Background: Leishmaniasis is a protozoan parasitic disease and a global health problem. The aim of this study is to diagnose the parasitic infection in humans for epidemiological identification and providing control programs using proprietary co-designed primers in three species of Leishmania.Materials and Methods: 30 common Leishmania isolates were gathered from different centers in Iran. Having been cultured in RPMI-1640 Medium, DNA was extracted and the gene ITS2-rRNA was amplified by PCR. The amplicons were examined by electrophoresis on agarose gel 2%. Also, in FLASH PCR method, a specific probe and florence colour were used to investigae the amplicon existence on sample.Results: The results of the investigations by PCR and FLASH PCR methods show that these methods are sensitive and specific for diagnosis of Leishmania.Conclusion: In this study, identification of Leishmania parasite using specific primer pairs was successful and TaqMan could be one of the most sensitive diagnostic methods to identify parasite load for the ITS2 region of Leishmania.

Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range, excellent sensitivity and specificity, has little to no postamplification processing. However, optimal benefit from these advantages requires a clear understanding of the many options available for running a real-time PCR experiment and its accurate application. Real-time PCR provides the amplification of a targeted DNA molecule and reports it using fluorescent reporter molecules during the amplification reaction. This review article discusses the principles of the real time reaction equipment utilized in the process, fluorescent detection systems, data analysis methods and several applications of this technique.

Background: The clinical importance of yeast infections has increased in recent decades. There are 10-15 pathogenic Candida species. The current morphological and physiological methods for identification of Candida species are generally not easy to interpret and may be expensive or time-consuming. In the present study, we introduce and use a new approach for the identification and differentiation of medically important yeast species of Candida. In this method, size polymorphism of the internal transcribed spacer regions, ITS1 and ITS2, of the ribosomal DNA in various Candida species is used as the basis of species recognition.Methods: The genomic DNA of 31 standard strains and 60 clinical isolates was extracted and PCR-amplified using two primer pairs (ITS1-ITS2 and ITS3-ITS4) separately. Both PCR products were mixed and analyzed after standard agarose gel electrophoresis. The species of the tested yeasts were identified by the electrophoretic patterns of the mixed PCR products of each sample, comparing the data obtained from the sequence analyses of ITS1 and ITS2 molecules.Results: By this method, with the exception of C. albicans and C. dubliniensis, we were able to clearly differentiate nearly all common pathogenic Candida species, including C. albicans, C. glabrata, C. gulliermondii, C. parapsilosis, C. tropicalis, C. krusei, C. kefyr, C. lusinaniae and C. rugosa. All standard and clinical strains were identified correctly, without expensive methods such as sequencing and capillary electrophoresis. Conclusion: It seems that the PCR-FSP method introduced in this study is the easiest molecular approach for the identification of a wide range of pathogenic Candida species and is applicable for diagnostic and epidemiological purposes in reference laboratories.

Trichomonas vaginalis is the most prevalent nonviral sexually transmitted disease in the world. Traditionally physicians make the diagnosis based on clinical grounds. Characteristics of the vaginal discharge, including color combination is not sufficient to diagnose trichomonas vaginalis infection reliably and laboratory diagnosis is necessary. In this study, a PCR targeting the beta-tubulin genes of the trichomonas vaginalis was developed for the detection of the organism in the vaginal swab samples. A vaginal swab specimen was obtained from 24 women visiting the women clinic of Shahid-Beheshti hospital of the Isfahan University of Medical Sciences. Each specimen was divided into two sections. One section was immediately examined by wet preparation microscopy test and the other section was placed in phosphate-buffered saline and transferred to biotechnology laboratory of Isfahan University of Medical Sciences, and the resulting suspension was used for a PCR reaction, specific for the trichomonas vaginalis The specificity of PCR method is similar to wet preparation. But the clinical observation is not sensitive enough to be used alone for diagnosing the parasite.24 samples were divided into two groups of 8 and 16 samples. The first group was examined by wet preparation microscopy and PCR tests. The second group was analyzed by clinical observation and PCR test.Specimens that were obtained from the first group had a positive test for both wet preparation and PCR test. In the second group, from the 16 specimens that were analyzed, 6 and 8 samples showed positive results with PCR and clinical observation, respectively. The results of this study indicate the high specificity of PCR method in detecting trichomonas vaginalis. The specificity of PCR method is similar to wet preparation. But the clinical observation is not sensitive enough to be used alone for diagnosing the parasite.

Introduction: The aim of this study was to setup, optimize and introduce a sensitive and specific PCR detection method for identification of Neisseria meningitidis DNA in clinical samples.Material and Methods: Capsular transport gene A (ctrA) was selected as a specific target sequence. This primer pair amplifies 101bp of the target gene. Neisseria meningitidis strain: ATCC; 13090 and Neisseria meningitides serogroup C were used as a standard organism for optimization experiments. A range of bacterial pathogens were used for specificity testing, including, Haemophilus influenzae Type b: ATCC; 49766, Escherichia coli: ATCC;35218, Enterobacter, Klebsiella pneumoniae,  Streptococcus pneumoniae, Staphylococcus aureus and Streptococcus group D.Phenol – chloroform method was used for DNA extraction. Amplified product was detected by gel agarose electrophoresis, stained by ethidiome bromide. Results: Our results confirmed amplification of the expected product. Specificity test proved no cross reaction with tested organisms. Sensitivity test detected 500fg of Neisseria meningitidis DNA as a final detection limit.Conclusion: As a conclusion, PCR is a method with high sensivity and specificity and specificity which can be performed within 3 hours, and therefore utilization of this test in the clinical laboratories can help rapid diagnosis of Neisseria meningitides in clinical samples.

Polymerase chain reaction (PCR) is a rapid and simple technique with high sensitivity and specificity. In the recent years, PCR has been used for rapid detection of viral nucleic acids, such as Human cytomegalovirus (HCMV), whereas, PCR optimization is an important task to be done, especially before it’s diagnostic application. Annealing temperature, ion concentration (especially Mg2+ ion) and the cycling program and enhancer compounds are important optimization parameters. Peripheral blood leukocytes (PBLs) were isolated from samples collected from renal transplant recipients suffering from severe and symptomatic CMV disease. PBLs DNA was extracted and used for PCR. Annealing temperature and MgCl2 concentration and cycling condition were optimized. Dimethyl sulfoxide (DMSO) and gelatin were checked as enhancer components. The optimized condition obtained through this study was: 1x PCR buffer (20 mM Tris-HCl pH 8.6, 50 mM KCl), 2.5 mM MgCl2, 0.2 mM of each dNTPs, 0.25 µM of each primers, 0.25 unit/25 µl Taq DNA polymerase, 5% DMSO, 500 µg/ml gelatin and 50-150 ng template DNA in 25 µl final volume. PCR was performed as: 95°C 5 min (pre-denaturation), 94°C 50 sec, 58°C 1 min, 72°C 1 min for 35 cycles and 72°C 5 min (final extension). Using these conditions, it was shown that optimized PCR was five fold more sensitive than initial PCR; which can be used for diagnostic application of HCMV in renal transplant patients.

In 1992, the analysis of PCR kinetics becomes possible by constructing a system that detects PCR products as they accumulate. This "realtime" system includes the intercalator ethidium bromide in each amplification reaction, an adapted thermal cycler to irradiate the samples with ultraviolet light and detection of the resulting fluorescence with a computer-controlled cooled CCD camera. Amplification produces increasing amounts of double-stranded DNA, which binds ethidium bromide, resulting in an increase in fluorescence. By plotting the increase in fluorescence versus cycle number, the system produces amplification plots that provide a more complete picture of the PCR process than assaying product accumulation after a fixed number of cycles. This methodology could be used to quantify population of beneficial soil bacteria and fungi in root or rhizospher of host plant for example ammonia-oxidizing bacteria, siderophore production bacteria and mycorrhiza as well as the investigation of therapeutic effect of different pesticide on the plant tissue, study of expression of different gene under environment stress condition the study of mechanism of resistance plants against plant pathogens, mutation is induced in organism genomes.

Background: Rapid diagnosis and differentiation of Brucella is of high importance due to the side effects of antibiotics for the treatment of brucellosis. This study aimed to identify and compare PCR-ELISA as a more accurate diagnositc test with other common molecular and serological tests.Methods: In this experimental and sectional study, during March 2014 to Sep 2015, 52 blood samples of suspected patients with clinical symptoms of brucellosis were evaluated in medical centers all over Iran with serum titers higher than 1: 80. Using two pairs of specific primers ofBrucella abortus, B. melitensis and DIG-dUTP, Fragment IS711 (The common gene fragment in B. melitensis and B. abortus) was amplified.DIG-ELISA was performed using specific probes of these 2 species of Brucella and patterns were subsequently analyzed, then positive responses were compared by detecting gel electrophoresis.Results: PCR-ELISA method detected all 28 samples from 52 positive samples. Its sensitivity was 6.0 pg concentration of genomic DNA of Brucella. In gel electrophoresis method, 22 samples of all positive samples were detected. PCR-ELISA was more efficient than PCR and bacterial culture method atP -value<0.05.Conclusion: PCR-ELISA molecular method is more sensitive than other molecular methods, lack of mutagenic color and also a semi-quantitative ability. This method is more effective and more accurate compared to PCR, serology and culture of bacteria.PCR-ELISA does not have false responses. The limitation of this method is detection of bacteria in the genus compared to Multiplex PCR and Gel Electrophoresis.