Title of Invention

A MULTIPLEX PCR METHOD FOR RAPID AND SPECIFIC IDENTIFICATION OF CYTOMEGALOVIRUS

Abstract A multiplex PCR method for rapid and specific identification of cytomegalovirus in which nucleotide sequences of morphological transforming region II, (ORF -79 ), UL-83 gene coding for the 65 k dalton phospho protein (pp 65 ) and Glycoprotein O gene (coding for the envelope protein Glycoprotein O) which has genome specific sequences for Cytomegalovirus,, are used as a target sequence, said method simultaneously amplifying at least three target genes in a single tube through a single reaction using four oligonucleotides for at least two target genes to be amplified, and at least two oligonucleotides for the third target gene.
Full Text

This invention relates to a multiplex PCR method for rapid and specific identification of cytomegalovirus.
Technical Field The present invention relates to a new multiplex polymerase chain reaction (PCR) method and PCR reaction mixture for rapid and specific identification of Cytomegalovirus, and more particularly, to a multiplex PCR method capable of detecting all the CMV strains thereof in a single PCR along with semiquantitatton of the viral load using genome-specific primers derived from the morphological transforming region II (mtr f i \ UL-83 gene and Glycoprotein O (gO gene) sequences of Cytomegalovirus.
Conventional method of identification includes Fluorescent Antibody Technique (FAT) which is highly subjective, less sensitive and requires a high antigen threshold. The other conventional method routinely used is virus isolation which is highly laborious and time consuming. This has provided impetus for the nucleic acid amplification methods such as Polymerase chain reaction (PCR) to be used. PCR methods are rapid and reliable in the detection of Cytomegalovirus..
Several primers are available for the detection of Cytomegalovirus. Though PCR has been widely used for rapid diagnosis of CMV infection, failures to detect CMV from specific populations owing to primer and target mismatches have also been attributed to the sequence variations in CMV genome.
Sequence variations occur in almost every region of the CMV genome as suggested by the restriction enzyme digestion assays. The diversity of organs and cell types infected by HCMV in vivo has led to hypothesis that HCMV disease and tissue iropism may be related to sequence variation among the strains.
Most of the PCRs developed for the detection of CMV target a single region of the CMV genome which may not be sufficient to detect the DNA in all the strains. The high degree of strain variations in the CMV genome and high degree of sensitivity required to detect the CMV DNA necessitates the use of multiplex PCR (mPCR) in which two or more

target sequences arc simultaneously amplified in the same reaction. mPCR targeting the Immediate early (BE) and Late antigen (LA) genes have been standardized and applied but it consists of a single round and utilizes Polyacrylamide gels with silver staining tor viewing the amplified products tor improved sensitivity which may be expensive as a diagnostic test. Researchers have standardized another Qi^driplex PCR but the value of its application onto the clinical specimens is not known.
To meet the necessity we combined the individual primer pairs targeting three regions of the CM V genome viz - the morphological transforming region H (mtr 11) - ORF 79-, the til ,-83 gene coding for the pp 65 antigen and-UL -74 gene coding for glycoprotein O (gO gene )in a multiplex format.
The use of three primer pairs also endows the assay with an element of quantitative discrimination.; sine© samples with small amounts of viral DNA are mare likely to lead to amplification of only one or two of the 3 targets.
To solve the above-described problems, it is a first object of the present invention to provide a multiplex polymerase chain reaction (PCR) method capable of amplifying &t least three target genes in a single PCR using a mimimini mttflber of primers.
It is a second object of the present invention to provide a PCR reaction mixture for use in the new PCR method in which detection of Cytomegalovirus genome and semiquantitation of the viral load can be achieved at the same time through a single PCR using a minimum number of primers.
ft is a third object of the present invention to provide the oombination of oligomidwtide primers for use in a new multiplex PCR method in ^vhich detection of Cytomegalovirus genome and semiquantitation of the viral load can be achieved at the same time through a single PCR using a minimum number of primers.
The first object of the present invention is achieved by a PCR method for simultaneously amplifying at least three target genes in a single tube through a single reaction using four

oligonucleotides for at least two target genes to be amplified, and at least two oligonucleotides for the third target gene.
The second object of the present invention is achieved by a PCR reaction mixture comprising four oligonucleotides for at least two target genes to he amplified, and at least two oligonucleotides for the third target gene in specific proportions simultaneously reacted in a single tube.
The third object of the present invention is achieved by optimizing the concentration of the primers along with the other components of the PCR reaction mixture for the simultaneous detection of all the three regions and to semiquantitate the viral load.
According to the present invention, a multiplex polymerase chain reaction (PCR) method is developed for identifying Cytomegalovirus and diagnosing infections caused by the same, in which nucleotide sequences of morphological transforming region II, (ORF -79), UL-83 gene coding for the 65 k dalton phospho protein (pp - 65 ) and Glycoprotein O gene (coding for die envelope protein Glycoprotein O) which has genome specific sequences for Cytomegalovirus, are used as a target sequence.
in other words, from the mtr II, UL-83 and g 0 region which contain both conserved and polymorphic sequences for Cytomegalovirus, a genome specific primer having the conserved sequence for the Cytomegalovirus is selected from literature.
These primers are selected such that PCM products obtained using the primers have different sizes for easy discrimination thereof by gel electrophoresis. All six primers are simultaneously applied to a single PCR to detect the Cytomegalovirus genome.
In general, one primer pair is used in a single reaction for detection of one target strain. Thus, 3 individual PCR reactions are required to detect three trargets using three separate PCRs. Thus the number of reactions squired can be reduced by ttwtiipkx PCR. In this asp&Vthe j^*h>ers werfc selected -having the same reaction conditions, which allows the primers to be simultaneously applied to the same PCR. As # result, detecting ifo?

primers can be realized. «*«VK
The multiples PCR method according to the present invention for detection and Nictitation of the Cytomegalovirus genome through a single reaction uses eight
pnmersmcludiflgau„iplexPCRForUL-S3andnestedrou„dSformtrHandgOg^ When standardizing the PCR, concentrations of template DNA, primers, dNTF, and MgCl2 reaction temperature, and reaction time, should he appropriate.
For the multiplex PCR method according to the present invention for detecting the Cytomegalovirus genome and semiquantification of the viral load through a single reaction by combination of appropriate primers, the reaction conditions should be fmther restricted. In addition, the primers should be selected such that the amptifieatioa products have different sizes and can be distinguished from one another oa a gel after the PCR.
The eight primers for use in detecting the Cytomegalovirus genome and semtquaatifcttton of the viral load according to the present invention are selected to be appropriate for die multiplex PVR method. All six primers of the first round amplification can be simultaneously applied to a single PCR, and thus the Cytomegalovirus genome of all the strains can be detected through a single PCR with high sensitivity. The PCR primers according to the present invention and the sizes of PCS products obtained using the primers are shown in Table 1.



The present invention will be described in greater detail by means of the following examples. The following examples are for illustrative purposes and are not intended to limit the scope of the invention.
Example 1; CMV AD 169 strain passaged onto WI-38 lung fibroblast cell line was obtained from CMC, Vellore. DNA of the AD 169 strain was isolated using the Biogene DNA extraction kit (BIOGENE Reagents Inc, CA, USA). Manufacturer's instruction was strictly followed. 100 j.il of the culture harvest of CMV AD 169 strain was lysed in the 200 ul of digestion buffer and 3-.jU of proteinase K and incubated at 56 deg. C for 5 minutes followed by adsorption of the DNA in the solution on to the silica column, washed twice with me washing solution provided and eluted with 100 uJ of elution buffer.
Example 2 : Preparation of Primers for use in the PCR
Example 3 : Determination of optimal primer concentration by testing various combination of primer dilutions of the three sets of primers of the first round and two sets of primers in the second round.
Example 4: The primer sets of the individual PCRs were combined and used in a nested mPCR reaction. For amplification a.50pl reaction was set with 240pM of each dNTPs, Ix PCR buffer ( 10m M Tris ~ HC1 (pH 8.3), 50mM KCl, 0.01% gelatin, 1.5raM MgCU.) and 2 units of Taq DNA polymerase. Two micromoles of each primer for gO gene , 0.1 uM of each primer of mtr H region and 0.2 uM of each primer of UL 83 were added. 10 ill of extracted DNA was added as template. The first round thermal profile consisted of an initial denataration at 94°C for 5 min for J cycle followed by 40 cycles each

consisting of denaturation at 94°C for 45 sec, annealing at 60"C for 45 sec sec and extension at 72°C for 45 followed by a single cycle of find extension at 72 deg. C for 10 min.
For the second round only the primers coding for mtr IT and gO gene were used. A SOp.1 reaction was set with 200uM of each dNTPs, Ix PCR buffer ( 10m M Tris - HC1 (pH 8.3), 50mM KCl, 0.01% gelatin, 1.5mM MgCl2) and 2 units of TaqDNA polymerase. 20 mM of each primer lor gO gene and 0.1 mM of mtr H gene was added and used. From the first round 2^1 of the amplified products were used as template for the second round. The profile consisted of an initial denaturation at 94^C for 5 min for 1 cycle followed by 20 cycles each consisting of denaturation at 94°C for 45 sec, annealing at 60°C for 45 sec and extension at 72°C for 45 see followed by a single cycle of final extension at 72°C fof 10 min.
Detection of the amplified product:
The amplified products were analyzed by gei electrophoresis using 4 per cent agarose containing 0.5 mg/ml of etbidiuro bromide. Electrophoresis was carried out at 100 V for 30-45 min. The gel was visualized on U V transilluminator at 302 am wavelength
Example 5 : SPECIFICITY OF mPCR:
The specificity of the mPCR was determined against relative viruses (HSV, VZV and EBV) and human DNA extracted from the leucocytes. The primers targeting the 3 regions of CMV were specific to CMV genome by mPCR as tbey did not amplify genomes of other organisms against which they were-tested.
Example 6: SBNSIT1 VITV OF mPCR:
The analytical sensitivity of the mPCR was determined using serial 10 - fold dilutions of DNA(0.5|xg initial concentration as determined by abosorbance at 260 nm> extracted from AD 169 infected diploid ce?l line (MRC -5 cell line supplied by National Centre for



costs along with savings in time and labour. The multiplex PCR method according to the present invention can identify all the strains of CMV through a single method, and thus it can he efficiently applied for the diagnosis of CMV infections.


We Claim:
1.A multiplex PCR method for rapid and specific identification of cytomegalovirus in which nucleotide sequences of morphological transforming region II, (ORF -79 ), UL-83 gene coding for the 65 k dalton phospho protein (pp 65 ) and Glycoprotein O gene (coding for the envelope protein Glycoprotein O) which has genome specific sequences for Cytomegalovirus,, are used as a target sequence, said method simultaneously amplifying at least three target genes in a single tube through a single reaction using four oligonucleotides for at least two target genes to be amplified, and at least two oligonucleotides for the third target gene.
2. A method as claimed in Claim 1 wherein The PCR primers and the sizes of PCR products obtained using the said primers are as illustrated in fable 1
3. A composition comprising a combination of oligonucleotides as set out in the preceding Claims 1 and 2,
4. A kit comprising a composition as claimed in Claim 3.
S.ArnuUiplex PCR method for rapid and specific identification of Cytomegalovirus, substantially as herein described with reference to, and as illustrated by, the lixampics.
5. A multiplex PCR method for rapid and specific identification of cytomegalovirus

substantially as herein described with reference to and as illustrated by the Examples, Dated this the 12th day of July 2GG5


Documents:

935-CHE-2005 CORRESPONDENCE OTHERS 22-07-2011.pdf

935-che-2005-claims.pdf

935-che-2005-correspodance others.pdf

935-che-2005-description(complete).pdf

935-che-2005-form 1.pdf

935-che-2005-form 26.pdf

EXAMINATION REPORT REPLY.PDF


Patent Number 246970
Indian Patent Application Number 935/CHE/2005
PG Journal Number 35/2009
Publication Date 28-Aug-2009
Grant Date 20-Aug-2009
Date of Filing 12-Jul-2005
Name of Patentee VISION RESEARCH FOUNDATION
Applicant Address SANKARA NETHRALAYA 18 COLLEGE ROAD CHENNAI 600 006 TAMIL NADU, INDIA
Inventors:
# Inventor's Name Inventor's Address
1 HAJIB NARAHARIRAO MADHAVAN SANKARA NETHRALAYA 18 COLLEGE ROAD CHENNAI 600 006 TAMIL NADU, INDIA
2 PARAMESWARAN SOWMYA SANKARA NETHRALAYA 18 COLLEGE ROAD CHENNAI 600 006 TAMIL NADU, INDIA
3 KULANDAI LILY THERESE SANKARA NETHRALAYA 18 COLLEGE ROAD CHENNAI 600 006 TAMIL NADU, INDIA
PCT International Classification Number C12O 01/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA