and the primer-probe target site were selected, and the GC ratio and melting temperature were taken into consideration. and probe sets were designed. In addition, primers and probe target sites were selected at the SNP sites separated between the measles virus and vaccine strain nucleotide sequences, and these were specifically distinguished and designed to be detected.

A detectable fluorescent label and a quencher are bound to the 5' and 3' ends of the probe for detecting measles virus and vaccine strain. Examples of fluorescent labeling factors include FAM, VIC, TAMRA, JOE, ROX, HEX, Cy3, Cy5, Texas Red, etc., and emission and exitation wavelengths are different depending on the type. Considering this, a fluorescent labeling factor used together in one PCR reaction must be selected and used by determining whether it can be detected separately, and different colors can be used.

In the present invention, using FAM and JOE, it is possible to simultaneously check whether the measles virus and the vaccine strain are in each fluorescence channel.

Fluorescence detection methods include an interchelating method, a TaqMan™ probe method, and a molecular beacon method. They use different principles to detect the increase in fluorescence, and the method used in the present invention is the TaqMan™ probe method. The TaqMan™ probe method is a method of adding an oligonucleotide (TaqMan™ probe), in which the 5' end is modified with a fluorescent labeling factor (FAM, etc.) and the 3' end with a quencher material (TAMRA, Blacke hole chencher, etc.), to the PCR reaction solution. Although the TaqMan™ probe specifically hybridizes to the template DNA in the annealing step, fluorescence generation is suppressed by the quencher on the probe, and the 5′ → 3′ exonuclease activity of Taq DNA polymerase hybridizes to the template in the extension step. As only the TaqMan™ probe is degraded, the fluorochrome is released from the probe, suppression by the quencher is released and fluorescence is emitted.

The set for detecting measles virus of the present invention can be performed using a conventional real-time PCR method and apparatus. At the end of PCR, the laser of the real-time PCR device detects the fluorescent labeling factor labeled on the probe of the amplified PCR product to realize a peak as shown in FIG. 1 . At this time, the result can be automatically analyzed by executing the program built into the device without the electrophoresis process. The analyzed result is displayed as Ct (Threshold cycle), so even an unskilled person can easily understand the analyzed result.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited to the following examples.

<Example 1> Preparation of primers and probes for detecting Measles virus and vaccine strains of Measles virus

After collecting the nucleotide sequence of the target gene, nucleoprotein (N gene), using BLASTsearch at the National Center for Biological Information (NCBI, https://www.hcbi.nlm.nih.gov/), it is compared with the nucleotide sequence in the database. Primers and probes were designed at the most specific site by analyzing homoly (Table 1). For the reporter of the probe for the multiplex, FAM was used for Measles virus, JOE was used for vaccine strains (Table 2), and MGBNFQ or black hole quencher (BHQ) was used for the quencher. Using primer design software (Primer Express, USA), the Tm (melting temperature) values of the primers and probes were designed to correspond to the ranges of 58 to 60°C and 68 to 70°C, respectively.

detection target primer
or probe Base sequence (5'→3') SEQ ID NO: Measles
virus first forward primer AGT AGA GCG GTT GGA CCC AG One first reverse primer GGT AGC TCA TTC TCA CTT TGA TCA C 2 first probe CCC AAG TAT CAT TTC 3 Vaccine strains
(genotype A) second forward primer CCC AAG GAG CTT AGC ATT GTT C 4 second reverse primer GAG GAA TCT CCA GGG ATT GGT AC 5 second probe ACA TCA GGA TCC GGT GGA GCC ATC AG 6

detection target fluorescent substance quencher Measles virus JOE BHQ1 Measles virus vaccine strains (genotype A) FAM MGBNFQ

<Experimental Example 1> Optimization of primer and probe concentration conditions for simultaneous detection of Measles virus and vaccine strains of Measles virus

An attempt was made to determine the concentration of the primers and probes prepared in Example 1, which did not affect the PCR reaction by competing with other primers or probes while exhibiting the best performance against the target pathogen.

By similarly adjusting ΔRn and slope of the amplification plot of each detection target gene region, the amplification efficiency of each target primer and probe is similarly matched, and non-specific amplification products are not generated and the amplification efficiency is maintained. We tried to find a condition that can minimize the concentration of the primer, and the optimized concentration conditions are shown in Table 3. According to the reaction solution composition shown in Table 4 and the reaction conditions described in Table 5, PCR was performed using a real-time PCR device (Applied Biosystems 7500 Fast Real-time PCR system, Life technologies, USA).

detection target Primer or probe final concentration Measles virus first forward primer 250nM first reverse primer 250nM first probe 150nM Vaccine strains
(genotype A) second forward primer 500nM second reverse primer 500nM second probe 100nM

reaction solution composition Addition (uL) First forward primer (10 pmol/uL) solution 0.5 First reverse primer (10 pmol/uL) solution 0.5 First probe (10 pmol/uL) solution 0.3 Second forward primer (10 pmol/uL) solution One Second reverse primer (10 pmol/uL) solution One Second probe (10 pmol/uL) solution 0.2 2X Real-time RT-PCR Master Mix* 10 template RNA 5 TE buffer 1.5 Sum 20

Temperature (℃) time number of cycles 50 30 minutes One 95 10 minutes One 95 15 seconds 40 60 1 minute

As a result, it was confirmed that similar performance was exhibited when the optimal multiplex concentration condition was compared with the singleplex condition in which each primer and probe were included alone.

<Experimental Example 2> Comparison of detection efficiency between the WHO standard diagnostic method and the primer and probe set of the present invention

When real-time RT-PCR is performed using the primer and probe set of the present invention, in order to confirm that it exhibits superior sensitivity compared to the known primer set in the WHO standard test method, the primer and probe set of the present invention and the known primer set RT-PCR was performed using

First, the RNA extracted from the sample confirmed as positive for measles was diluted stepwise, and then, according to the reaction solution composition shown in Table 4 and the reaction conditions shown in Table 5, a real-time PCR device (Applied Biosystems 7500 Fast Real-time) PCR was performed using the PCR system, Life technologies, USA). As a primer set for detecting Measles virus known in the WHO standard diagnostic method, the primer set shown in Table 6 below was used, and PCR was performed in the same manner as in the manual. After the PCR reaction was completed, the PCR reaction was electrophoresed and compared with the results of the present invention.

detection target primer Base sequence (5'→3') density Measles virus forward primer TGG CAT CTG AAC TCG GTA TCA C 300 nM reverse primer TGT CCT CAG TAG TAT GCA TTG CAA 300 nM probe CCG AGG ATG CAA GGC TTG TTT CAG A 250 nM

As a result of comparative evaluation using two types of positive samples, when PCR was performed with the primer and probe set of the present invention, detection was confirmed in a cycle earlier than that of the primer and probe set known to WHO, indicating that the performance was superior. It was confirmed (FIG. 2).

<Experimental Example 3> Confirmation of the detection efficiency of Measles virus vaccine strains of the primer and probe set of the present invention

In order to confirm the sensitivity in detecting Measles virus vaccine strains when real-time RT-PCR was performed using the primer and probe set of the present invention, RNA of a vaccine strain commercialized using the primer and probe set of the present invention PCR was performed using

After stepwise dilution of commercialized Measles virus vaccine strain RNA (Vircell, Spain), according to the reaction solution composition shown in Table 4 and the reaction conditions shown in Table 5, a real-time PCR device (Applied Biosystems 7500 Fast Real- PCR was performed using the time PCR system, Life technologies, USA).

As a result, when PCR was performed with the primer and probe set of the present invention, the Measles virus vaccine strain was able to detect up to 10 RNA copies/uL, confirming that the sensitivity of the primer and probe set of the present invention was excellent (FIG. 3).

<110> KCDC <120> Primers and probes for Measles virus and Measles virus vaccine strain and detection method using them <130> M19-6485 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> RNA <213> Measles virus <400> 1 agtagagcgg ttggacccag 20 <210> 2 <211> 25 <212> RNA <213> Measles virus <400> 2 ggtagctcat tctcactttg atcac 25 <210> 3 <211> 15 <212> RNA <213> Measles virus <400> 3 cccaagtatc atttc 15 <210> 4 <211> 22 <212> RNA <213> Measles virus <400> 4 cccaaggagc ttagcattgt tc 22 <210> 5 <211> 23 <212> RNA <213> Measles virus <400> 5 gaggaatctc cagggattgg tac 23 <210> 6 <211> 25 <212> RNA <213> Measles virus <400> 6 ggtagctcat tctcactttg atcac 25

Claims (12)

A primer set comprising a primer of SEQ ID NO: 1 and SEQ ID NO: 2 and a probe of SEQ ID NO: 3 for detecting measles virus. delete A primer set comprising a primer of SEQ ID NO: 4 and SEQ ID NO: 5 and a probe of SEQ ID NO: 6 for detecting a measles virus strain. delete A composition for detecting measles virus comprising the primer set of claim 1 A composition for detecting a measles virus vaccine strain comprising the primer set of claim 3 The composition according to claim 5 or 6, wherein the sample is isolated from blood, serum, sputum, urine or tissue in vivo. A multiplex kit for simultaneous detection of a measles virus and a measles virus vaccine strain comprising the primer set of claim 1 and the primer set of claim 3. The multi-kit according to claim 8, wherein the sample is isolated from blood, serum, sputum, urine or tissue in vivo.
A method for detecting measles virus in an isolated sample, comprising the steps of:
(a) preparing the primer set of claim 1;
(b) isolating RNA from the sample;
(c) performing a reverse transcription reaction using the isolated RNA;
(d) performing amplification by adding the primer set of claim 1 to the sample on which the reverse transcription reaction has been performed; and
(e) analyzing the sequence of the amplified product; A method for detecting a measles virus vaccine strain in an isolated sample, comprising the steps of:
(a) preparing the primer set of claim 3;
(b) isolating RNA from the sample;
(c) performing a reverse transcription reaction using the isolated RNA;
(d) performing amplification by adding the primer set of claim 3 to the sample on which the reverse transcription reaction has been performed; and
(e) analyzing the sequence of the amplified product; The method of claim 10 or 11, wherein the separated sample is separated from blood, serum, sputum, urine, or living tissue.

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