One-step reverse transcriptase polymerase chain reaction for the diagnosis of respiratory syncytial virus in children

Human respiratory syncytial virus (HRSV) is the main cause of acute lower respiratory tract infections in infants and children. Rapid diagnosis is required to permit appropriate care and treatment and to avoid unnecessary antibiotic use. Reverse transcriptase (RT-PCR) and indirect immunofluorescence assay (IFA) methods have been considered important tools for virus detection due to their high sensitivity and specificity. In order to maximize use-simplicity and minimize the risk of sample cross-contamination inherent in two-step techniques, a RT-PCR method using only a single tube to detect HRSV in clinical samples was developed. Nasopharyngeal aspirates from 226 patients with acute respiratory illness, ranging from infants to 5 years old, were collected at the University Hospital of the University of Sao Paulo (HU-USP), and tested using IFA, one-step RT-PCR, and semi-nested RT-PCR. One hundred and two (45.1%) samples were positive by at least one of the three methods, and 75 (33.2%) were positive by all methods: 92 (40.7%) were positive by one-step RT-PCR, 84 (37.2%) by IFA, and 96 (42.5%) by the semi-nested RT-PCR technique. One-step RT-PCR was shown to be fast, sensitive, and specific for RSV diagnosis, without the added inconvenience and risk of false positive results associated with semi-nested PCR. The combined use of these two methods enhances HRSV detection. (C) 2007 Elsevier B.V. All rights reserved.

Taenia saginata: Differential diagnosis of human taeniasis by polymerase chain reaction-restriction fragment length polymorphism assay

Speciation of Taenia in human stool is important because of their different clinical and epidemiological features. DNA analysis has recently become possible which overcomes the problems of differentiating human taeniid cestodes morphologically. In the present study, we evaluated PCR coupled to restriction fragment length polymorphism to differentiate Taenia solium from Taenia saginata eggs present in fecal samples from naturally infected patients. A different Dral-RFLP pattern: a two-band pattern (421 and 100 bp) for T saginata and a three-band pattern (234, 188, and 99 bp) for T solium was observed allowing the two species to be separated.. The lower detection limit of the PCR-RFLP using a non-infected fecal sample prepared with a given number of T saginata eggs was 34 eggs in 2 g stool sediment. The 521 bp mtDNA fragment was detected in 8 out of 12 Taenia sp. carriers (66.6%). of these, three showed a T solium pattern and five a T saginata pattern. (c) 2005 Elsevier B.V. All rights reserved.

Double-Tagging Polymerase Chain Reaction with a Thiolated Primer and Electrochemical Genosensing based on Gold Nanocomposite Sensor for Food Safety

A novel material for electrochemical biosensing based on rigid conducting gold nanocomposite (nano-AuGEC) is presented. Islands of chemisorbing material (gold nanoparticles) surrounded by nonreactive, rigid, and conducting graphite epoxy composite are thus achieved to avoid the stringent control of surface coverage parameters required during immobilization of thiolated oligos in continuous gold surfaces. The spatial resolution of the immobilized thiolated DNA was easily controlled by merely varying the percentage of gold nanoparticles in the composition of the composite. As low as 9 fmol (60 pM) of synthetic DNA were detected in hybridization experiments when using a thiolated probe. Moreover, for the first time a double tagging PCR strategy was performed with a thiolated primer for the detection of Salmonella sp., one of the most important foodborne pathogens affecting food safety. Ibis assay was performed by double-labeling the amplicon during the PCR with a -DIG and -SH set of labeled primers. The thiolated end allows the immobilization of the amplicon on the nano-AuGEC electrode, while digoxigenin allows the electrochemical detection with the antiDIG-HRP reporter in the femtomole range. Rigid conducting gold nanocomposite represents a good material for the improved and oriented immobilization of biomolecules with excellent transducing properties for the construction of a wide range of electrochemical biosensors such as immunosensors, genosensors, and enzymosensors.

Identification of Eimeria mitis and Eimeria praecox in broiler feces using polymerase chain reaction

There are few reports concerning the epidemiology of Eimeria praecox and Eimeria mitis in Brazil. In the present experiment, the polymerase chain reaction (PCR) was used to identify these species in 156 samples of broiler chicken feces from several Brazilian states and the Federal District. Oocysts present in feces samples were purified by sodium chloride flotation followed by addition of DNAzol reagent (Invitrogen®) for extraction of genomic DNA. DNA was precipitated and stored following DNAzol reagent manufacture's instructions. The primers and PCR conditions were as described by Schnitzler et al. (1999). In the 156 field samples analyzed by PCR, 70 and 45 were positive for E. praecox and E. mitis, respectively. In this study we have shown that DNA extraction using DNAzol followed by PCR can be a useful tool in epidemiological studies, since it provides fast and reliable detection of Eimeria sp. in field samples.

Polymerase chain reaction and real-time PCR for diagnosing of Leishmania infantum chagasi in dogs

A importância do cão como reservatório de L. infantum chagasi no meio urbano tem estimulado a realização de inúmeros trabalhos de avaliação de técnicas de diagnóstico, uma vez que este procedimento, quando realizado corretamente, torna-se um importante passo na prevenção da doença em humanos. Dentre os métodos de diagnóstico, as técnicas moleculares têm adquirido destaque. Objetivou-se neste trabalho verificar o desempenho da Reação em Cadeia da Polimerase (PCR) e da PCR em tempo real (qPCR) para diagnóstico da Leishmaniose Visceral Canina (LVC) utilizando diferentes amostras biológicas. Para tanto foram utilizados 35 cães provenientes de uma área endêmica para LVC, onde foram utilizados para o diagnóstico molecular, aspirado de medula óssea, fragmentos de linfonodo e baço. Neste estudo a qPCR foi capaz de detectar um maior número de animais positivos quando comparada com a PCR. Já entre as diferentes amostras biológicas utilizadas não foi observada diferença significativa na detecção de DNA de L. infantumchagasi por meio da PCR e qPCR. Mesmo assim, considerando a facilidade de obtenção, o linfonodo pode ser considerada como a melhor amostra para diagnóstico molecular da infecção por L. infantum chagasi.

Hemoculture and polymerase chain reaction using primers TCZ1/TCZ2 for the diagnosis of canine and feline trypanosomiasis

Introduction. American trypanosomiasis, also known as Chagas disease, is a zoonosis caused by Trypanosoma cruzi (T. cruzi). Dogs and cats participate actively in this parasite's transmission cycle. This study aimed at evaluating the occurrence of T. cruzi in dogs and cats from Botucatu, SP, Brazil, as well as at evaluating the technique of hemoculture in LIT (liver infusion tryptose) medium by polymerase chain reaction (PCR). Methods. Blood samples were collected from 50 dogs and 50 cats in Botucatu-SP, Brazil. For hemoculture, the samples were inoculated in LIT medium, and readings were performed for four months. Upon completion of such period, all the hemocultures were processed for parasitic DNA extraction. The PCR reactions were performed by using primers TCZ1/TCZ2. Results. Ten dogs and ten cats (20%) were positive to PCR, and four dogs and three cats (7%) were positive to hemoculture. Only in a one cat sample (1%) there was confirmation of positive hemoculture by PCR for T. cruzi. Conclusions. Results showed that PCR was a suitable tool for the confirmation of the parasite detection in hemoculture samples, and that dogs and cats from Botucatu, SP, Brazil, are maintaining the role of household reservoirs of T. cruzi, which reinforces the need for constant epidemiologic surveillance for this zoonosis.

Clinical and Microbiologic Evaluation, by Real-Time Polymerase Chain Reaction, of Non-Surgical Treatment of Aggressive Periodontitis Associated With Amoxicillin and Metronidazole

Background: The aim of the present study is to evaluate the clinical and microbiologic changes resulting from non-surgical periodontal treatment associated with amoxicillin and metronidazole in individuals with aggressive periodontitis. Methods: Fifteen individuals with aggressive periodontitis received non-surgical periodontal treatment and 45 days after completion of treatment were treated with antibiotics. Clinical data and samples of subgingival plaque were collected at baseline, 45 days after the non-surgical periodontal treatment, and 1 month after the use of antimicrobial agents. After 3 and 6 months, only clinical data were collected. The presence and quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), and Dialister pneumosintes were determined by real-time polymerase chain reaction. Results: All clinical parameters, with the exception of clinical attachment level (CAL), had significantly (P<0.05) improved at the end of the third month after non-surgical therapy associated with antibiotics. There was significant (P<0.05) reduction in the quantities of Td and Tf. After 1 month, there were significant (P<0.05) reductions in the frequencies of Pg and Tf. Conclusion: Non-surgical mechanical treatment associated with the use of amoxicillin and metronidazole led to an improvement in all clinical parameters studied, except for CAL, and significantly reduced the amount of subgingival Tf and Td. J Periodontal 2012;83:744-752.

Use of the polymerase chain reaction to detect Mycobacterium leprae in urine

Leprosy is an infectious disease caused by Mycobacterium leprae. The polymerase chain reaction (PCR) has been applied to detect M. leprae in different clinical samples and urine seems to be attractive for this purpose. PCR was used to improve the sensitivity for diagnosing leprosy by amplifying a 151-bp PCR fragment of the M. leprae pra gene (PCR-Pra) in urine samples. Seventy-three leprosy patients (39 males and 34 females, 14 to 78 years old) were selected for leprosy diagnosis at a reference laboratory in Maringa, PR, Brazil. Of these, 36 were under anti-leprosy multidrug therapy with dapsone and rifampicin for tuberculoid (TT) and dapsone, rifampicin and clofazimine for borderline (BB) and lepromatous (LL) forms. The control group contained 50 healthy individuals without any clinical history of leprosy. DNA isolated from leprosy patients' urine samples was successfully amplified by PCR-Pra in 46.6% (34/73) of the cases. The positivity of PCR-Pra for patients with the TT form was 75% for both patients under treatment and non-treated patients (P = 0.1306). In patients with the LL form, PCR-Pra positivity was 52 and 30% for patients under treatment and non-treated patients, respectively (P = 0.2386). PCR-Pra showed a statistically significant difference in detecting M. leprae between the TT and LL forms of leprosy in patients under treatment (P = 0.0033). Although the current study showed that the proposed PCR-Pra has some limitations in the detection of M. leprae, this method has the potential to be a useful tool for leprosy diagnosis mainly in TT leprosy where the AFB slit-skin smear is always negative.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria

Detection of multidrug-resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes 2 or more weeks to identify by culture. RIF-resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative polymerase chain reaction (qPCR) is a novel approach that takes ≤2 days. However, qPCR identification of resistant isolates, particularly for isolates with mixed RIF-susceptible and RIF-resistant bacteria, is reader dependent and limits its clinical use. The aim of this study was to develop an objective, reader-independent method to define rpoB mutants using beacon qPCR. This would facilitate the transition from a research protocol to the clinical setting, where high-throughput methods with objective interpretation are required. For this, DNAs from 107 M. tuberculosis clinical isolates with known susceptibility to RIF by culture-based methods were obtained from 2 regions where isolates have not previously been subjected to evaluation using molecular beacon qPCR: the Texas–Mexico border and Colombia. Using coded DNA specimens, mutations within an 81-bp hot spot region of rpoB were established by qPCR with 5 beacons spanning this region. Visual and mathematical approaches were used to establish whether the qPCR cycle threshold of the experimental isolate was significantly higher (mutant) compared to a reference wild-type isolate. Visual classification of the beacon qPCR required reader training for strains with a mixture of RIF-susceptible and RIF-resistant bacteria. Only then had the visual interpretation by an experienced reader had 100% sensitivity and 94.6% specificity versus RIF-resistance by culture phenotype and 98.1% sensitivity and 100% specificity versus mutations based on DNA sequence. The mathematical approach was 98% sensitive and 94.5% specific versus culture and 96.2% sensitive and 100% specific versus DNA sequence. Our findings indicate the mathematical approach has advantages over the visual reading, in that it uses a Microsoft Excel template to eliminate reader bias or inexperience, and allows objective interpretation from high-throughput analyses even in the presence of a mixture of RIF-resistant and RIF-susceptible isolates without the need for reader training.^

Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid `real-time' polymerase chain reaction

We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples. This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction. The time from sample collection to result was less than 1h. RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment.

Angiomatoid fibrous histiocytoma: comparison of fluorescence in situ hybridization and reverse transcription polymerase chain reaction as adjunct diagnostic modalities

Angiomatoid fibrous histiocytoma (AFH) is a rare soft tissue neoplasm of intermediate biologic potential and uncertain differentiation, most often arising in the extremities of children and young adults. Although it has characteristic histologic features of a lymphoid cuff surrounding nodules of ovoid cells with blood-filled cystic cavities, diagnosis is often difficult due to its morphologic heterogeneity and lack of specific immunoprofile. Angiomatoid fibrous histiocytoma is associated with recurrent chromosomal translocations, leading to characteristic EWSR1-CREB1, EWSR1-ATF1, and, rarely, FUS-ATF1 gene fusions; fluorescence in situ hybridization (FISH), detecting EWSR1 or FUS rearrangements, and reverse transcription-polymerase chain reaction (RT-PCR) for EWSR1-CREB1 and EWSR1-ATF1 fusion transcripts have become routine ancillary tools. We present a large comparative series of FISH and RT-PCR for AFH. Seventeen neoplasms (from 16 patients) histologically diagnosed as AFH were assessed for EWSR1 rearrangements or EWSR1-CREB1 and EWSR1-ATF1 fusion transcripts. All 17 were positive for either FISH or RT-PCR or both. Of 16, 14 (87.5%) had detectable EWSR1-CREB1 or EWSR1-ATF1 fusion transcripts by RT-PCR, whereas 13 (76.5%) of 17 had positive EWSR1 rearrangement with FISH. All 13 of 13 non-AFH control neoplasms failed to show EWSR1-CREB1 or EWSR1-ATF1 fusion transcripts, whereas EWSR1 rearrangement was present in 2 of these 13 cases (which were histopathologically myoepithelial neoplasms). This study shows that EWSR1-CREB1 or EWSR1-ATF1 fusions predominate in AFH (supporting previous reports that FUS rearrangement is rare in AFH) and that RT-PCR has a comparable detection rate to FISH for AFH. Importantly, cases of AFH can be missed if RT-PCR is not performed in conjunction with FISH, and RT-PCR has the added advantage of specificity, which is crucial, as EWSR1 rearrangements are present in a variety of neoplasms in the histologic differential diagnosis of AFH, that differ in behavior and treatment.

Development and evaluation of the polymerase chain reaction (PCR) method for diagnosis of spring viremia of carp virus (SVCV)

Aquaculture has been expanded rapidly to become a major commercial and food-producing sector worldwide in recent decade. In parallel, viral diseases rapidly spread among farms causing enormous economic losses. The accurate detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Spring Viraemia of Carp Virus (SVCV) is a very severe pathogen of carp fishes in different parts of the world and is categorized as a reportable listed disease in the annual published list of World Organization for animal Health (OIE). The objective of this study was to develop and evaluate RT- PCR test for detecting SVC virus and also the sensitivity and specificity of this test. A semi nested RT- PCR was designed using combination of three primers: two external (SVCF , SVCR) and one internal (SVCS) primers which based on conserved region of G gen. The specificity of designed primers (only external ones) by examination on Viral Hemorrhagic Septicemia Virus (VHSV) and Infectious Hematopoietic Necrosis Virus (IHNV) was confirmed. For optimizing of the PCR test, primer concentration, primer annealing temperature, cycle number and Mgcl2 concentration were surveyed. Also for validity test, prevention of false negative and Assurance of its accuracy, a competitive internal control (mimic) designed and its suitable concentration was defined. Evaluation of the sensitivity of designed test were conducted first by comparing the different commercially available RNA isolation guidelines, two guidelines: isotiocyanate phenol–chloroform based protocols (RNX–Plus Iran, Iq2000 kit Taiwan ) and two column based protocols (Cinna pure RNA Iran , high pure viral RNA kit, Roche Germany ). The results indicated that the column based protocols (Roche method and Cinna pure), yield 36.77 ng/μl and 16/47 ng/μl RNA concentration respectively, which were significantly higher than other protocols(P<0.05). Then for evaluation of extracted RNA sensitivity, Serial dilution of SVCV strain 56.70 grown in EPC (1.9×105 TCID50/ml) was examined To compare sensitivity. Extracted RNA from serial dilution with stone's primers and commercial IQ-2000 kit were examined simultaneously. The result indicated that designed semi- nested RT- PCR was able to recognize SVC virus to 10-4 dilution and stone's primer recognize to 10-3 dilution whereas Iq-2000 commercial kit did not recognized in any dilution. In high virus titer in designed test two DNA band (462 bp and 266 bp) produced, and by decreasing virus titer 462 bp was omitted. In low virus titer or lack of virus, just DNA band (mimic) 729 bp can propagate. After designing and optimizing PCR test, a total of 400 suspected cultured Cyprinus carpio with high mortality from 4 aquaculture zone of Khuzestan province were collected and tested for SVCV during 2012- 2013 using developed PCR method and IQ- 2000. The results indicated that SVC virus was not observed in samples using both methods.

An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy.

Survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction

This work reports a survey of Leptospira spp in pampas deer (Ozotoceros bezoarticus) in the Pantanal wetlands of the state of Mato Grosso do Sul, Brazil by serology and polymerase chain reaction (PCR). Seventy pampas deer were captured in the dry season and surveyed using PCR, microscopic agglutination test (MAT) (n = 51) and by both techniques (n = 47). PCR detected infections in two pampas deer and MAT detected infections in three. Through sequencing and phylogenetic analyses, the PCR-amplified fragment detected in deer was identified as Leptospira interrogans. Serovars Pomona and Butembo were detected using MAT and the highest titre was 200 for serovar Pomona. Epidemiological aspects of the findings are discussed.