Detection and dynamics of porcine circovirus 2 shedding in semen using conventional and real-time PCR

The dynamics of porcine circovirus type 2 (PCV2) shedding in semen of naturally infected boars was studied. Semen was collected serially each 15 or 20 days during 62 days from 5 boars from a herd and from 11 boars from an artificial insemination center. All boars were positive for PCV2 DNA by nested polymerase chain reaction of raw semen in at least two sampling dates, and most of them had detectable shedding in all sampling dates. Real-time quantitative PCR was performed in 23 samples. All samples showed low amounts of PCV2 DNA, ranging from 98 to 652 PCV2 copies/mL. No differences between the frequencies of PCV2 DNA shed in semen were found considering herds and age of boars. PCV2 shedding in the semen can occur continuously or intermittently up to 60 days in naturally infected boars at 12 to 42 months old in absence of PCV2 clinical signs. These results demonstrate sporadic and long-term shedding patterns of low amounts of PCV2 DNA in semen from naturally infected boars.

Detection and quantification of Roundup Ready® soybean residues in sausage samples by conventional and real-time PCR

The increasing presence of products derived from genetically modified (GM) plants in human and animal diets has led to the development of detection methods to distinguish biotechnology-derived foods from conventional ones. The conventional and real-time PCR have been used, respectively, to detect and quantify GM residues in highly processed foods. DNA extraction is a critical step during the analysis process. Some factors such as DNA degradation, matrix effects, and the presence of PCR inhibitors imply that a detection or quantification limit, established for a given method, is restricted to a matrix used during validation and cannot be projected to any other matrix outside the scope of the method. In Brazil, sausage samples were the main class of processed products in which Roundup Ready® (RR) soybean residues were detected. Thus, the validation of methodologies for the detection and quantification of those residues is absolutely necessary. Sausage samples were submitted to two different methods of DNA extraction: modified Wizard and the CTAB method. The yield and quality were compared for both methods. DNA samples were analyzed by conventional and real-time PCR for the detection and quantification of Roundup Ready® soybean in the samples. At least 200 ng of total sausage DNA was necessary for a reliable quantification. Reactions containing DNA amounts below this value led to large variations on the expected GM percentage value. In conventional PCR, the detection limit varied from 1.0 to 500 ng, depending on the GM soybean content in the sample. The precision, performance, and linearity were relatively high indicating that the method used for analysis was satisfactory.

Molecular diagnosis of strongyloidiasis in tropical areas: a comparison of conventional and real-time polymerase chain reaction with parasitological methods

This study aimed to evaluate the use of conventional polymerase chain reaction (cPCR) and real-time quantitative PCR (qPCR) in the diagnosis of human strongyloidiasis from stool samples in tropical areas. Stool samples were collected from individuals and were determined to be positive for Strongyloides stercoralis (group I), negative for S. stercoralis (group II) and positive for other enteroparasite species (group III). DNA specific to S. stercoralis was found in 76.7% of group I samples by cPCR and in 90% of group I samples by qPCR. The results show that molecular methods can be used as alternative tools for detecting S. stercoralis in human stool samples in tropical areas.

Bioeffects of albumin-encapsulated microbubbles and real-time myocardial contrast echocardiography in an experimental canine model

Myocardial contrast echocardiography has been used for assessing myocardial perfusion. Some concerns regarding its safety still remain, mainly regarding the induction of microvascular alterations. We sought to determine the bioeffects of microbubbles and real-time myocardial contrast echocardiography (RTMCE) in a closed-chest canine model. Eighteen mongrel dogs were randomly assigned to two groups. Nine were submitted to continuous intravenous infusion of perfluorocarbon-exposed sonicated dextrose albumin (PESDA) plus continuous imaging using power pulse inversion RTMCE for 180 min, associated with manually deflagrated high-mechanical index impulses. The control group consisted of 3 dogs submitted to continuous imaging using RTMCE without PESDA, 3 dogs received PESDA alone, and 3 dogs were sham-operated. Hemodynamics and cardiac rhythm were monitored continuously. Histological analysis was performed on cardiac and pulmonary tissues. No hemodynamic changes or cardiac arrhythmias were observed in any group. Normal left ventricular ejection fraction and myocardial perfusion were maintained throughout the protocol. Frequency of mild and focal microhemorrhage areas in myocardial and pulmonary tissue was similar in PESDA plus RTMCE and control groups. The percentages of positive microscopical fields in the myocardium were 0.4 and 0.7% (P = NS) in the PESDA plus RTMCE and control groups, respectively, and in the lungs they were 2.1 and 1.1%, respectively (P = NS). In this canine model, myocardial perfusion imaging obtained with PESDA and RTMCE was safe, with no alteration in cardiac rhythm or left ventricular function. Mild and focal myocardial and pulmonary microhemorrhages were observed in both groups, and may be attributed to surgical tissue manipulation.

Detection of replication-defective hepatitis A virus based on the correlation between real-time polymerase chain reaction and ELISA in situ results

ELISA in situ can be used to titrate hepatitis A virus (HAV) particles and real-time polymerase chain reaction (RT-PCR) has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10(5) copies/mL) (p = 0.0002), while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001). At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.

MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR) ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonellaspecies, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

Evaluation of the use of real-time PCR for human T cell lymphotropic virus 1 and 2 as a confirmatory test in screening for blood donors

INTRODUCTION: HTLV-1/2 screening among blood donors commonly utilizes an enzyme-linked immunosorbent assay (EIA), followed by a confirmatory method such as Western blot (WB) if the EIA is positive. However, this algorithm yields a high rate of inconclusive results, and is expensive. METHODS: Two qualitative real-time PCR assays were developed to detect HTLV-1 and 2, and a total of 318 samples were tested (152 blood donors, 108 asymptomatic carriers, 26 HAM/TSP patients and 30 seronegative individuals). RESULTS: The sensitivity and specificity of PCR in comparison with WB results were 99.4% and 98.5%, respectively. PCR tests were more efficient for identifying the virus type, detecting HTLV-2 infection and defining inconclusive cases. CONCLUSIONS: Because real-time PCR is sensitive and practical and costs much less than WB, this technique can be used as a confirmatory test for HTLV in blood banks, as a replacement for WB.

Detection of human bocavirus and human metapneumovirus by real-time PCR from patients with respiratory symptoms in Southern Brazil

The introduction of newer molecular methods has led to the discovery of new respiratory viruses, such as human metapneumovirus (hMPV) and human bocavirus (hBoV), in respiratory tract specimens. We have studied the occurrence of hMPV and hBoV in the Porto Alegre (PA) metropolitan area, one of the southernmost cities of Brazil, evaluating children with suspected lower respiratory tract infection from May 2007-June 2008. A real-time polymerase chain reaction method was used for amplification and detection of hMPV and hBoV and to evaluate coinfections with respiratory syncytial virus (RSV), influenza A and B, parainfluenza 1, 2 and 3, human rhinovirus and human adenovirus. Of the 455 nasopharyngeal aspirates tested, hMPV was detected in 14.5% of samples and hBoV in 13.2%. A unique causative viral agent was identified in 46.2% samples and the coinfection rate was 43.7%. For hBoV, 98.3% of all positive samples were from patients with mixed infections. Similarly, 84.8% of all hMPV-positive results were also observed in mixed infections. Both hBoV and hMPV usually appeared with RSV. In summary, this is the first confirmation that hMPV and hBoV circulate in PA; this provides evidence of frequent involvement of both viruses in children with clinical signs of acute viral respiratory tract infection, although they mainly appeared as coinfection agents.

Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR), nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.

Fast test for assessing the susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by real-time PCR

Mycobacterium tuberculosis is the bacterium that causes tuberculosis (TB), a leading cause of death from infectious disease worldwide. Rapid diagnosis of resistant strains is important for the control of TB. Real-time polymerase chain reaction (RT-PCR) assays may detect all of the mutations that occur in the M. tuberculosis 81-bp core region of the rpoB gene, which is responsible for resistance to rifampin (RIF) and codon 315 of the katG gene and the inhA ribosomal binding site, which are responsible for isoniazid (INH). The goal of this study was to assess the performance of RT-PCR compared to traditional culture-based methods for determining the drug susceptibility of M. tuberculosis. BACTEC TM MGIT TM 960 was used as the gold standard method for phenotypic drug susceptibility testing. Susceptibilities to INH and RIF were also determined by genotyping of katG, inhA and rpoB genes. RT-PCR based on molecular beacons probes was used to detect specific point mutations associated with resistance. The sensitivities of RT-PCR in detecting INH resistance using katG and inhA targets individually were 55% and 25%, respectively and 73% when combined. The sensitivity of the RT-PCR assay in detecting RIF resistance was 99%. The median time to complete the RT-PCR assay was three-four hours. The specificities for tests were both 100%. Our results confirm that RT-PCR can detect INH and RIF resistance in less than four hours with high sensitivity.

Simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene using quenched-FRET real-time PCR

Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation.

Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6)

HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.

QUANTITATIVE REAL-TIME PCR (Q-PCR) FOR SPUTUM SMEAR DIAGNOSIS OF PULMONARY TUBERCULOSIS AMONG PEOPLE WITH HIV/AIDS

Objective: To assess quantitative real-time polymerase chain reaction (q-PCR) for the sputum smear diagnosis of pulmonary tuberculosis (PTB) in patients living with HIV/AIDS with a clinical suspicion of PTB.Method: This is a prospective study to assess the accuracy of a diagnostic test, conducted on 140 sputum specimens from 140 patients living with HIV/AIDS with a clinical suspicion of PTB, attended at two referral hospitals for people living with HIV/AIDS in the city of Recife, Pernambuco, Brazil. A Löwenstein-Jensen medium culture and 7H9 broth were used as gold standard.Results: Of the 140 sputum samples, 47 (33.6%) were positive with the gold standard. q-PCR was positive in 42 (30%) of the 140 patients. Only one (0.71%) did not correspond to the culture. The sensitivity, specificity and accuracy of the q-PCR were 87.2%, 98.9% and 95% respectively. In 39 (93%) of the 42 q-PCR positive cases, the CT (threshold cycle) was equal to or less than 37.Conclusion: q-PCR performed on sputum smears from patients living with HIV/AIDS demonstrated satisfactory sensitivity, specificity and accuracy, and may therefore be recommended as a method for diagnosing PTB.

Rapid detection of Van genes in rectal swabs by real time PCR in Southern Brazil

INTRODUCTION: Laboratory-based surveillance is an important component in the control of vancomycin resistant enterococci (VRE). METHODS: The study aimed to evaluate real-time polymerase chain reaction (RT-PCR) (genes vanA-vanB) for VRE detection on 115 swabs from patients included in a surveillance program. RESULTS: Sensitivity of RT-PCR was similar to primary culture (75% and 79.5%, respectively) when compared to broth enriched culture, whereas specificity was 83.1%. CONCLUSIONS: RT-PCR provides same day results, however it showed low sensitivity for VRE detection.