KR101099041B1 - Multiplex PCR primers and detection method for diagnosing herpesviruses - Google Patents

Abstract

The present invention relates to multiplex PCR (multiplex polymerase chain reaction-based PCR) that can quickly identify various types of herpesvirus (Herpesvirus), which is one of the important infectious diseases for immunocompromised patients. More specifically, four types of herpesviruses to be amplified, namely, human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and rosin virus (HHV-6: Human Herpesvirus 6 and HHV-). 7: Human Herpesvirus 7) Amplifying the target genes of the four herpesviruses by using a oligonucleotide having a specific sequence in the target gene of the virus as a primer and simultaneously reacting in one container. It is a multiplex PCR method characterized by the above-mentioned. As a result, the target genes of the four herpes viruses can be detected simultaneously by only one PCR, which can be effectively used for the rapid diagnosis of the herpes virus, and the economic cost of the diagnosis can be reduced.

Herpesviruses, Multiplex PCR

Description

Multiplex PCR primers and detection method for diagnosing herpesviruses

The present invention provides four types of herpesviruses, namely, human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and rosin virus (HHV-6: Human Herpesvirus 6 and HHV-7). : Multiplex PCR primers for simultaneous diagnosis of Human Herpesvirus 7), kits comprising the same and multiplex PCR methods.

Herpesviruses are widespread in nature and most people are infected with one or more herpesviruses. Herpesviruses belong to the herpesviridae family, and they are classified into three subfamily, α, β, and γ, depending on the biological properties of the cell culture. The subfamily α-herpesvirinae belongs to the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), and the varicella-Zoster virus (VZV), and the β-herpesvirinae subfamily to human cytomegalovirus (HCMV) and rosin Viruses (HHV-6: Human Herpesvirus 6 and HHV-7: Human Herpesvirus 7). γ-herpesvirinae subfamily include Epstein-Barr virus (EBV) and Kaposi's sarcoma virus (KSHV) (Hara et al, 2002). Most of these viruses are latent in normal humans, but are activated when the host's immune status is suppressed or deficient, causing infections.In particular, these viruses cause fatal infectious complications in patients with reduced immunity such as organ transplant patients and HIV infected persons. The main viruses are known as HCMV and EBV. Recently, HHV-6 and HHV-7 are also known as viruses that are often a problem in organ transplant patients and AIDS patients with reduced immunity. [Hara et al , 2002; Wada et al , 2007].

The clinical symptoms of these viruses represent a variety of clinical features ranging from asymptomatic infection to severe infection or death. Like other herpesviruses, HCMV is known to lie in vivo throughout life after infection, and causes neonatal infections such as jaundice, pneumonia, and encephalitis due to nervous system and liver damage. It is known to cause pneumonia, hepatitis and retinitis in patients and AIDS patients [Sassenscheidt et al , 2006]. The age of the initial infection of EBV depends on the hygienic environment, but serological tests are prevalent around the world, with nearly 100% positive rates in adults in their late 20s. EBV is a causative agent of infectious mononucleosis, a virus that causes bikitym and nasopharyngeal carcinoma, and is known to cause fever, pharyngitis, lymphadenopathy, lymphocytosis [Kang et al , 2007; Ikegaya et al , 2008]. HHV-6 is most likely to occur in infants between 6 and 24 months of age, and occurs in about 20% of children, with major clinical symptoms leading to skin lesions such as roseola or exanthema subitum [ Clark, 2002; Deback et al , 2008]. In addition, HHV-6 is known to cause HCMV-like symptoms in immunocompromised patients when infected with CD4 + T cells. HHV-7 is also known to cause sudden rashes like HHV-6. Ashshi et al , 2003; Murray et al , 2003; Deback et al , 2008]. However, some treatments for these viral infections have been used, but effective vaccines have not yet been developed, and the only way to prevent contact with secretions such as blood and saliva of infected people is currently the best preventive measure. GCV (gancyclovir) drug treatment reduces HCMV in vivo, and forscarnet therapy is effective for retinitis, and foscarnet and cidoforvir, antiviral drugs for HHV-6 and HHV-7, are known to be more effective than other drugs. However, the reactivation of these viruses and the timing of drug administration have not been established [Qamruddin et al , 2001; Clark DA 2002]. EBV is also a therapeutic agent for acyclovir that reduces viral secretion from the oropharyngeal gland, but is not effective for other diseases such as mononucleosis.

As such, a faster and more accurate diagnosis of the causative virus is of great importance for early prevention and effective treatment of the viral infection and reactivation. Various methods are used to diagnose the virus of herpes virus. The detection method of the virus is known to isolate the virus using cell culture together with the ELISA and the IFA using the fluorescent antibody, which is a serological method, but the detection time by the detection of the antigen and antibody can be detected. In this case, the cost of producing the antibody is high, the storage is difficult, and the isolation of the virus using cell culture is an accurate diagnostic method, but it requires a high skill and time of the experimenter. Therefore, in order to overcome this limitation, quantitative and qualitative PCR methods for detecting specific genes of the virus have been developed by many researchers, but all of the above herpes viruses, which are still a problem in immunocompromised patients, are simultaneously diagnosed. The multiplex PCR method is not developed.

Therefore, the present inventors have developed a multiplex PCR method and a primer for performing single-tube-multiplex PCR, which can more quickly detect a herpes virus, which is a problem in immunocompromised patients, and more easily and accurately, the specific genes of the viruses. So that it can be detected.

The present invention aims to provide a simpler, more accurate way to detect four herpesviruses, namely HCMV, EBV, HHV-6 and HHV-7 simultaneously.

In order to achieve the above object, the present invention provides a primer set of SEQ ID NO: 1 and SEQ ID NO: 2, a primer set of SEQ ID NO: 3 and SEQ ID NO: 4, a primer set of SEQ ID NO: 5 and SEQ ID NO: 6 And a single tube-multiplex PCR method for detecting Herpesvirus, using a primer set of SEQ ID NO: 7 and SEQ ID NO: 8.

The present invention also provides a primer set of SEQ ID NO: 1 and SEQ ID NO: 2, a primer set of SEQ ID NO: 3 and SEQ ID NO: 4, a primer set of SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: 7 and Provided is a set of single-tube-multiplexed PCR primers for detecting herpesvirus, characterized in that it consists of a primer set of SEQ ID NO: 8.

The present invention also provides a single tube-multiplex PCR premix kit comprising a primer set, a reaction buffer, and a DNA polymerase for detecting herpesvirus. More preferably, the PCR primer set is a primer set of SEQ ID NO: 1 and SEQ ID NO: 2, a primer set of SEQ ID NO: 3 and SEQ ID NO: 4, a primer set of SEQ ID NO: 5 and SEQ ID NO: 6, and And a set of primers of SEQ ID NO: 7 and SEQ ID NO: 8.

In addition, the present invention provides a composition for detecting herpes virus, which comprises a primer set, a reaction buffer and a DNA polymerase, a DNA staining reagent, and a carrier used for multiplex PCR. More preferably, the primer set is a primer set of SEQ ID NO: 1 and SEQ ID NO: 2, a primer set of SEQ ID NO: 3 and SEQ ID NO: 4, a primer set of SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: : 7 and SEQ ID NO: 8, characterized in that the composition for the detection of herpesvirus.

Preferably, the herpesviruses are human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) and rosinvirus (HHV-6: Human Herpesvirus 7). At least one herpesvirus selected from the group consisting of:

According to the method of the present invention, it is possible to detect four herpes virus target genes simultaneously by only one PCR, so that the herpesvirus can be diagnosed more quickly and efficiently. Accordingly, it is possible to construct an efficient virus diagnosis system capable of quickly and rapidly diagnosing various causes of immunocompromised diseases.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited by the following examples.

Example

Example 1 Isolation of Four Herpes Viruses

DNA from reference strains of human cytomegalovirus (HCMV), Epistein-Barr virus (EBV), Human Herpesvirus 6 (HHV-6), and Human Herpesvirus 7 (HHV-7) was determined by ABI (Advanced Biotechnologies Inc, www.abionlone). .com). Specifically, the standard stocks of HCMV are AD169 (Catalog No. 08-701-000, Genbank No. BK000394), and the standard stocks of EBV are B95-8 (Catalog No. 08-702-000, Genbank No. AJ507799), HHV. -6 standard stocks are Z-29 (Catalog No. 08-703-000, Genbank No. AF157706) and HHV-7 standard stocks are H7-4 (Catalog No. 08-765-000, Genbank No. AF037218) Was used.

Example 2: Multiplex PCR Primer Preparation

Specific amplification of four herpes (HCMV, EBV, HHV-6 and HHV-7) viruses according to the present invention, and after agarose gel electrophoresis, each virus is classified according to the size of PCR results Primers were prepared based on the Nucleocapsid gene. Each primer is shown in Table 1 below.

virus Name of the primer Primer sequence Amplicon size (bp) SEQ ID NO: HCMV CMV-F ACG CCG CTG TCG AGT ATA GAT 307 One CMV-R CAA CGT TTT TGA CCT CGA AGA T 2 EBV EBV-F TTC TCT TCC CCG ATG ATT GGT A 420 3 EBV-R GCA TAG GAG TGC GAA CAT GGA 4 HHV-6 HHV6-F GCG AAT CTC ACA GAG CAG GAT 158 5 HHV6-R GGC ACA CCC GAA GGA ATA A 6 HHV-7 HHV7-F ATT TTA TTG TCG CGT CAC CAG A 701 7 HHV7-R TGG TGC GTA CAC TGT AAG CTC A 8

Example 3: Multiplex PCR Premix Kit Preparation

A multiplex PCR kit was prepared to perform a PCR reaction using DNA isolated from plasma samples of immunocompromised patients. Single-tube-multiplexed PCR premixes with Taq polymerase were prepared for primers for HCMV, EBV, HHV-6 and HHV-7 (CMV-F / R, EBV-F / R, HHV6-F / R, HHV7- F / R).

Example 4: Multiplex PCR

Solent's 2x Multiplex PCR pre-Mix (Catalog No. SG6400) specific primers for HCMV, EBV, HHV-6 and HHV-7 (CMV-F / R, EBV-F / R, HHV6- PCR was performed by adding 10 pmol of F / R and HHV7-F / R) and 1 μl (concentration range: 10 ¹ ng to 10 ⁻⁵ ng) of DNA samples for the standard strains of the virus. First, predenaturation at 95 ° C. for 15 minutes, followed by denaturation at 95 ° C. for 20 seconds, annealing at 55 ° C. for 40 seconds, and extension at 72 ° C. for 1 minute After repeating, further reaction was performed at 72 ° C. for 7 minutes. After PCR reaction, the amplified DNA was subjected to electrophoresis using 2.5% agarose gel containing EtBr, and observed under UV light.

Figure 1 shows the results of the multiplex PCR experiment according to the present invention, the multiplex PCR for each standard strain using the primer set of the present invention for the gene isolated from the standard strains of four herpes virus The result is. As a result of the experiment, each virus showed PCR results of different sizes. In Figure 1, lane M is 100bp of labeled DNA, lane 1 is HCMV standard strain AD169 (307 bp), lane 2 is EBV standard strain B95-8 (420 bp), lane 3 is HHV-6 standard strain Z-29 ( 158 bp) and lane 4 are the result of HHV-7 standard strain H7-4 (701 bp). Therefore, it was possible to specifically distinguish four herpes virus target genes in a single PCR reaction.

Subsequently, FIG. 2A to FIG. 2D show the results of experiments on the sensitivity of the multiplex PCR technique for each herpes virus, that is, the detection limit, and diluting the gene isolated from each standard strain by 10-fold unit. This is the result of the same PCR technique. As a result, PCR of different sizes for HCMV (FIG. 2A, 307 bp), EBV (FIG. 2B, 420 bp), HHV-6 (FIG. 2C, 158 bp) and HHV-7 (FIG. 2D, 701 bp), respectively. The result was obtained. In each result, lane M is 100bp of labeled DNA, lane 1 is 10 ng of standard stock, lane 2 is 1 ng of standard stock, lane 3 is 10 ^-1 ng of standard stock, and lane 4 is 10 ng. ^-2 ng standard strain, lane 5 is ^10-3 ng standard strain, lane 6 is ^10-4 ng standard strain and lane 7 is ^10-5 ng standard strain. Experimental detection limits for each virus showed that HCMV (FIGS. 2A, 307 bp) and EBV (FIGS. 2B, 420 bp) were ^10-3 ng, HHV-6 (FIGS. 2D, 158 bp) and HHV-7 ( 2e, 701 bp) showed a minimum detection limit of 10 ^-4 ng, and the lowest detection limit concentration of each herpes virus was converted into a copy number by the following formula. In the following formula, 6.02 * 10 ²³ represents the number of avogadro, and the viral genome size represents the number of base pairs.

As a result, HCMV showed approximately 4 * 10 ³ (copies), EBV had approximately 5 * 10 ³ (copies), and HHV-6 and HHV-7 had approximately 6 * 10 ² (copies).

In addition, the results of multiplex PCR performed by mixing all the genes isolated from the standard strains of four herpes viruses (HCMV, EBV, HHV-6 and HHV-7) are shown in FIG. 3. As a result, it was observed that HCMV (307 bp), EBV (420 bp), HHV-6 (158 bp) and HHV-7 (701 bp) showed PCR products of four different sizes in one lane. In FIG. 3, lane M is 100 bp of labeled DNA, and lane P is each standard strain for four herpesviruses.

Example 5: Application of Multiplex PCR

DNA isolated from 45 plasma samples obtained from hematopoietic stem cell transplant patients was used to confirm the utility of the multiplex PCR prepared in Example 3. DNA isolation and multiplex PCR of these 45 clinical specimens were performed in the same manner as described above, and HCMV and EBV DNA were measured by real-time quantitative test to confirm the usefulness of the multiplex PCR prepared in Example 3. It was.

As a result of performing multiplex PCR, as shown in FIG. 4, bands were formed at 307 bp in 17 lanes (26, 27, 31 to 45), and HCMV was detected. The remaining three herpesviruses (EBV, No bands representing HHV-6 and HHV-7) were detected. HCMV DNA of these 45 plasma samples was measured using a commercially available real-time quantitative assay kit (artus ^® CMV LC PCR kit, Qiagen, Germany), and the HCMV detection limit of the multiplex PCR performed in Example 4 4 * There were 17 clinical specimens measured at 10 ³ copies (concentration 10 ⁻³ ng) or more, of which 15 (Fig. 4, lanes 31 to 45) were detected by HCMV by multiplex PCR performed in Example 4. Confirmed. EBV was also measured using a commercially available real-time quantitative assay kit (artus ^® EBVTM PCR kit, Qiagen, Germany), and detected by real-time quantitative assay as in the result of multiplex PCR prepared in Example 3. It wasn't. The experimental results confirmed that the multiplex PCR according to the present invention can be usefully used for diagnosis.

1 is a result of performing a multiplex PCR technique using a primer pair of the present invention for genes isolated from the standard strains of four herpesviruses according to the present invention.

Figure 2a is a result of performing multiplex PCR by diluting the genes isolated from the standard strain AD169 for HCMV in 10-fold units each.

Figure 2b is a result of performing multiplex PCR by diluting the genes isolated from the standard strain B95-8 for EBV in 10-fold units each.

Figure 2c shows the results of multiplex PCR by diluting the genes isolated from the standard strain Z-29 for HHV-6 in 10-fold units, respectively.

Figure 2d is a result of performing multiplex PCR by diluting the genes isolated from the standard strain H7-4 for HHV-7 in 10-fold units, respectively.

Figure 3 is a result of performing multiplex PCR by mixing all the genes isolated from the standard strains of four herpes virus according to the present invention.

4 is a result of performing multiplex PCR measured by real-time quantitative test in order to confirm the usefulness of the multiplex PCR according to the present invention.

Claims (8)

Primer sets of SEQ ID NO: 1 and SEQ ID NO: 2, primer sets of SEQ ID NO: 3 and SEQ ID NO: 4, primer sets of SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: 7 and SEQ ID NO: 8 Human cytomegalovirus (HCMV), epstein barr virus (EBV) and rosin virus (HHV-6: Human Herpesvirus 6, characterized by using an primer set and annealing at 55 ° C. And HHV-7: Human Herpesvirus 7) A single tube-multiplex PCR method for detecting at least one herpesvirus selected from the group consisting of: delete Primer sets of SEQ ID NO: 1 and SEQ ID NO: 2, primer sets of SEQ ID NO: 3 and SEQ ID NO: 4, primer sets of SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: 7 and SEQ ID NO: 8 Characterized in that it consists of a primer set, an annealing temperature of 55 ° C cytomegalovirus (HCMV: Human cytomegalovirus), Epstein bar virus (EBV: epstein barr virus) and rosin virus (HHV-6: Human Herpesvirus 6, And HHV-7: Human Herpesvirus 7). A set of single-tube-multiplexed PCR primers for detecting one or more herpesviruses selected from the group consisting of: delete In a single tube-multiplex PCR premix kit comprising a primer set for detecting herpesvirus, a reaction buffer and a DNA polymerase, PCR primer sets include a primer set of SEQ ID NO: 1 and SEQ ID NO: 2, a primer set of SEQ ID NO: 3 and SEQ ID NO: 4, a primer set of SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: 7 and sequence No. 8, characterized in that it consists of a primer set of 8, human cytomegalovirus (HCMV), epstein barr virus (EBV) and rosin virus (HHV-6: A single tube-multiplex PCR premix kit for detecting one or more herpesviruses selected from the group consisting of Human Herpesvirus 6, and HHV-7: Human Herpesvirus 7). delete In a composition for detecting herpesviruses comprising a primer set used in multiplex PCR, a reaction buffer and a DNA polymerase, a DNA staining reagent, and a carrier, the primer set comprises a primer set of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: : A primer set of 3 and SEQ ID NO: 4, a primer set of SEQ ID NO: 5 and SEQ ID NO: 6, and a primer set of SEQ ID NO: 7 and SEQ ID NO: 8, and the annealing temperature is One selected from the group consisting of human cytomegalovirus (HCMV), epstein barr virus (EBV) and rosinvirus (HHV-6: Human Herpesvirus 6, and HHV-7: Human Herpesvirus 7) at 55 ° C. Composition for detecting herpes virus above. delete

Images