Multiplex Polymerase Chain Reaction Technique Research Articles

Genotypic analysis of Escherichia coli strains that cause urosepsis in the Aegean region.

The aim of this study was to characterize strains genotypically, to determine their phylogenetic relationships, to investigate the presence of the papG gene, and to compare their antibiotic susceptibility test results. Seventy pathogenic E. coli strains were isolated from both urine and blood cultures of patients with the preliminary diagnosis of urosepsis who were referred to the Ege University Faculty of Medicine, Bacteriology Laboratory of Medical Microbiology Department in İzmir. All of these strains were examined for the papG gene and phylogenetic groups with the multiplex polymerase chain reaction technique. Pulsed-field gel electrophoresis and multilocus sequence typing (MLST) were used for epidemiologic analysis. Phylogenetically, it was found that 16 belonged to group B2, 31 belonged to group D, 15 belonged to group A, and 7 belonged to group B1. One strain was not identified as belonging to a group. papG genes were found in 26 of 70 E. coli strains. Thirty urosepsis pathogenic E. coli strains were analyzed with MLST. Twenty-two strains were identified as new STs. These findings are extremely important for Turkey and these new 22 strains should be investigated in more detail because they are new and have the potential to lead to infections.

Sero-prevalence and Molecular Detection of Brucella Species in Pig Producers of Punjab, India

The current study was carried out to determine the sero-prevalence and molecular detection of Brucella species in persons involved in pork production in Punjab state of India. The sample size was selected using survey toolbox and a total of 123 blood samples were collected from pig farmers and slaughter house workers. The serum samples were tested using Rose Bengal Plate Test (RBPT), Standard Tube Agglutination test (STAT) and indirect ELISA. Polymerase chain reaction (PCR) on blood samples using B4 and B5 primers was carried out on all sero-positive and 20 randomly selected sero-negative samples. The Bruce ladder Multiplex PCR technique was further used for confirming the species of Brucella positive samples. The results observed that 1 (0.81%) and 4 (3.25%) subjects were positive in RBPT/STAT and ELISA, respectively. Polymerase Chain Reaction confirmed the presence Brucella species in blood of one of the sero-positive samples and Bruce ladder multiplex PCR confirmed the species to be B. abortus. Chi-square test was applied on the results to determine the significant difference among various groups. A significant difference was found in the prevalence rates in different districts in Punjab. The results indicate that B. abortus is circulating in the persons involved in pork production in Punjab state of India. The preventive and control measures need to be enforced to prevent the occupational exposure of brucellosis to pig producers in Punjab (India).

Rapid multiplex polymerase chain reaction for simultaneous detection of Vibrio harveyi, V. parahaemolyticus, and V. vulnificus in pacific white shrimp (Litopenaeus vannamei)

Context: A comparatively small number of species, e.g., Vibrio parahaemolyticus and V. vulnificus , cause disease in both aquatic animals and humans. V. harveyi is marine animal pathogen and rarely causes infections in humans; however, it might become a reservoir of antibiotic-resistant bacteria forms and virulence genes. Aims: 1) to develop rapid multiplex polymerase chain reaction (PCR) assay for the simultaneous detection of V. harveyi, V. parahaemolyticus , and V. vulnificus by using vhhP2, tl , and rpoS genes as the respective target genes and 2) to evaluate specificity and determined detection of multiplex PCR technique. Materials and Methods: The multiplex PCR assay was developed and evaluated for specificity on 36 isolates of V. harveyi , 30 isolates of V. parahaemolyticus , and 14 isolates of V. vulnificus , along with other species of Vibrio and non- Vibrio bacterial isolates. Sensitivity of test was described as detection limit of pathogens in lowest amount of sample (CFU/mL or CFU/g) was determined by diluted DNA extracts of the pure cultures and spiked pacific white shrimp (Litopenaeus vannamei) samples Results: This developed multiplex PCR was proved as an accurate method, which was specific for three Vibrio species. The detection limits of V. harveyi , V. parahaemolyticus , and V. vulnificus in pure cultures and spiked shrimp samples ranged 1.05-4.8 × 10 3 CFU/mL and 1.9-7 × 10 4 CFU/g, respectively. Conclusions: This rapid multiplex PCR assay can decrease amount and process of sample preparation, which was time-consuming, and had preferable accuracy. This developed technique will be suitable and useful for food-borne pathogen detection in shrimp and horizontal gene transfer study among different Vibrio species in aquatic animals.

High frequency of multidrug-resistant Staphylococcus aureus with SCCmec type III and Spa types t037 and t631 isolated from burn patients in southwest of Iran.

Methicilin resistance Staphylococcus aureus (MRSA) infections are the major challenges in hospitals, especially in the burn units. The use of molecular typing methods is essential for tracking the spread of S. aureus infection and epidemiological investigations. The aim of this study was to find the profile of the spa types and also the prevalence of each SCCmec type of S. aureus strains in a central burn hospital in southwest of Iran. A total of 81 non-duplicate S. aureus were isolated from burn patients between April 2011 and February 2012. The susceptibility of the isolates against 13 different antibiotics was tested by disk agar diffusion (DAD) method. MRSA strains were identified by amplification of mecA gene. Multiplex-polymerase chain reaction (PCR) technique was used to determine the SCCmec types of MRSA strains and all the S. aureus isolates were typed by spa typing method. Detection of mecA gene showed that 70 (86.4%) of the isolates were MRSA. The highest rate of resistance was observed for penicillin (97.5%) and erythromycin (77.8%). None of the isolates were resistant to vancomycin. Sixty-seven of the 70 MRSA isolates harbored only SCCmec type III and three untypeable isolates. Five different spa types were detected. The most common spa types were t037 (42.5%) and t631 (34.5%) and were only found in MRSA isolates. Only SCCmec type III was found in burn patients which emphasizes the HA-MRSA origin of these strains. Only five different spa types identified in this study are in accordance with one SCCmec type which indicates that a limited number of bacterial colons are circulated in the burn unit in this hospital.

Association of interferon lambda-1 with herpes simplex viruses-1 and -2, Epstein-Barr virus, and human cytomegalovirus in chronic periodontitis.

Periodontal tissues facilitate the homing of herpes viruses that elicit the immune-inflammatory response releasing the interferons (IFN). IFN lambda-1 (λ1) can suppress the replication of viruses, and induces the antiviral mechanism. The aim of the present study was to evaluate the association between IFN-λ1 and periodontal herpes viruses in the immunoregulation of chronic periodontal disease. The cross-sectional study design included 30 chronic periodontitis patients with a mean age of 42.30±8.63years. Gingival crevicular fluid collected was assessed for IFN-λ1 using enzyme-linked immunosorbent assay and four herpes viruses were detected using multiplex polymerase chain reaction technique. IFN-λ1 levels were compared between virus-positive and -negative patients for individual and total viruses. Fifty per cent (n=15) of patients were positive for the four herpes viruses together; 50% (n=15), 30% (n=9), 26.7% (n=8), and 40% (n=12) were positive for herpes simplex virus (HSV)-1, Epstein-Barr virus, HSV-2, and human cytomegalovirus, respectively. The mean concentrations of IFN-λ1 in virus-positive patients (14.38±13.95) were lower than those of virus-negative patients (228.26±215.35). INF-λ1 levels in individual virus groups were also lower in virus-positive patients compared to virus-negative patients, with P<0.001. These results suggest that IFN-λ1 could have antiviral and therapeutic value against the viruses in the pathogenesis of chronic periodontitis.

High allele frequency of CYP2C9*3 (rs1057910) in a Negrito’s subtribe population in Malaysia; Aboriginal people of Jahai

Background: CYP2C9 gene polymorphisms modulate inter-individual variations in the human body’s responses to various endogenous and exogenous drug substrates. To date, little is known about the CYP2C9 gene polymorphisms among the aboriginal populations of the world, including those in Malaysia.Aim: To characterise and compare the CYP2C9 polymorphisms (CYP2C9*2, CYP2C9*3, CYP2C9*4 and CYP2C9*5) between one of Malaysia’s aboriginal populations, Jahai, with the national major ethnic, Malay. To also compare the allele frequencies from these two populations with available data of other aboriginal populations around the world.Subjects and methods: The extracted DNA of 155 Jahais and 183 Malays was genotyped for CYP2C9 polymorphisms using a nested multiplex allele-specific polymerase chain reaction technique. The results were confirmed by DNA direct sequencing.Results: Genotyping results revealed that CYP2C9*2, CYP2C9*4 and CYP2C9*5 were absent in Jahais, while only the latter two were absent in Malays. The CYP2C9*3 allelic frequency in Jahais was 36.2%, making them the most frequent carriers of the allele thus far reported in any ethnic group from Southeast Asia.Conclusions: The high frequency of CYP2C9*3 and the absence of CYP2C9*2 in Jahais suggest that genetic drift may be occurring in this ethnic group. This is the first study to determine the CYP2C9 polymorphisms in an aboriginal population in Malaysia.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye.

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio.

Changes in pneumococcal nasopharyngeal colonization among children with respiratory tract infections before and after use of the two new extended-valency pneumococcal conjugated vaccines

Background: The 10-valent pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine (PHiD–CV) and the 13-valent pneumococcal conjugate vaccine (PCV13) replaced the 7-valent PCV (PCV7) in May 2010 in Korea. We investigated the dynamics of pneumococcal nasopharyngeal (NP) colonization in children with a respiratory illness before and after use of PHiD–CV and PCV13. Methods: From March 2009 to December 2012 NP secretions were obtained from 2176 children aged < 5 years with respiratory diseases. We used the multiplex polymerase chain reaction (PCR) technique to determine pneumococcal serotypes. Results: Among the samples, 468 (21.5%) specimens were positive by multiplex PCR. The overall pneumococcal colonization rate remained stable during the 2009–2012 periods. The serotypes present in PCV7 and serotype 19A decreased in frequency from 36.8% and 26.4% in 2009 to 10.1% and 11.4% in 2012, respectively (χ2 for trend, P < 0.001 and P = 0.007, respectively). The frequency of non-PCV13 serotypes increased from 36.8% in 2009 to 78.5% in 2012 (χ2 for trend, P < 0.001). There was no significant difference in carriage rates of each serotype between groups of children that received PCV7, PHiD–CV, or PCV13. Conclusions: Compared with the period of PCV7 vaccination, overall carriage rate was not affected by the introduction of new PCVs. However, serotype distribution now consists mostly of non-vaccine serotypes. PCVs affect mucosal immunity against Streptococcus pneumoniae (SP) in NP carriage; but, global SP colonization seems to be maintained by replacement.

Prevalence of Escherichia coli Pathotypes among Children with Diarrhoea in Zaria, Nigeria

Diarrhoea is one of the foremost public health problems worldwide especially among Children under five years in developing countries. Only few studies have investigated the epidemiology and virulence of Escherichia coli pathotypes in South-Eastern and South-Western Nigeria leaving the Northern part of the country unstudied. In this study, a total of 100 isolates of E. coli (45%) were obtained from the stool of 222 diarrhoea patients who were children below five (5) years attending Ahmadu Bello University Teaching Hospital, Shika, and Institute of Child Health, Banzazzau; an annex of Ahmadu Bello University Teaching Hospital, Shika-Zaria, Nigeria. The isolation and Original Research Article Sani et al.; BJMMR, 7(1): 17-24, 2015; Article no.BJMMR.2015.303 18 biochemical identification of the E. coli isolates were performed using standard microbiological procedures. A multiplex polymerase chain reaction (PCR) technique was used to differentiate the five (5) major diarrhoeagenic Escherichia coli pathotypes (EHEC, ETEC, EPEC, EIEC and EAEC) in one reaction condition, by using different diarrhoeagenic E. coli primers for different virulent genes found in E. coli. From the result obtained, only one (1) percent of the isolates was found to harbor the virulence gene out of the 100 E. coli isolated from the diarrhoea stools of children employed in this study.

Incidence of alpha-globin gene defect in the Lebanese population: a pilot study.

Background. It is well established that the Mediterranean and Arab populations are at high risk for thalassemias in general and for alpha-thalassemia in particular. Yet, reports on alpha-thalassemia in Lebanon are still lacking. In this study, we aim at assessing the incidence of alpha-thalassemia in the Lebanese population. Methods. 230 newborns' dried blood cards remaining from routine neonatal screening at the American University of Beirut Medical Center were collected for DNA extraction. Samples were screened for the 21 most common α-globin deletions and point mutations reported worldwide, through multiplex Polymerase Chain Reaction (PCR) and Reverse-Hybridization technique. Results. Upon analyses, the carrier rate of α-thalassemia was found to be 8%. Two mutations detected the −α 3,7 single gene deletion found in 75% of cases and the nongene deletion α2 IVS1 [−5nt] in the remaining samples. Conclusion. This study is the first dedicated to investigate α-thalassemia trait incidence in Lebanon. Data obtained demonstrates a high carrier rate in a relatively, highly consanguineous population; it also highlighted the presence of two common mutations. These results may be of an important impact on premarital and newborn screening policies in our country.

Effect of CYP1A1 and GSTM1 genetic polymorphisms on bone tumor susceptibility.

Tumor gene polymorphisms are often associated with individual susceptibility to genetic diseases. Cytochrome P4501A1 (CYP1A1) and glutathione S-transferase mu 1 (GSTM1) gene polymorphisms are closely related to the susceptibility of the body to chemical carcinogens in the environment. Therefore, we explored the relationship between CYP1A1 and GSTM1 gene polymorphisms and susceptibility to bone tumors. Multiplex-polymerase chain reaction (PCR), allelic-specific PCR, and PCR-restriction fragment length polymorphism techniques were used to analyze CYP1A1 and GSTM1 gene polymorphisms in 52 bone tumor patients and 100 healthy subjects. The allelic variation frequency of the CYP1A1 gene at exon 7 (Ile 462 Val) in bone tumor patients was 0.462, which was significantly higher than that in the normal controls (0.223). The frequency of the absence of the GSTM1 homozygous genotype in the patients (0.65) was also markedly higher than that in the control group (0.41). Subjects with CYP1A1 Val/Val homozygous mutations and absence of the GSTM1 homozygous genotype were at markedly increased risk of developing bone tumors [ORs 4.15 (95%CI: 1.268-13.30) and 2.35 (95%CI: 1.15-4.85), respectively]. The OR for the combined effect of the CYP1A1 and GSTM1 gene polymorphisms was 8.55 (95%CI: 1.75-41.50). CYP1A1 and GSTM1 polymorphisms are genetic risk factors in patients with bone tumors, and the allelic variation of these genes increases the risk of bone tumor occurrence.

Étude prospective de l’écologie virale hivernale dans un service de réanimation pédiatrique

RésuméLe but de cette étude prospective était d’évaluer l’épidémiologie des virus respiratoires chez les enfants hospitalisés dans une unité de réanimation et de soins continus pédiatriques pendant 3 mois d’hiver, en 2012–2013. Ont été inclus tous les enfants admis en réanimation pédiatrique du centre hospitalier universitaire (CHU) de Caen et ayant bénéficié d’une recherche d’infection par un virus respiratoire à partir d’un prélèvement nasal analysé par amplification génique (PCR) multiplex. Sur 105 enfants admis, 84 répondaient aux critères d’inclusion. Trente-sept enfants présentaient un ou plusieurs symptômes respiratoires à l’admission. Cinquante-quatre échantillons étaient positifs (64,3 %) avec 70 virus détectés. Le virus dont la prévalence était la plus élevée était le virus respiratoire syncytial (VRS) (n = 28 ; 40,0 %), suivi par le rhinovirus (n = 24 ; 34,3 %). Parmi les enfants sans symptômes respiratoires, 42,6 % étaient infectés par un ou plusieurs virus. Aucune différence de durée d’hospitalisation, de durée de ventilation mécanique n’a été mise en évidence en fonction du virus détecté. La principale limite de cette étude est l’analyse par PCR, beaucoup plus sensible que d’autres méthodes de détection, notamment chez des sujets asymptomatiques sur le plan respiratoire. Cette étude révèle cependant un taux élevé d’infections respiratoires causées par des virus chez les enfants qui ne présentent pas de symptômes respiratoires à leur admission en unité de réanimation et soins continus. Elle suggère un intérêt du dépistage de ces infections virales à l’admission des enfants et un bénéfice à l’extension des mesures d’isolement pour tous les enfants en réanimation pédiatrique.

Detection of deletion mutation in the central hotspot region of the dystrophin gene using multiplex polymerase chain reaction technique

Duchenne Muscular Dystrophy (DMD), the most common genetic neuromuscular disease of childhood is caused by deletions, duplications or point mutations of the dystrophin gene located at Xp 21.2. DMD is an X-linked recessive genetic disease which is characterized by progressive muscle weakness and degeneration of skeletal muscle. Most of the deletions occur in two hotspot regions present at the 5' end or the central region of dystrophin gene. The study focused on the deletion mutations in the central region of the dystrophin gene specifically exons 34, 42, 44, 47, 48, 51, 52. Multiplex polymerase chain reaction was standardized for these exons in control samples of normal female. Two clinically diagnosed DMD patients were analyzed using this standardized protocol to detect deletions in these particular exons. The multiplex PCR conditions for exon 34, 42, 44, 47, 48, 51 and 52 in the central region of dystrophin gene were standardized. Out of the two patient samples analyzed in which patient 1 showed deletion of exon 47 and 52 in the dystrophin gene. Deletion analysis of patient 2 did not show any deletion in the above mentioned exons. This resulted in diagnosing deletion mutation in exon 47 and 52 of dystrophin gene. How to cite this paper : Fargade, S.A., Shelke, M.R. and Darade, R.V. (2014). Detection of deletion mutation in the central hotspot region of the dystrophin gene using multiplex polymerase chain reaction technique.

Multiplex PCR in Species Authentication: Probability and Prospects—A Review

Food forgery is one of the most articulated socio-economic concerns which contributed to increase people’s awareness on what they eat and how and where it is produced. Consumers are anxious about the consequences of food falsification on their choices, religious rituals, health, and hard-earned fortunes. The recent scandals of horse and rat meats in Europe and China have given us a brainstorming apprehension on the detection, differentiation, and identification of meat products. To restore consumers’ trust and protect wildlife in natural habitats, researchers and policy-making and policy-implementing authorities have massively monitored all steps in the production of foods and food materials. Analytical approaches based on lipids, proteins, and DNA have been proposed for the authentication of meat species under pure and complex matrices. However, protein and lipid-based methods are less effective since the target biomarkers could be modified throughout the processing treatments. On the other hand, DNA-based species identification schemes have gained wider acceptance and reliability because of the superior stability and universality of DNA in all tissues and cells. We systematically presented here major species detection schemes with special emphasis on multiplex polymerase chain reaction (PCR) of both end-point and real-time platforms. We believe this short but comprehensive review would serve as a reference guide for the developers and users of multiplex PCR and others DNA-based techniques.

Comparison of a Simple-Probe Real-Time PCR and Multiplex PCR Techniques for HPA-1 to HPA-6 and HPA-15 Genotyping

We aimed to compare HPA-1 to HPA-6 and HPA-15 genotyping results obtained by a simple-probe real-time polymerase chain reaction (PCR) technique with the multiplex PCR technique. Five hundred DNA samples from the Thai National Stem Cell Donor Registry (TSCDR) of the National Blood Centre, Thai Red Cross Society were included. Human platelet antigen (HPA) genotyping was performed by simple-probe real-time PCR and multiplex PCR techniques. HPA-1, HPA-2, HPA-3, and HPA-4 genotyping results obtained by both techniques were in agreement. The misinterpretation of HPA-5, HPA-6, and HPA-15 genotypes was found in eight samples by simple-probe real-time PCR and HPA genotypes were confirmed by DNA sequencing. Two samples of HPA-5 were misinterpreted as HPA-5a5a instead of HPA-5a5b due to an NM_002203.3:c.1594A>C mutation (rs199808499) near the HPA-5 polymorphism (5' side). Five samples of HPA-6a6b were misinterpreted as HPA-6b6b because of an NM_000212.2:c.1545G>A mutation (rs4634) adjacent to the HPA-6 polymorphism (3' side). Interestingly, one sample of HPA-15a15b was misinterpreted as HPA-15b15b due to an NM_133493.1:c.2118C>A mutation near the HPA-15 polymorphism (3' side). HPA genotyping results by two PCR techniques were compared. Incorrect assignments were found due to genetic variations near each HPA single nucleotide polymorphism. Therefore, to avoid false assignation, the use of two genotyping techniques is recommended.

Performance of a rapid multi-analyte 2-photon excitation assay in children with acute respiratory infection

The purpose of this study is to evaluate the diagnostic performance of the novel 2-photon excitation-based mariPOC© Assay (ArcDia Laboratories, Turku, Finland) for antigen detection of respiratory viruses versus real-time polymerase chain reaction (PCR). The mariPOC Assay and 2 multiplex real-time PCR techniques were performed on nasopharyngeal samples from pediatric patients with suspicion of acute respiratory infection admitted to a children's hospital in Spain during October 2011 to January 2013. A total of 233 samples were studied. Sensitivities and specificities (95% confidence interval) of the mariPOC Assay were for respiratory syncytial virus (RSV), 78.4% (69.7–85.6) and 99.2% (96.3–100.0); influenza virus (IFV) A, 66.7% (26.2–94.0) and 99.6% (97.9–100.0); IFV-B, 63.6% (33.6–87.2) and 100.0% (98.7–100.0); human metapneumovirus (hMPV), 60.0% (34.5–81.9) and 100.0% (98.6–100.0); adenovirus (ADV), 12.5% (0.6–48.0) and 100.0% (98.7–100.0), respectively. The mariPOC Assay is a highly specific method for simultaneous detection of 8 respiratory viruses but has sensitivities that range from moderately high for RSV to moderate for IFV and hMPV and low for ADV.

Application of PCR and real-time PCR for monitoring cyanobacteria, Microcystis spp. and Cylindrospermopsis raciborskii in Macau freshwater reservoir

Freshwater algal blooms have become a growing concern world-wide. They are caused by a high level of cyanobacteria, predominantly Microcystis spp. and Cylindrospermopsis raciborskii, which can produce microcystin and cylindrospermopsin, respectively. Longtime exposure to these cyanotoxins may affect public health, thus reliable detection, quantification, and enumeration of these harmful algae species has become a priority in water quality management. Traditional manual enumeration of algal bloom cells primarily involves microscopic identification which limited by inaccuracy and time-consumption.With the development of molecular techniques and an increasing number of microbial sequences available in the Genbank database, the use of molecular methods can be used for more rapid, reliable, and accurate detection and quantification. In this study, multiplex polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) techniques were developed and applied for monitoring cyanobacteria Microcystis spp. and C. raciborskii in the Macau Storage Reservoir (MSR). The results showed that the techniques were successful for identifying and quantifying the species in pure cultures and mixed cultures, and proved to be a potential application for water sampling in MSR. When the target species were above 1 million cells/L, similar cell numbers estimated by microscopic enumeration and qPCR were obtained. Further quantification in water samples indicated that the ratio of the estimated number of cell by microscopy and qPCR was 0.4–12.9 for cyanobacteria and 0.2–3.9 for C. raciborskii. However, Microcystis spp. was not observed by manual enumeration, while it was detected at low levels by qPCR, suggesting that qPCR is more sensitive and accurate. Thus the molecular approaches provide an additional reliable monitoring option to traditional microscopic enumeration for the ecosystems monitoring program.

Sex Determination of Fetus Prenatal from Maternal Plasma in Goats Using Duplex PCR

This work was carried out the Genetic Engineering Lab, College of Agriculture, Basrah University, Iraq. This study included determines the sex of the fetus prenatal by analyzing free fetal DNA cells in maternal plasma by using duplex PCR. blood were collected from 5 pregnant females prepared the plasma and extract the DNA by using heatbased direct method for plasma and then investigate the sequences GAPDH and DYS14 through the use of multiplex polymerase chain reaction technique and the results were 2 (40%) males show a double bands one at 97 bp representing GAPDH and the second at 198 bp representing DYS14 and 3 (60%) females show a single band at 97 bp representing GAPDH.

Development of an efficient multiplex semi-nested PCR for convenient use in urine samples for diagnosis of toxoplasmosis

BACKGROUND: Toxoplasmosis is a protozoan parasitic infection. The cerebrospinal, ocular and congenital forms of the disease are complicated and life threatening. Diagnosis of toxoplasmosis is difficult due to invasive sample requirement, complications in pathogenesis, immunology, and interpretation of test results. Using urine as a non invasive sample source for molecular diagnosis of toxoplasmosis is the main object of this research. Attempts were made to diagnose toxoplasmosis using polymerase chain reaction (PCR) technique on the parasite’s deoxyribonucleic acid (DNA) in urine. METHODS: Parasite: The Toxoplasma gondii (T. gondii) RH strain was obtained from peritoneal exudates of small white laboratory mice inoculated with 5x105 tachyzoites three days before aspiration. Urine samples with defined numbers of tachyzoites per ml were used as laboratory samples. The target was a 529 bp segment (AF146527 gene bank) of the T. gondii genome with 200-300 repeats as target in PCR. Selected primers were designed on its sequence. RESULTS: A multiplex semi-nested PCR technique was developed for obtaining a method for precise diagnosis of toxoplasmosis, time and budget saving and to diminish the DNA cross contamination risk. It was sensitive to detect 2 this tachyzoites DNA per final sample. CONCLUSION: With further development, nested PCR can be a useful and non-invasive method for diagnosis of cerebrospinal, ocular, and congenital toxoplasmosis.

Chlamydia trachomatis among HIV infected patients using PCR technique

Background and Objectives: Chlamydia trachomatis is a sexually transmitted organism and an important public health problem in the sexually active age group. Limited studies are found regarding the prevalence of Chlamydia trachomatis in Nepal. Moreover, no study in Nepal reports the association of Chlamydia and HIV infection. The current study attempts to determine the burden of Chlamydia on HIV positive patients. Material and Methods: A total of 117 HIV positive patients visiting a HIV clinic in Kathmandu, were screened for Chlamydia infection. For this, Urine samples were collected and analyzed using the Multiplex polymerase chain reaction technique (MPCR) and Agarose gel electrophoresis. DNA isolation was performed using QIAamp DNA and Blood mini kit handbook protocol. Results: C. trachomatis was detected in 4.27% of the total 117 HIV patients. Out of positive cases 60% were males and 40% were females. However, Chlamydia is found more prevalent among females (6.89%) than in males (3.4%). Eighty percent of positive cases were asymptomatic. Conclusion: Chlamydia infection was found less commonly among studied patients and most of those cases were asymptomatic. So there is difficulty in timely detection of C. trachomatis and track the clinical sequel, which might be devastating. Hence, routine checkup is recommended for all suspected cases for timely management of the disease Janaki Medical College Journal of Medical Sciences (2013) Vol. 1 (2): 3-10 DOI: http://dx.doi.org/10.3126/jmcjms.v1i2.9261