Polymerase Chain Reaction for Detection and Genotyping of Molluscum Contagiosum Virus in Diyala Province

Background: Molluscum contagiosum virus (MCV): is a viral skin infection which may infect themucous membrane and skinoccasionally, It is caused by Molluscipox virus from family Poxviridae. Molluscum contagiosumvirus (MCV) was first described and later assigned its name by Bateman in the beginning of the nineteenth century. Aim: This study was done to confirm the clinical diagnosis of MCV by laboratory test through using PCR assay andto know the predominate type of MCV that found in Diyala Province. Patients, and methods:The present study was conducted for the period from1 November 2011 to 30 April of 2012 in outpatient clinic of Baquba Teaching Hospital in Baquba city. The study aimed to confirm the clinical diagnosis of MCV by laboratory test through using PCR assay and to know the domain subtype of MCV that found in Diyala province Sixty (60) patients were diagnosed with clinical lesions of MCV on different areas of the body, their age ranged from(1-80 years) including 40(66.7%) males and 20(33.3%) females, and the lesion samples were taken and examined by PCR Results: After testing by PCR, 51(85%) of patients gave positive results for MCV,30(58.8%) patients gave positive results for MCV type 1( 26.7%) and 2 (73.3). The results showed 23(45.1%) with age group (31-40 years), included 36(70.6%) were males and 15(29.4%) females, no significant difference showed between the MCV infection and either the sex or age . The results revealed that MCV type 2 was more prevalent 22(73%) compared with MCV type 1(26.7) ,most of type 2 (73.3) infected males 14(46.5%) , and found in age group (31-40 years),while the MCV type 1 was equally affecting males and females , consisted of (100%) in age ( ≤10 years) ,with significant difference recorded between the types and age, but no significant difference between the types and the sex. Conclusions: 85%of examined patients with MC showed postive PCR results for MCV prevalence of MCV type 1 with high in children of age group (≤10) .MCV type 2 was predominately seen in (31-40) patient age group. key word : Molluscum Contagiosum Virus, PCR, Subtype. College of Science/ Diyala University/ Diyala/ Iraq. Polymerase Chain Reaction for Detection and Genotyping of Molluscum Mohammed Khalifa AL –Azawi Contagiosum Virus in Diyala Province Diyala Journal of Medicine 34 Vol. 4, Issue 1, April 2013 Introduction Molluscum contagiosum (MC): is a viral skin infection which may infect the mucous membrane occasionally. It is caused by Molluscipox virus from family Poxviridae. Molluscum contagios umvirus (MCV) was first described and later assigned its name by Bateman in the beginning of the nineteenth century cited by [1] In 1841 Henderson and Paterson described the intracytoplasmic inclusion bodies, now known as molluscum or Henderson-Paterson bodies.In the early twentieth century, Juliusberg, Wile, and Kingery were able to extract filterable virus from lesions and show transmissibilitycited by [2]. MCV has no animal reservoir, infecting only humans and there are four types of MCV, MCV-1,MCV-2,MCV-3 and MCV-4. MCV -1 was the most prevalent predominantly seen in children and MCV -2 was seen usually in adults and often sexually transmitted [3]. The diagnosis of MCV was usually done clinically. They need for laboratory diagnosis of MCV was speculative,because a spontaneous healing was observed in cases where no underlying immune defect is present, the disease was considered as a selflimiting condition. However, there were several lines of reasoning where medical intervention and treatment might be beneficial. Though molluscum cannot be cultured in the laboratory.Histological examination of a curetted or biopsied lesion can also used in the diagnosis in cases that are not clinically clear. The thick white central core can be expressed and smeared on a slide and left unstained or stained with Geimsa or Gram stains to demonnnstrate the large brick-shaped inclusion bodies. Electron microscopy has also been used to demonstrate MCV structures. Immunohistochemical methods' using a polyclonal antibody allows recognition of molluscum contagiosum virus in fixed tissue. In-situ hybridization for MCV DNA has also been utilized [4,5]. The best option for the definitive diagnosis of MCV was PCR-based assays. An additional benefit of molecular diagnosis was the results provide information about the type of the infecting molluscum strain. No molecular data have been reported in the literature regarding prevalence of MCV types in Iraq . Thus, in this study we attempted to document the feasibility of DNA amplification-based assay in laboratory.The aims of this study To confirm the clinical diagnosis of MCV by laboratory test through using PCR assay, and to know the predominate type of MCV that found in Diyala Province. Patients and Methods A.Collection of samples:This study was conducted in outpatient Clinic of Dermatology of Baquba Teaching Hospital as across section study including all patients attending in the period between 1 November 2011 to 3o April 2012. The collection of patients sample was done in dermatology unit after diagnosis by dermatologists the cases and sample was submmited to PCR diagnosis. The demographic information include age, sex, address, educational status, and last the number and the distribution of lesions present was recorded. The lesion from each patient was curetted and placed in 5 ml phosphate buffered saline, pH 7.1, and immediately transported to the laboratory. The samples were stored at -37 ̊C until the extraction of the DNA. Conventional Polymerase chain reaction was used to detect the Molluscum contagiosum virus, and resection enzyme Bam.HI to type of virus . Sixty (60) samples Polymerase Chain Reaction for Detection and Genotyping of Molluscum Mohammed Khalifa AL –Azawi Contagiosum Virus in Diyala Province Diyala Journal of Medicine 35 Vol. 4, Issue 1, April 2013 were selected depending on the size of lesions of the patient, with lesion not less than 30mg depended on method as described by geneaid compony B. DNA Extraction:Sixty samples were selected depending on their size and after neglecting the PBS, the DNA extraction and purification as instructed by the Geneaid company was done. C. Primer selection: Table (1) showed two sets of primers that were used in the study as suggested by [6]. Table (1): primers used in the study. Set No. Primer No .of bp. Company 1 F1(5-GGCGCGTAGCCGAGCGG-3) R1(5 CTTCCGGGCTTGCCGCCGGGCAG-3) 393-bp Bioneer 2 KU (5-GGAGGAGTGCCCATCAAGAAT-3) OR (5-GCTTTTCAGTTTTTGTGCGA-3) 575-bp Bioneer NO. bp: Number of base pair The first primers F1 and R1 amplify 393base pair (bp) portion of p43K polypeptide from MCV genome whereas KUF and OR primers amplify 575-bp-long region from p43K polypeptide of MCV genome. KU and OR primers amplified region containing BamH1 restriction site in sub-type genome of MCV, thus allowing accurate subtyping of the infecting strain. D.Polymerase Chain Reaction (PCR) Conventional PCR was the method that applicated on our samples to obtain result. Doubled polymerization process have been performed twice , first PCR to use a primer of first set ( F1 and R1) which was used for the diagnosis of molluscum contagiosum and saw any positive or negative lesions, the second set another detection to MCV and find out with kind of molluscum contagiosum was prevalent by using restricted enzyme Bam.H1 after amplified region allowing to digestion. Doubled amliphcation had already been done by (7) with simple modification . 1-The first thermo cycle PCR process include :1-Two micro liters of isolated DNA was added to 0.2 of a PCR Pre mix. .PCR Pre mix.kit was selected from bioneer (bioneer/ korea). 2This mixture(Table 3.5.) containing 10 μM Tris-HCl (pH 8.3), 30 μM KCl, 1.5 mM MgCl2,each deoxynucleoside triphosphate at a concentration of 250 μM,1U of Taq polymerase, 30 pmol (picomol ) of primersF1 and R1,and the mixture was complete to 20 micoleter Deionize distill water ( D.D water) . 3-The samples were used with a thermal phases involving initial denaturation at 95°C for 1 min and 40 cycles consisting of denaturation at 95°C for 1mint , annealing at 58°C for 1 min, and extension at 72°C for 1 min, after complete the thermo cycle, extension at 72°C for 5 mint and finally hold the reaction at 4°Cfor 5 mint . 4The amplification reactions were visualized on a 1.5 % agarose. Polymerase Chain Reaction for Detection and Genotyping of Molluscum Mohammed Khalifa AL –Azawi Contagiosum Virus in Diyala Province Diyala Journal of Medicine 36 Vol. 4, Issue 1, April 2013 2.B.Asecond PCR amplification and Bam.HI digestion of amplified products:1-Three microliters of isolated DNA was added to 25μl of a Green Master Mix ,PCR reaction kit was selected from the (promega /USA). 2-This mixture containing 10 m MTris-HCl ( pH 8.5 ), 3 mM MgCl2, each deoxynucleoside triphosphate(dATP, dGTP, dCTP, dTTP) at a concentration of 400 μM, 1.25 U of Taq polymerase, 30picomol( pmol) of primers KuF and OR1, and the mixtures was completed to 50 microliter Deionized distilled water ( D.D water) . 3-The samples was used with a thermal phases involving initial denaturation at 95°C for 1 min and 40 cycles consisting of denaturation at 95°C for 1mint , annealing at 58°C for 1 min,and extension at 72°C for 1 min, after complete the rmocycleextention at 72°C for 5 mintand, hold the reaction at 4°Cfor 5 mint . 4The amplification reactions were visualized on a( 2%) agarose gel. 3.1Ethanol Precipitation Of DNA : Ethanol precipitation Of DNA carried out according to the method of.(8) the salt concentration of the viral DNA samples was balance by addition of (MgCl2) to final concentration of 0.01M and ( NaCl2)to final concentration 0.2M . The DNA sample put in to small volume (300) in eppendr of tube. Three volumes of cold (20°C) absolute ethanol were added to one volume of salt –adjusted DNA sample . The content mixed gently by using micropipette. The DNA ethanol mixture was then kept at (-20°C) overnight and the precipitation DNA was pelleted by centrifugation at 10,000 RPM(Rondom Per Cycle) for 30 minute in eppendr of centrifuge at 4 ° C . The supernatant was gently aspirated and the pellet was re suspended in cold ethanol 70% ethanol . The DNA suspension was centrifuged as above and the pellet was drain in temperature room before the pellet was resuspended in TE buffer(PH 7.8) 4.Enzyme and Buffer :One restriction endonuclease enzyme were obtained from promegacompany /USA. That enzyme was Bam.H1 . Bam. H1. consist of (Restriction Enzyme 10X Buffer, Acetylated BSA, 10μg/μl. 5.Digestion :About 40μl of purified DNA were ethanol precipitation ,pelleted and draind as described previously .The DNA digestion with Bam.H1 Restriction Enzyme, 10u/μl). 1. In a sterile tube, assemble the following components in the order listed below. Table (2): Method of digestion by Bam.H1.