KR20020089746A - A size-modifying-anchor primer, method for rt-pcr using the size-modifying-anchor primer and method for diagnosis using the same method - Google Patents

Abstract

The present invention relates to a size-induced anchor primer, which is a DNA fragment having an anchor region having a sequence complementary to an RNA region at 3 'end and a tail region having a random sequence at 5' end. The present invention also relates to a method for selectively amplifying DNA selectively from an RNA template in a sample containing both DNA and RNA by using the anchor primer for basic RT-PCR, and more specifically, (a) anchor Preparing RNA for use as a primer and template; (b) using the anchor primer and performing a reverse transcription reaction without undergoing a pre-denaturing step; (c) after the reverse transcription reaction, adding a PCR mixture comprising a primer corresponding to the anchor primer directly to the reaction vessel and performing a PCR reaction. The present invention also relates to a method for diagnosing the infection of a specific organism using the above method.

Description

A SIZE-MODIFYING-ANCHOR PRIMER, METHOD FOR RT-PCR USING THE SIZE-MODIFYING-ANCHOR PRIMER AND METHOD FOR DIAGNOSIS USING THE SAME METHOD}

The present invention relates to an anchor primer consisting of an anchor portion and a tail portion, an RT-PCR method for discriminating DNA and RNA using the same, and a diagnosis method for infecting a specific organism using the same.

Conventional reverse transcription-polymerase chain reaction (RT-PCR) has been widely used for the detection of RNA in the clinical field, including research and diagnosis of viral infections. Reverse transcriptase, a key enzyme of the reverse transcription (RT) reaction, can be polymerized using not only RNA but also DNA as a template. As a result, despite large sample throughput, fast reaction rates and sensitivity, the products amplified by RT-PCR from RNA and DNA templates could not be discerned (Nelson et al., 1996, J Virol Methods., 56, 139). Freeman et al., 1999, Biotechniques., 17, 112-125; Stenman et al., 1999, Nature Biotechnoloy., 17, 720-722; Yun et al., 1999, J Pathol., 187, 518-522; Gozlan et al., 1996, J Clin Microbiol., 34, 2085-2088; Bitsch et al., 1993, J Infect Dis., 167, 740-743).

Two approaches have been established to address this problem (Nelson et al., 1996, J Virol Methods., 56, 139-148; Freeman et al., 1999, Biotechniques., 17, 112-125; Stenman et al., 1999, Nature Biotechnoloy , 17, 720-722). One is to use pure RNA extract which does not contain DNA as a template. However, complete removal of DNA is extremely cumbersome and requires separate confirmation of DNA contamination in the sample by reaction without reverse transcriptase (Yun et al., 1999, J Pathol., 187, 518-522; Gozlan et al. 1996 , J Clin Microbiol., 34, 2085-2088). The second method is to select the target to be amplified within the region containing the intron (splicing) sequence (Nelson et al., 1996, J Virol Methods., 56, 139-148; Freeman et al., 1999, Biotechniques., 17, 112-125; Bitsch et al., 1993, J Infect Dis., 167, 740-743). However, this method also cannot be applied if there is no intron region in the target region, as in the case of late viral genes (Britt et al., 1996, J Infect Dis., 167, 740-743).

In the present invention, in order to solve the above problems of the prior art, based on the difference in the reaction temperature between the RT reaction and the PCR reaction as well as the structural difference between the RNA and DNA, DNA and RNA can be discriminated regardless of the presence of introns. It is an object of the present invention to provide an RT-PCR method.

In addition, the above-described method is specifically applied to glycoprotein B (gB, gpUL55), which is a late gene of hCMV without intron, to provide a diagnostic method for a specific organism.

It is also an object of the present invention to provide a sized anchor primer that can be used in the above methods.

1 is a view of the design of the primer for SMART-PCR of the present invention,

(a) shows the relative position of the primers in the gB gene of hCMV AD169 ,

(b) is a diagram showing the secondary structure of the target RNA region,

Figure 2 is a diagram showing the process of the SMART-PCR of the present invention,

3 is a diagram illustrating a manufacturing process of a standard DNA and RNA template.

(a) is a diagram regarding standard DNA (pSP72 / -SP6 / gB),

(b) is a diagram regarding standard RNA,

(c) is a diagram showing formaldehyde modified gel electrophoresis of in vitro transcripts,

4 is a diagram illustrating an optimization process of SMART-PCR.

(a) is a diagram showing the effect of MgCl ₂ concentration on reverse transcription,

(b) is a diagram showing a SMART-PCR result under an optimal factor,

5 is a diagram showing the results of competitive amplification of RNA against DNA in SMART-PCR,

Fig. 6 shows the results of SMART-PCR on nucleic acids extracted from reference strains AD169, Davis and Towne .

<Brief Description of Reference Symbols>

gB / F: Forward primer for gB amplification gB / R: Reverse primer for gB amplification

pcF: common forward primer used for RNA and DNA amplification

pcR: reverse primer for DNA

SMA: Size-Modifying-Anchor primer consisting of 3 ′ anchor portion and 5 ′ nonspecific tail portion

RT: reverse transcription POL: polymerisation

Amp ^r : ampicillin resistance gene Ori: origin of replication

SP6: SP6 promoter BMV RNA: RNA size marker

Trans: in vitro transcription

MRC-5: SMART-PCR results from total nucleic acids extracted from MRC-5

AD169, Davis and Towne : SMART-PCR using total nucleic acid extracted from MCR-5 infected from each hCMV strain.

The modified anchor primer of the present invention is a DNA fragment having an anchor portion having a sequence complementary to an RNA region at 3 'end and a tail region having a random sequence at 5' end. .

The RNA region to be bound to the anchor is preferably present in a single strand during reverse transcription reaction with a reaction temperature of 37 to 45 ℃. Thus, during the reverse transcription reaction, the anchor primers bind only to RNA, rather than to two strands of DNA, thereby selectively cDNA synthesis only from the RNA template.

The base number of the anchor site alone may vary depending on the sequence, but it is preferable that the estimated Tm of the site is near or above the temperature of the reverse transcription reaction and does not exceed the annealing temperature of the PCR reaction. That is, it is preferable that the estimated Tm of only an anchor site | part is about 36-48 degreeC, and about 11-13 bases are preferable. This allows specific binding to RNA without denaturation during reverse transcription, followed by a high temperature at the tail when performing PCR reactions, so that the anchor site alone does not bind to the template, but the entire anchor primer containing the tail By allowing only the binding to the template, the genomic DNA originally included is not able to be a template but only cDNA synthesized from RNA can act as a template.

The total Tm of the anchor primer may be a temperature such that the anchor region alone cannot be used as a template at the binding temperature of the PCR reaction. Preferably it is about 69-74 degreeC.

The tail region is preferably nonspecifically bound to the DNA or RNA template during the reverse transcription reaction and also does not form self-priming during the reverse transcription reaction and subsequent PCR reaction.

In addition, a method of selectively amplifying DNA from an RNA template (hereinafter, also referred to as Size Modifying Anchor-mediated RT-PCR or SMART-PCR) in a sample containing RNA of the present invention may be used as (a) the anchor primer and template. Preparing RNA for use; (b) using the anchor primer and performing a reverse transcription reaction without undergoing a pre-denaturing step; (c) after the reverse transcription reaction, adding a PCR mixture including a forward primer (primer 1) corresponding to the anchor primer directly to the reaction vessel and performing a PCR reaction.

The preparation of the anchor primers and RNA in step (a), the reverse transcription reaction in (b) and the PCR reaction in (c) can be carried out by conventional methods. For example, the reverse transcription reaction of step (b) uses a 20 μl reverse transcription reaction mixture containing 25 picomoles of the anchor primer, and the reaction conditions are 5 minutes at 42 ° C. and 60 minutes at 90 ° C. By denaturation for minutes and cooling at 4 ° C. for 5 minutes, wherein the PCR reaction of step (c) is carried out at 95 ° C. for 10 minutes, 40 times of 94 ° C., 30 seconds, 68 ° C., 10 seconds, 72 It may be carried out under the reaction conditions consisting of a final extension of 30 ℃ and 72 ℃ 10 minutes.

The reverse transcription reaction mixture of step (b) preferably comprises about 0.5 to 2.5 mM, more preferably about ₁ to 1.25 mM of MgCl ₂ . This is because when the concentration of MgCl ₂ exceeds 2.5 mM, nonspecific binding to the DNA of the anchor occurs, and reverse transcription does not occur at a concentration of less than 0.5 mM.

In addition, the method for diagnosing the infection of a specific organism of the present invention is characterized by using a method of selectively amplifying DNA from an RNA template in a sample containing the RNA (SMART-PCR).

The method comprises, for example, (i) (a) preparing RNA for use as the anchor primer and template; (b) performing the reverse transcription reaction using the anchor primer, without undergoing a pre-denaturing step, and (c) after the reverse transcription reaction, directly anchoring the anchor to the reaction vessel. Amplifying a specific DNA fragment by adding a PCR mixture comprising a forward primer corresponding to the primer (primer 1) and performing a PCR reaction; And (ii) diagnosing the infection of a specific organism according to the formation of the specific DNA fragment.

The specific organism may include any one that makes an RNA genome or RNA transcript, including a virus. For example, human cytomegalovirus can be included.

The specific DNA fragment can be used as long as it can be used as a marker indicating the presence of the specific organism. Particularly, the specific DNA fragment exists from the same species or the same strain and is derived from a highly homologous moiety. For example, among glycoproteins B (gB, gpUL55) of human cytomegalovirus, it may be a site with high homology between AD169 (GenBank accession no. X04606) and Towne (GenBank accession no. M22343) (FIG. 1, See Table 1).

The primer (primer 1) corresponding to the anchor primer is a forward primer specifically binding to the 5 ′ end of the DNA fragment to be amplified. The anchor primer and the corresponding primer (primer 1) include, for example, a base fragment of SEQ ID NO: 9 (pcF) and a base fragment of SEQ ID NO: 2 or 4 complementary to gB, one of the late genes of human cytomegalovirus. Each can be.

As a result of the above method, when the specific DNA fragment to be amplified is synthesized, it can be recognized that the human cytomegalo virus is present, and if it is not synthesized, it can be recognized as not present.

The PCR of step (i) (c) of the method may include primer 2 which specifically binds to the gene from which the DNA to be amplified is derived as a reverse primer. Such primers include, for example, base fragments of SEQ ID NO: 10 (pcR) that are complementary to gB, one of the late genes of human cytomegalovirus.

By adding the primer 2, the PCR reaction competitively synthesizes DNA fragments amplified by the forward primer 1 and the anchor primer set and the forward primer 1 and the reverse primer 2 set, the amount of RNA and DNA to function as a template. It is determined by the relative amount.

DNA fragments synthesized from the RNA template or DNA template can be classified by their length. Therefore, in this case, it is natural that the position of the primer can be properly adjusted so that the length can be discerned.

The results of the RNA / DNA fractional amplification can be used to diagnose the presence of a particular organism in the clinic. For example, when primer 1 is the nucleotide fragment of SEQ ID NO: 9 (pcF), primer 2 is SEQ ID NO: 10 (pcR), and anchor primer is SEQ ID NO: 2 or 4, it will be used as a diagnostic marker indicating the infection state of hCMV Can be. If only DNA fragments are amplified from DNA, it can be assumed that hCMV infection is incubated. DNA fragments amplified from RNA indicate gB gene expression, indicating that the infection is active. In addition, when only DNA fragments amplified from RNA are amplified, it can be seen that the mRNA amount of the gB gene is 8 times the gene of the genomic DNA.

The SMART-PCR method of the present invention is described in more detail with reference to FIG. 2 as follows. This reaction proceeds in two steps in a single tube. First, reverse transcription reactions (RT) occur in the presence of DNA, RNA or DNA / RNA templates with anchor primers (SMA). The anchor portion of the anchor primers binds to a single stranded region of RNA (FIG. 2, R-1), but does not bind to a DNA region because its complementary sequence is two strands at reverse transcription temperature (FIG. 2, D). -One).

After the reverse transcription reaction is complete, a PCR mixture comprising forward primer 1 (pcF) and reverse primer 2 (pcR) is added into the same tube for subsequent PCR reactions. The forward primer 1 (pcF) initiates the synthesis of a second strand of DNA (FIG. 2, R-2). As a result, the product will contain a sequence complementary to the anchor and tail of the anchor primer (SMA), and the Tm between the second strand of DNA and the anchor primer (SMA) is sufficient to allow binding to occur even at the binding temperature of the PCR. It becomes high (FIG. 2, R-3). As a result, the anchor primers can be used as reverse primers for subsequent PCR reactions. In the case of DNA templates, forward primer 1 (pcF) and reverse primer 2 (pcR) will bind to their complementary sequences during the first PCR cycle, such as a conventional PCR reaction, to initiate polymerization. The anchor portion of the anchor primer cannot bind because the hybridization temperature of only the anchor portion is lower than the binding temperature of PCR.

The cDNA synthesized by the anchor primer (SMA) has no sequence complementary to primer 2 (pcR) because the anchor portion is about 300 bp upstream of reverse primer 2 (pcR) (FIGS. 1A, 2, R). -2, 3). Thus, in the subsequent PCR reaction, primer 2 (pcR) selectively hybridizes to DNA, while the anchor primer hybridizes selectively to RNA. Primer 1 (pcF) is commonly used for both RNA and DNA amplification during PCR reactions (FIGS. 1A, 2, R-3, D-3). As a result, primer 1 (pcF) and anchor primer (SMA) amplified the short product (156 bp) only from the RNA template, while primer 1 (pcF) and primer 2 (pcF) produced the long product (425 bp) only from the DNA template. Will be amplified. The amplified products can be easily fractionated by their size by conventional agar gel electrophoresis.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only, and the present invention is not limited to these examples.

Example

To specifically describe the modified anchor primer of the present invention, a method for selectively amplifying a DNA fragment from an RNA template in a mixed DNA and RNA template using the same, and a method for diagnosing the presence of a specific organism using the method, the human Cytomegalovirus (human cytomegalovirus, also referred to as hCMV) was selected as the specific organism. Anchor primers were designed from glycoprotein gB, one of the late genes of hCMV, and SMART-PCR was performed based on this, and a method of diagnosing the infection and clinical condition of hCMV was performed through SMART-PCR.

The hCMV belongs to the human herpesvirus family having a double stranded DNA genome. hCMV tends to remain latent infection after resolution of active infection in the body of a normal healthy person. Since such reactivation of latent hCMV leads to fatality in recently transplanted organ transplant patients or patients infected with HIV virus, clinical diagnosis is important. To this end, RT-PCR has been proposed as an apparatus for diagnosing the active state by the selective detection of mRNA which does not exist in the incubation period. It has been reported that immediate-early genes, including introns, can be easily distinguished by the size of their products and thus may be technically useful targets. However, the effectiveness of using these mRNAs as markers for active hCMV infection has been questioned since the early genes are transcribed even in non-productive latent infections. In contrast, transcription of late mRNAs occurs exclusively during active infection. Therefore, the development of late mRNA detection method will be useful for the classification of the latent and active phase. Nevertheless, late genes are not easily distinguished from genomic DNA by conventional RT-PCR because they are devoid of introns. In this example, the suitability of SMART-PCR was evaluated using gB of hCMV, one of the late genes without introns.

Materials and methods used in this example are as follows.

[material]

hCMV strain AD169 (ATCC No. VR-538), Davis (ATCC No. VR-807), Towne (ATCC No. VR-977), and human lung fibroblast MRC-5 (ATCC No. CCL-171) are American Type Purchased from Culture Collection (Rockville, MD, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, penicillin, streptomycin and PCR SuperMix High Fidelity were purchased from GibcoBRL (Rcokville, MD, USA). Wizard Plus Minipreps kit, AMV reverse transcriptase and cloning vector pSP72 were purchased from Promega (Madison, WI, USA). SP6 RNA polymerase, guanidium thiocaynate and DNase-free RNase were purchased from Roche MB (Mannheim, Germany). RNeasy Mini kit and DNase I were purchased from QIAGEN (Valencia, CA, USA). DNA blunting / ligation kits were purchased from TaKaRA-Biomedicals (Kyoto, Japan). Ampli Taq Gold polymerase was purchased from PE Applied Biosystems (Foster City, CA, USA). SYBR green II was purchased from FMC BioProducts (Rockland, ME, USA). All restriction enzymes were purchased from New England BioLabs (Beverly, MA, USA). Other chemicals were purchased from Sigma (St. Louis, MO, USA).

[Preparation of Cells and Viruses]

MRC-5 cells were maintained at 37 ° C. in a 5% CO ₂ incubator in DMEM supplemented with 10% heat inactivated fetal bovine serum, 100 IU / ml penicillin and 50 μg / ml streptomycin. When MRC-5 grew to 95% confluence, cells were infected with hCMV strains AD169 , Davis and Towne . When cytopathic effects became clear 5 to 7 weeks after infection, the supernatants and cells were collected by simple centrifugation and stored at -80 ° C.

[Lysis and binding (LB) buffer]

The resin binding solution of the Wizard Plus Minipreps kit was modified to promote rupture of the viral particles. The resin binding solution comprising guanidium chloride was mixed with 25 mM sodium citrate, 0.5% sarcosyl and 0.1M β-mercaptoethanol (final concentration). This modified LB buffer was used as rupture and binding buffer during viral nucleic acid extraction.

[Cell lysis (CL) buffer]

CL buffer is 4M guanidium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sacosyl and 0.1M β- in 0.1M tris-hydrochloride buffer (pH 6.4). Mercaptoethanol. This buffer was used for nucleic acid extraction from cells.

[Preparation of DNA and RNA Standards]

To minimize unwanted RNA transcription from pSP72, the SP6 promoter sequence of pSP72 was deleted (pSP72 / -SP6) by digesting pSP72 with Xho I and Nde I. The gB gene was obtained by PCR of viral DNA recovered from MRC-5 infected with AD169 using a PCR SuperMix High Fidelity polymerase. A primer set for gB amplification based on the sequence obtained from GenBank (access no. X04606) was synthesized (Table 1, FIG. 1A).

Table 1. Base sequences and positions of primers used in these examples

name Standing column location gB / F 5'-CCCGC G G C AT G CATG GAATC CAGGA TC-3 ' 145-171 gB / R 5'-GCACC TAG GG AT CCA GTTTA ACCCC GTATA TC-3 ' 2927-2958 pcF 5'-GTCAG CTTCC CAGCC TCAAG ATCTT C-3 ' 1985-2010 pcR 5'-CGAAG GGGTT TTTGA GGAAG GTGGC AAC-3 ' 2383-2410

In Table 1, base sequence numbers are assigned relative to AD169 (GenBank accession no. X04606). gB / F and gB / R are for amplification of the complete glycoprotein B gene. The thick, underlined base was modified from the original sequence to make BamH I and Sph I restriction sites. pcF and pcR were used for DNA amplification. pcF is also used as an anchor primer for RNA amplification with anchor primers (SMA).

PCR was performed using a thermal cycler (PCRsprint, Hybaid, Middlesex, UK) and the conditions were as follows: denaturation at 94 ° C. for 10 minutes, 30 seconds at 94 ° C., 20 seconds at 65 ° C., 72 ° C. 16 minutes for 3 minutes, 30 seconds at 94 ° C., 30 seconds at 65 ° C., 3 minutes + 15 seconds / time at 72 ° C., 12 times and 10 minutes at 72 ° C. for 10 minutes. The amplified 2.8 kb PCR product was digested with BamH I and Sph I and previously digested with pSP72 or the same restriction enzyme and cloned into pSP72 / -SP6. As a result, pSP72 and pSP72 / -SP6 / gB (FIGS. 3A, b) were recovered from E. coli DH5α, and the DNA concentration was spectroscopically determined at 260 nm (using a 10 mm optical path quartz cuvette). A ₂₆₀ value of 1 when the estimated DNA 50㎍ / ml standard DNA pSP72 / a calculated copy number of -SP6 / gB is approximately 2.0x10 ¹⁰ copies / ㎕, and its final concentration by diluting 1.0x ¹⁰ 10 copies / Adjusted to μl.

RNA standards were prepared using SP6 RNA polymerase by in vitro transcription of linear pSP72 / gB previously digested with BamH I. The RNA copy number was about 1.9 × ^{10 10} copies / μl based on A ₂₆₀ = 40 μg / ml RNA calculated above. Final concentrations of RNA standards were also adjusted to ^1.0 × ^{10 10} copies / μl.

[Virus Nucleic Acid (DNA / RNA) Extraction]

Viral RNA was extracted by a modified method from the Wizard Plus Minipreps kit. Alkaline rupture and neutralization steps included in the manufacturer's instructions were not performed. Instead, rupture of the virus and binding of resin and nucleic acid were performed in one reaction in the LB buffer prepared as above. Viral DNA was extracted from previously prepared supernatants. To remove RNA contamination, 400 μl of supernatant was treated with 20 units / ml of DNase free-RNase at 37 ° C. for 1 hour. The sample was then mixed with 1 ml of LB buffer and incubated in a rotary mixer for 10 minutes. The remaining steps followed the manufacturer's instructions except for the final step. DNA was eluted by adding 50 μl of DEPC-treated distilled water at 55 ° C. for 10 minutes to a mini-column and centrifuging at 12,000 g for 2 minutes.

Viral RNA was extracted from infected cells using the RNeasy Mini kit and DNaseI. DNaseI treatment was performed as directed in the manufacturer's instructions. The absence of DNA contamination was confirmed by PCR using primers pcF and pcR (see Table 1). PCR was performed under the following conditions: denaturation for 10 min at 94 ° C., 94 ° C. 30 sec, 65 ° C. 10 sec, 72 ° C. 30 sec 40 times and final extension for 10 min at 72 ° C. Viral DNA was removed by repeated DNaseI treatment until not detected by PCR.

For the extraction of total nucleic acid, a method modified from the Wizard Plus Minipreps kit was used. The alkaline rupture and neutralization steps in the manufacturer's instructions were replaced by a cell rupture step using the CL buffer prepared above to facilitate the release of nucleic acid from the cell. 400 μl of CL buffer was added to previously prepared cell pellets, and the samples were incubated for 10 minutes at room temperature in a rotary mixer. The sample was then mixed with 1 ml of resin binding solution and further purification steps were performed according to the manufacturer's instructions.

Example 1

[Design of Anchor Primer]

Glycoprotein B (gB, gpUL55) of hCMV was selected as the target for optimization of SMART-PCR of the present invention. One region of gB gene with high homology between AD169 (GenBank accession no. X04606) and Towne (GenBank accession no. M22343) was selected for amplification. To amplify this portion, a common forward primer pcF (SEQ ID NO: 9) and a reverse primer pcR (SEQ ID NO: 10) were selected (Table 1, Figure 1A).

Size modifying anchor primers (SMA primers) in SMART-PCR are key components and serve as two functions as initiators starting cDNA synthesis during reverse transcription (RT) and subsequent primers during subsequent PCR. (FIG. 2). This primer consists of an anchor portion at the 3 'end and a tail portion at the 5' end (FIGS. 1A and 1B). The anchor portion allows the anchor primer (SMA primer) to specifically bind to RNA during the reverse transcription reaction to initiate cDNA synthesis. The tail portion increases the hybridization temperature of the anchor primers, allowing them to function as reverse primers for subsequent PCR reactions that proceed at higher temperatures than reverse transcription reactions. The sequence complementary to the anchor was designed to amplify the short product by positioning it in the same direction about 300bp upstream from the pcR. The anchor binding sequence was allowed to be selected from RNA regions that remained single stranded during reverse transcription in order to minimize nonspecific binding to DNA. For this purpose, the secondary structure of the target RNA at the reverse transcription reaction temperature (42 ° C.) was drawn using RNA draw (v1.1b2), and an open loop region was selected as the anchor binding site (FIG. 1B). . Unlike RNA in single stranded state, the DNA corresponding to this region is double stranded at reverse transcription temperature (37-45 ° C). Therefore, there is no free DNA base to be used for binding the anchor portion of the anchor primers during the reverse transcription reaction.

The sequences and calculated Tm values of the selected anchor primer candidates (SMA candidates or SMACs) are shown in Table 2.

Table 2. Sequence and Tm values of the SMA candidate primers of this example

name Standing column Tm value of anchor (℃) TM value (℃) of primer SMAC1 5'-GTCGTCGTCGTAT TATTTTCCAGC -3 ' 36.60 69.96 SMAC2 5'-GTCGTCGTCGTAT TATTTTCCAGCGG -3 ' 45.07 72.72 SMAC3 5'-GTCGTCGTCGTAT TATTTTCCAGCG -3 ' 40.55 71.47 SMAC4 5'-GTCGTCGTCGTATAT TTTTCCAGCGG -3 ' 43.80 72.72 SMAC5 5'-GTCGTCGTCGT GGTATTTTCCAGC -3 ' 47.08 73.92 SMAC6 5'-GTCGTCGTCGCGGTAT TATTTTCCAGC -3 ' 36.60 74.26

In Table 2 above, the thick, underlined sequences are complementary to the anchor and are located within the open loop region (base positions from 2116 to 2128 of GenBank accession no. X04606). The anchor portion is 11 to 13 bases in length, and its calculated Tm value varies depending on its sequence and length, but is calculated by the method of Sugimoto et al. (Sugimoto et al., 1996, Nuc Acids Res., 24). , 4501 to 4505. After adding a random tail at the 5 'end of the anchor portion, the final Tm value was calculated to be 69.6 to 73.92 ° C (calculated by Allawi et al.). (Allawi et al., 1997, Biochemistry., 36, 10581-10594) .The irrelevant random tail sequences are non-specifically hybridized or self-priming to DNA or RNA templates during reverse transcription or subsequent PCR reactions. Designed.

Example 2

[Perform of SMART-PCR]

1. Preparation of gB Standard DNA / RNA Templates Used for SMART-PCR

The complete gB gene of hCMV strain AD169 was cloned into pSP72 (pSP72 / -SP6 / gB) without the SP6 promoter and used as standard DNA (FIG. 3A). At the same time a similar construct (pSP72 / gB) comprising SP6 was prepared, used as template for in vitro transcription and the product used as standard RNA (FIG. 3B). Suitable RNA transcripts were specifically synthesized by in vitro transcription in the presence of pSP72 / gB, but not in the presence of pSP72 / -SP6 / gB. The size of the transcript was confirmed by 1% formaldehyde modified gel electrophoresis and stained with SYBR green II (FMC BioProducts, Rockland, ME, USA), as expected, 2.8kb (Fig. 3c).

2. Reaction Condition of SMART-PCR

SMART-PCR was performed in two stages in a single tube. Reverse transcription reaction mixture (20 μL) was prepared with buffer (10 mM Tris-Cl and 50 mM KCl, pH 8.3), 1 mM NTPs (dATP, dTTP, dCTP and dGTP respectively), 25 pmole anchor primer, 2 unit AMV reverse transcriptase (Promega Company, Madison, WI, USA) and template (control or extracts). The conditions for reverse transcription carried out by a thermal cycler (PCRsprint, Hybaid, Middlesex UK) are as follows. Reverse transcription for 60 minutes at 42 ° C., denaturation at 90 ° C. for 5 minutes and cooling at 4 ° C. for 5 minutes.

A characteristic of the reaction is that it does not undergo a full denaturation step prior to the reverse transcription reaction in order to prevent denaturation and strand separation of the DNA, which may cause nonspecific DNA amplification.

After the reverse transcription reaction step, 30 μl of PCR mixture was added directly to the reaction tube. The PCR mixture was buffered (10 mM Tris-Cl and 50 mM KCl, pH 8.3), 50 pmole pcF, 25 pmole pcR and 1 unit Ampli Taq Gold polymerase (PE Applied Biosystems, Foster City, CA, USA). In the initial starting phase of PCR, a hot start polymerase was used to eliminate the opportunity for the anchor portion to bind nonspecifically to DNA. The conditions of the PCR reaction were as follows: denaturation at 95 ° C. for 10 minutes and activation of Ampli Taq Gold, 94 ° C. 30 sec, 68 ° C. 10 sec, 72 ° C. 30 sec 40 times and final extension at 72 ° C. for 10 min. Electrophoresis of the final product was performed on a 2% agar gel prestained with 0.5 μg / ml ethidium bromide in TAE buffer.

3. Results of SMART-PCR

SMART-PCR with RNA template resulted in a short product produced exclusively except for SMAC3 (data not shown). In the case of SMAC3, although the estimated Tm of the anchor portion is higher than SMAC1 (Table 2), it may be because during the reverse transcription reaction, the 3 ′ end portion is covered by its tail sequence, preventing the anchor from binding to RNA. Thus, SMAC3 was excluded as a primer. In addition, SMAC1 was also excluded due to the low hybridization capacity for RNA templates. It was determined that the anchor portion of all other SMAC primers could effectively bind to the RNA and be used as anchor primers.

In order to investigate the specificity of the SMAC primers, SMART-PCR was performed in the presence of DNA only. The short product was weakly amplified with SMAC2 and SMAC4 and strongly amplified with SMAC5 and SMAC6. For SMAC5 and SMAC6, these short products were often amplified even when not undergoing reverse transcription reactions (data not shown). Based on the above results, SMAC2 and SMAC4 were selected as anchor primers. In addition, these primers were used to further improve reaction conditions to increase specificity for RNA during reverse transcription reactions.

Example 3

[Optimization of SMART-PCR Response]

DNA can be partially denatured even if double stranded, allowing SMAC2 and SMAC4 to amplify the short product from DNA. In addition, magnesium ions have been known to weaken the repulsive force between nucleic acid strands caused by the phosphorus skeleton, which is a negative charge, thereby increasing the Tm value.

Based on these two facts, it was attempted to increase the binding specificity of SMAC2 and SMAC4 to RNA by adjusting the concentration of MgCl _{2 in} the reverse transcription reaction mixture.

As a result, when SMART-PCR was performed at various MgCl ₂ concentrations (1 to 5 mM) during reverse transcription using SMAC4, the MgCl ₂ concentration is preferably about 1 to 1.25 mM (FIG. 4A). Non-specific binding to anchor DNA occurs at concentrations above 2.5 mM (FIG. 4A), and reverse transcription does not occur at concentrations below 0.5 mM. The ability of SMAC4 to bind to RNA and the efficiency of polymerization by reverse transcriptase did not change at different MgCl ₂ concentrations (FIG. 4A).

SMAC2, like SMAC4, was also hybridized to RNA templates as effectively and specifically. However, SMAC2 has a higher anchor Tm value than SMAC4 (Table 2). High Tm values can be detrimental to obtaining the specificity of RNA for DNA because it can lead to nonspecific amplification of short products from DNA. In this regard, SMAC4 is a more preferred anchor primer for SMART-PCR than SMAC2. Hereinafter, SMAC4 was used for the optimization experiment, and the concentration of MgCl ₂ during the reverse transcription reaction was 1 mM.

The binding temperature of the PCR reaction was optimized with SMAC4 and PCR primer sets (pcF and pcR). Nonspecific amplification by the anchor primers can occur when the binding temperature is below 68 ° C. and above this temperature the specificity is too low.

SMART-PCR was performed in the presence of DNA and RNA and the following optimized factors were used: SMAC4, 1 mM MgCl ₂ during reverse transcription and binding temperature 68 ° C. during PCR. Under these conditions, the short product was observed when the reverse transcription reaction was performed in the presence of RNA (FIG. 4B, RNA +, RT +). This indicates that the short product was produced by the anchor primers during the reverse transcription reaction. On the other hand, the long product was amplified in the presence of DNA regardless of the presence of reverse transcriptase (FIG. 4B, DNA +, RT +/−). This means that the anchor portion of the anchor primer did not bind DNA during reverse transcription and PCR reactions. When performed in the presence of DNA and RNA, only the long product was detected in the absence of reverse transcription and both bands were observed in reverse transcription (Figure 4b, DNA +, RNA +). Both products were recovered after electrophoresis and the sequence was confirmed by DNA sequencing.

Example 4

[Diagnostic method of hCMV infection using SMART-PCR]

To this end, in order to evaluate the sensitivity of SMART-PCR SMART-PCR was performed with 10 to ¹⁰ copies of DNA or RNA as a 10-fold serial dilution. The detection limit was found to be about 10 ² copies for both RNA and DNA.

As expected from the clinical state, when both DNA and RNA are present, there is competition between anchor primers and pcR during PCR. In this regard, the limit of detection for RNA in the presence of DNA was investigated. The SMART PCR-fixed amount of DNA (1x10 ⁵ copies) with the change (2-fold serial dilutions in the range of ^2-7 2 ⁴ x10 ^5), the amount of RNA that was carried out in the presence.

S As a result, the product has been detected by the short to the ratio of 1/128 of RNA / DNA, two distinct bands were easily observed when the ratio is higher than 1/32 (right lane in Fig. 5, RNA 2 ^-5 ).

This relative quantification pattern can be used as a status marker for hCMV infection in clinical conditions. If only the long product was amplified, the infection of hCMV would be incubated. If the short product is observed, this means that gB gene expression is taking place and thus the infection is active. Moreover, if the amount of mRNA of gB is higher than 8 times the viral genomic DNA, only this short product will be detected (Figure 5, lanes to the left of RNA 2 ³ ).

Example 5

[Diagnosis of hCMV Infection Using SMART-PCR of MRC-5 Infected with hCMV]

In order to further confirm the suitability of the present invention for the diagnosis of active viral infection, SMART-PCR was performed with DNA, RNA and total nucleic acids (DNA and RNA) extracted from MRC-5 infected with hCMV strains AD169, Davis and Towne . . Total nucleic acid extracted from uninfected MRC-5 was used as a negative standard. All strains showed the same pattern as the DNA and RNA standards (Figure 4b, Figure 6). The short product bands were only amplified when they were amplified with reverse transcription (Figure 6, RNA +, RT +). The long product bands were observed in the presence of DNA regardless of the reverse transcription reaction (FIG. 6, DNA +). Both bands were observed in the presence of total nucleic acid (FIG. 6, DNA +, RNA +, RT +).

Taken together, these results clearly show that SMART-PCR can distinguish mRNA of the intron-free gB gene from its corresponding DNA.

According to the RT-PCR (SMART-PCR) using the anchor primer of the present invention, it is possible to distinguish the product amplified from RNA and DNA regardless of the presence of introns.

According to the diagnosis method of the infection using the RT-PCR (SMART-PCR) method using the anchor primer of the present invention, the infection can be confirmed even for the gene without intron.

In addition, according to the RT-PCR (SMART-PCR) using the anchor primer of the present invention, RNA does not need to be separated from DNA, and RNA and DNA can be simultaneously separated and detected in a single tube, and the relative amount thereof can be detected. You can decide.

Claims (14)

The modified anchor primer, which is a DNA fragment having an anchor region having a sequence complementary to an RNA region at 3 'end and a tail region having a random sequence at 5' end. The modified anchor primer of claim 1, wherein the binding RNA region is present in a single strand during reverse transcription at a reaction temperature of 37 ° C. to 45 ° C. 3. The modified anchor primer according to claim 1, wherein the anchor portion comprises 11 to 13 bases, and the estimated Tm of only the anchor portion is about 36 to 48 ° C. The modified anchor primer of claim 1, wherein the total estimated Tm of the anchor primer is about 69-74 ° C. The size modified anchor primer of claim 1, wherein the tail region does not hybridize specifically to a DNA or RNA template and does not form self-priming during reverse transcription and subsequent PCR reactions. The size-modified anchor primer of claim 1, wherein the size-modified anchor primer has a nucleotide sequence of SEQ ID NO: 2 (SMAC2) or 4 (SMAC4). A method for selectively amplifying DNA from an RNA template in a sample containing RNA comprising the following steps. (a) preparing RNA for use as the anchor primer and template of any one of claims 1 to 5; (b) using the anchor primer and performing a reverse transcription reaction without undergoing a pre-denaturing step; (c) after the reverse transcription reaction, adding a PCR mixture including a forward primer (primer 1) corresponding to the anchor primer directly to the reaction vessel and performing a PCR reaction. The method of claim 7, wherein The reverse transcription reaction of step (b) uses 20 μl of a reverse transcription reaction mixture containing 25 picomol of the anchor primer, and the reaction conditions are denatured for 60 minutes at 42 ° C. and 5 minutes at 90 ° C. denaturation) and cooling at 4 ° C. for 5 minutes, PCR reaction conditions of step (c) is 10 minutes at 95 ℃, 40 times 94 ℃ 30 seconds, 68 ℃ 10 seconds, 72 ℃ 30 seconds and 72 ℃ 10 minutes, characterized in that consisting of the final extension of 10 minutes A method for selectively amplifying DNA from an RNA template in a sample containing. The method of claim 7 or 8, wherein the reverse transcription reaction mixture of step (b) comprises amplifying DNA selectively from an RNA template in a sample containing RNA, characterized in that it comprises about ₁ to 1.25 mM MgCl ₂ How to let. Method of diagnosing the infection of a specific organism using the method of claim 7 or 8. The method of claim 10, wherein the method is (1) The specific organism is a human cytomegalovirus, the anchor primer of step (a) is the nucleotide fragment of SEQ ID NO: 4, and the RNA to be used as a template is the mRNA of human cytomegalo virus, and (c) The primer corresponding to the anchor primer of the step is a step of selectively amplifying DNA derived from the mRNA of the human cytomegalo virus using the base fragment of SEQ ID NO: # (pcF); And (2) diagnosing the infection of human cytomegalovirus according to the formation of the selectively amplified DNA product. The method of claim 10, wherein the method is (1) The specific organism is a human cytomegalovirus, the anchor primer of step (a) is the nucleotide fragment of SEQ ID NO: 4, and the RNA to be used as a template is the mRNA of human cytomegalo virus, and (c) The primer corresponding to the anchor primer of step is a nucleotide fragment of SEQ ID NO: # (pcF), the PCR mixture is derived from the mRNA and DNA of the human cytomegalo virus further comprising a nucleotide fragment of SEQ ID # # (pcR) Selectively amplifying the DNA; And (2) diagnosing the infection of human cytomegalovirus and its clinical condition according to whether the selectively amplified DNA product is formed and its aspect. Human cytomegalovirus consisting of SEQ ID NO: # (pcF) base fragment specifically complementary to glycoprotein B, one of the late genes of human cytomegalovirus, and SEQ ID NO: 4, a sized anchor primer hCMV) diagnostic primer set. Base fragment of SEQ ID NO: # (pcF) complementary to gB (glycoprotein B), one of the late genes of human cytomegalovirus; The nucleotide fragment of SEQ ID ## (pcR); A set of primers for diagnosing human cytomegalovirus (hCMV) consisting of a size modified anchor primer of SEQ ID NO: 4 (SMA).