Conventional Polymerase Chain Reaction Technique Research Articles

Determinación molecular de Leptospira spp. en semen y líquido preseminal y estudio serológico de caballos criollos en el departamento de Cundinamarca (Colombia)

El propósito de este estudio fue detectar serológica (prueba de microaglutinación) y molecularmente (el ADN de Leptospira spp. por PCR convencional-reacción de la polimerasa en cadena) la exposición o presencia de Leptospira spp., a partir de muestras de suero sanguíneo, líquido preseminal y semen en caballos reproductores criollos colombianos representativos de esta raza, en 44 criaderos del departamento de Cundinamarca. En Colombia no existen estudios relacionados con la presencia de leptospiras en semen equino, y dentro de los planes sanitarios en esta especie no se vacuna contra esta enfermedad. Se evaluaron muestras de un total de 107 animales, escogidos por muestreo por conveniencia, de las cuales al analizar el suero fueron seroreactivas 35 (32,7 %), correspondientes al serogrupo Leptospira interrogans, serovariedades Canícola, Pomona y Grippotyphosa. Se detectó el DNA de Leptospira spp., por PCR convencional en dos muestras de semen que representaron el 1,9 % del total de muestras. Mediante la técnica de campo oscuro se encontraron en la mayoría de las muestras de líquido preseminal diferentes formas bacterianas, algunas de ellas compatibles con leptospira. También se observaron células de descamación, cristales y uratos. Adicionalmente, no se obtuvo aislamiento de la bacteria en el medio EMJH y no se detectó ADN mediante la técnica de PCR convencional a partir del líquido preseminal.

Rapid detection of tomato leaf curl Bengaluru virus through loop mediated isothermal amplification assay.

A loop-mediated isothermal amplification (LAMP) technique was employed to develop a simple and rapid method for the detection of tomato leaf curl Bangalore virus (ToLCBaV) in diseased plants of tomato (Solanum lycopersicum). Six sets of primers were designed for LAMP technique targeting the conserved AC1 region and successfully detected ToLCBaV. No reaction was detected in the tissues of healthy plants by either the LAMP or the polymerase chain reaction (PCR). The LAMP products can be visualized by presence or absence of turbidity and staining (0.2μL for 25μL LAMP product) directly in the tube with nucleic acid stain dye which allowed easy detection. Sensitivity of LAMP assay is 100 times of conventional PCR technique. Although, both the LAMP and the PCR methods were capable of detecting ToLCBaV in infected tissues of tomato, the LAMP method would be more useful than the PCR method for detection of ToLCBaV infection in tomato plants because it is more rapid, simple and accurate method.

Rickettsia typhi in rodents and R. felis in fleas in Yucatán as a possible causal agent of undefined febrile cases.

Rickettsia typhi is the causal agent of murine typhus; a worldwide zoonotic and vector-borne infectious disease, commonly associated with the presence of domestic and wild rodents. Human cases of murine typhus in the state of Yucatán are frequent. However, there is no evidence of the presence of Rickettsia typhi in mammals or vectors in Yucatán. The presence of Rickettsia in rodents and their ectoparasites was evaluated in a small municipality of Yucatán using the conventional polymerase chain reaction technique and sequencing. The study only identified the presence of Rickettsia typhi in blood samples obtained from Rattus rattus and it reported, for the first time, the presence of R. felis in the flea Polygenis odiosus collected from Ototylomys phyllotis rodent. Additionally, Rickettsia felis was detected in the ectoparasite Ctenocephalides felis fleas parasitizing the wild rodent Peromyscus yucatanicus. This study’s results contributed to a better knowledge of Rickettsia epidemiology in Yucatán.

Principles and applications of polymerase chain reaction in medical diagnostic fields: a review

Recent developments in molecular methods have revolutionized the detection and characterization of microorganisms in a broad range of medical diagnostic fields, including virology, mycology, parasitology, microbiology and dentistry. Among these methods, Polymerase Chain Reaction (PCR) has generated great benefits and allowed scientific advancements. PCR is an excellent technique for the rapid detection of pathogens, including those difficult to culture. Along with conventional PCR techniques, Real-Time PCR has emerged as a technological innovation and is playing an ever-increasing role in clinical diagnostics and research laboratories. Due to its capacity to generate both qualitative and quantitative results, Real-Time PCR is considered a fast and accurate platform. The aim of the present literature review is to explore the clinical usefulness and potential of both conventional PCR and Real-Time PCR assays in diverse medical fields, addressing its main uses and advances.

Impact of variants in the VEGF gene on progression of proliferative diabetic retinopathy

The development of diabetic retinopathy is associated with the duration of diabetes and HbA1c levels. However, the familial aggregation of diabetic retinopathy is consistent with genetic susceptibility. Recently, a -634C/G polymorphism in the vascular endothelial growth factor (VEGF) gene was shown to be associated with diabetic retinopathy. To clarify the contribution of the VEGF gene in the development of diabetic retinopathy we analyzed variants in this gene among 469 Japanese patients with type 2 diabetes. DNA from each patient was typed for -634C/G and -2578C/A polymorphisms using conventional polymerase chain reaction techniques. The vitreous fluid samples were obtained from 40 patients with PDR for measurement of VEGF levels. We found a significantly higher frequency of the A allele in the group with proliferative diabetic retinopathy (PDR) than in the control group at -2578C/A polymorphism (p = 0.036). Moreover, if the subjects were grouped according to the duration of diabetes and status of diabetic retinopathy (a first group consisting of subjects with longer duration (>20 y) of diabetes without any retinopathy (n = 102), and a second group of those with shorter diabetes (<15 y) but having retinopathy (n = 35), the genotype distribution at -2578 C/A polymorphism was again significantly higher in the second group (p = 0.005) and differed significantly (p = 0.002) in a recessive model. The risk of the AA for PDR was 7.7 (95%, CI: 1.8-30.9). The AA genotype at -2578C/A polymorphism in the VEGF gene is associated with proliferative diabetic retinopathy. No significant association with -634 C/G polymorphism was confirmed.

Polymerase Chain Reaction-Guided Diagnosis of Mycotic Keratitis: A Prospective Evaluation of Its Efficacy and Limitations

To assess the utility of polymerase chain reaction (PCR) in diagnosing fungal keratitis and compare its sensitivity and specificity with those of the conventional microbiologic techniques used in the authors' laboratory. A prospective nonrandomized investigation was undertaken at a tertiary-care ophthalmic facility to evaluate 40 eyes of 40 patients with presumed fungal keratitis, both fresh and treated. Besides routine bacterial culture and sensitivity, corneal scrapings were evaluated by fungal culture, potassium hydroxide (KOH) wet mount, Gram's stain, and PCR. The conventional PCR technique was followed with minor modifications to suit the setup, using primers targeted to 28S rRNA sequence, which is common to all fungi that cause corneal infections in tropical climes. Of the 40 presumed cases of mycotic keratitis (30 untreated), PCR showed positivity in 50%, culture in 25%, hyphae in KOH in 40%, and Gram's staining in 35%. The sensitivities of PCR, KOH, and Gram's were 70%, 60%, and 40% and specificities 56.7%, 66.7%, and 66.7%, respectively. Among 10 of 40 eyes pretreated with antifungal agents, PCR was positive in 50%, but culture in 30%. The time taken for PCR assay was 4 to 8 hours, whereas positive fungal cultures took at least 5 to 7 days. PCR not only proved an effective rapid method for the diagnosis of fungal keratitis, but was also more sensitive in our hands than KOH wetmount and Gram's smear. Barring the potential limitations, PCR remains a promising tool for faster diagnoses of fungal keratitis.

Evaluation of detection of Legionella spp. in water samples by fluorescence in situ hybridization, PCR amplification and bacterial culture

AbstractOne hundred water samples (32 from clinical units and 68 from private households) were examined for Legionella by culture, fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). Twenty-four samples were positive by culture (22 L. pneumophila; 2 non-pneumophila species), 36 by FISH (32 L. pneumophila; 4 non-pneumophila species) and 75 by PCR (41 positive for L. pneumophila; 26 positive for L. pneumophila and a non-pneumophila species; 8 positive for non-pneumophila species).PCR and FISH results were compared to bacterial culture as the “gold standard” method by calculating sensitivities and specificities, respectively: PCR assays, 96% and 47%; FISH assays, 67% and 72%, respectively. In comparison with FISH the lower specificity of PCR is probably caused by dead Legionella bacteria and/or free Legionella DNA in potable water, and the higher sensitivity of PCR may be explained by the detection limit of fluorescence microscopy. In conclusion, the relatively high specificity, sensitivity and quickness of the FISH assay offer significant advantages over conventional PCR and culture-based techniques.

Detection of human immunodeficiency virus-1 nucleic acid on inactivated filter paper disks by polymerase chain reaction and microtiter plate assay.

Human immunodeficiency virus type 1 (HIV-1) in cultured cells, peripheral blood samples and sera were adsorbed on filter paper disks and inactivated by heat or ethanol. Two procedures, the polymerase chain reaction (PCR) and microtiter plate assay (HMPA) were used to detect the nucleic acid. The sensitivity after different heat treatments with nested PCR for HIV-1 DNA (or nested reverse transcription-PCR for HIV-1 RNA) was identical regardless of whether the samples were examined immediately or one month later. Inactivation by ethanol treatment resulted in a slight loss of sensitivity. The HMPA proved to be as reliable and specific as the conventional PCR technique. We conclude that the heat-treated filter paper disk assay is suitable for identifying HIV nucleic acid in clinical samples sent to the laboratory from a distance, e.g. in an envelope.