Conventional Real-time Polymerase Chain Reaction Research Articles

PCR protocol for detection of Vibrio ordalii by amplification of the vohB (hemolysin) gene

Vibrio ordalii is the causative agent of atypical vibriosis and has the potential to cause severe losses in salmonid aquaculture. To prevent and control outbreaks, a rapid, reproducible, sensitive, and effective diagnostic method is needed. We evaluated a new conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) protocol using a primer set (VohB_Fw-VohB_Rv) designed to amplify a 112 bp fragment flanking the vohB gene (coding for hemolysin production), against 24 V. ordalii strains isolated from different fish species, the V. ordalii type strain, and 42 representative related and unrelated bacterial species. The primer set was species-specific, recognizing all V. ordalii strains evaluated, with no cross-reaction with the other bacterial species. A sensitivity of 103 copies of the vohB gene was obtained with a standard curve. When the VohB_Fw-VohB_Rv qPCR protocol was applied to Atlantic salmon seeded tissues (kidney, liver, spleen, and muscle), the detection limit ranged from 5.27 × 102 to 4.13 × 103 V. ordalii CFU ml-1, i.e. 62 to 145 copies of the vohB gene, using the previously calculated standard curve. The conventional PCR also detected V. ordalii, but the total reaction time was 1 h longer. When the qPCR protocol was applied to naturally infected cage-cultured Atlantic salmon samples, 5 of 8 fish tested positive for V. ordalii, but only one of them was diagnosed as positive by direct cultivation on agar. We conclude that the PCR protocol evaluated is fast, specific, and sensitive enough to detect V. ordalii in infected tissues and is an important tool for secure diagnosis of atypical vibriosis, and is therefore helpful for the control of the disease through the prompt detection within fish populations.

Comparison between Conventional and Real-Time PCR Assays for Diagnosis of Visceral Leishmaniasis

The diagnosis of visceral leishmaniasis (VL) is a challenging issue and several studies worldwide have evaluated the different tools to reach a diagnostic solution. The polymerase chain reaction (PCR) has proven to be effective in detecting the genome of Leishmania species in different biological samples. In this study, we compared the conventional PCR and real-time PCR using the Sybr Green system and their application in molecular diagnosis of visceral leishmaniasis in peripheral blood as a biological sample. The genus-specific conserved region of kinetoplast DNA (kDNA) was the target of amplification. We studied 30 samples from patients with suspect of visceral leishmaniasis who were treated by the Medical Clinic of Santa Casa de Belo Horizonte Hospital, Brazil. Among the samples studied, 19 had a confirmed diagnosis for VL by serology and/or by clinical findings. Among these 19 samples, 63% (n = 12) presented positive results for serology and 79% (n = 15) positive results in both PCR methodologies. This fact suggests that the PCR technique can assist in the diagnosis of visceral leishmaniasis in patients who do not have detectable antibodies by serology but can present the genome of the parasite circulating in whole blood. Also, it was possible to observe that there was conformity between the results of the techniques of cPCR and qPCR using the Sybr Green system in 100% of samples analyzed. These data suggest that both PCR techniques were equally effective for detection of the genome of the parasite in the patient's blood.

Detection of viruses in the peritoneum of the Iranian patients on peritoneal dialysis

Despite reductions in overall infectious peritonitis rate due to improvement in peritoneal dialysis (PD) systems, peritonitis is still a major complication of PD and a major cause of failure of this method of dialysis. In this context, viral infections have rarely been investigated, although up to 20% of cases remain culture negative (non-bacterial, non-fungal). The present study aimed to screen stored PD effluent samples for viral infection to assess the incidence in a cross-sectional cohort of patients on PD. Nucleic acid extracted from PD samples (n=205) were analysed using either conventional polymerase chain reaction (PCR) or real-time PCR for polyomaviruses (JCV, BKV, KIV, WUV, MCV, SV40, and LPV), human cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex viruses (HSV-1 and 2), varicella-zoster virus, adenoviruses, enteroviruses, parechovirus and human norovirus. Of the 205 PD effluent samples analysed, 10 (4.8%) were found to be positive for viral infections (BKV: 2.9%; CMV: 1.9%). One out of ten positive patients were clinically well at the time of PD effluent collection although five patients who tested positively for CMV or BKV was diagnosed with sterile peritonitis. This study has described that BKV and CMV may be present in the peritoneum of patients on PD. BKV and CMV should be considered as a potential cause of aseptic peritonitis in patients on PD. Key words: Peritoneal dialysis (PD), peritonitis, polymerase chain reaction (PCR), BK polyomavirus, cytomegalovirus (CMV).

A New High-Speed Droplet-Real-Time Polymerase Chain Reaction Method Can Detect Bovine Respiratory Syncytial Virus in Less than 10 Min

ABSTRACTThe polymerase chain reaction (PCR) has been widely used for diagnosis of infectious diseases of domestic animals. Rapid detection of respiratory pathogens of cattle is useful for making therapeutic decisions. Therefore, we developed a new genetic-based method called droplet-real-time PCR, which can detect bovine respiratory syncytial virus (BRSV) within 10 min. Our droplet-real-time PCR markedly reduced the reaction time of reverse transcription-PCR while maintaining the same sensitivity as conventional real-time PCR, and it can be used as a rapid assay for detection of BRSV. Furthermore, our method is potentially applicable for rapid diagnosis of almost all infectious diseases, including highly pathogenic avian influenza virus.

A specific polymerase chain reaction method to identify Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is a multidrug-resistant nosocomial pathogen that is difficult to identify unequivocally using current methods. Accordingly, because the presence of this microorganism in a patient may directly determine the antimicrobial treatment, conventional polymerase chain reaction (PCR) and real-time PCR assays targeting 23S rRNA were developed for the specific identification of S. maltophilia. The PCR protocol showed high specificity when tested against other species of Stenotrophomonas, non-fermentative Gram-negative bacilli and 100 clinical isolates of S. maltophilia previously identified using the Vitek system.

Genetic Markers Associated to Dyslipidemia in HIV-Infected Individuals on HAART

This study evaluated the impact of 9 single nucleotide polymorphisms (SNPs) in 6 candidate genes (APOB, APOA5, APOE, APOC3, SCAP, and LDLR) over dyslipidemia in HIV-infected patients on stable antiretroviral therapy (ART) with undetectable viral loads. Blood samples were collected from 614 patients at reference services in the cities of Porto Alegre, Pelotas, and Rio Grande in Brazil. The SNPs were genotyped by conventional polymerase chain reaction (PCR) and real-time PCR. The prevalence of dyslipidemia was particularly high among the protease inhibitors-treated patients (79%). APOE (rs429358 and rs7412) genotypes and APOA5 −1131T>C (rs662799) were associated with plasma triglycerides (TG) and low-density-lipoprotein cholesterol levels (LDL-C). The APOA5 −1131T>C (rs662799) and SCAP 2386A>G (rs12487736) polymorphisms were significantly associated with high-density-lipoprotein cholesterol levels. The mean values of the total cholesterol and LDL-C levels were associated with both the APOB SP Ins/Del (rs17240441) and APOB XbaI (rs693) polymorphisms. In conclusion, our data support the importance of genetic factors in the determination of lipid levels in HIV-infected individuals. Due to the relatively high number of carriers of these risk variants, studies to verify treatment implications of genotyping before HAART initiation may be advisable to guide the selection of an appropriate antiretroviral therapy regimen.

Quantitative detection of Proteus species by real-time polymerase chain reaction using SYBR Green I

A SYBR Green real-time polymerase chain reaction (PCR) method for rapid detection of Proteus species was developed and evaluated. Of 322 clinical and food samples tested, 75 samples were positive for Proteus species by using conventional PCR and real-time PCR assays. The results were consistent with standard culture methods and the Vitek auto-microbe system, indicating a 100 % specificity obtained by both PCR assays. For the real-time PCR method, the minimum detectable level was 10 colony forming units (CFU) /ml, which was a 103 multiple higher than the conventional PCR method. Correlation coefficients of standard curves which were constructed using the threshold cycle (Ct) versus copy numbers of Proteus showed good linearity (R 2 = 0.997). In conclusion, several significant advantages such as higher sensitivity and rapidness were observed by using the SYBR Green real-time PCR method for identifying Proteus species.

Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood

Species of Candida frequently cause life-threatening infections in neonates, transplant and intensive care unit (ICU) patients, and others with compromised host defenses. The successful management of systemic candidiasis depends upon early, rapid diagnosis. Blood cultures are the standard diagnostic method, but identification requires days and less than half of the patients are positive. These limitations may be eliminated by using real-time polymerase chain reaction (PCR) to detect Candida DNA in the blood specimens of patients at risk. Here, we optimized a PCR protocol to detect 5-10 yeasts in low volumes of simulated and clinical specimens. We also used a mouse model of systemic candidiasis and determined that candidemia is optimally detectable during the first few days after infection. However, PCR tests are often costly, labor-intensive, and inconvenient for routine use. To address these obstacles, we evaluated the innovative microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.), which has the potential for full automation and rapid turnaround. Eleven and nine of 16 specimens from individual patients with culture-proven candidemia tested positive for C. albicans DNA by conventional and microfluidic real-time PCR, respectively, for a combined sensitivity of 94%. The microfluidic platform offers a significant technical advance in the detection of microbial DNA in clinical specimens.

Infectious laryngotracheitis virus in chickens

Infectious laryngotracheitis (ILT) is an important respiratory disease of chickens and annually causes significant economic losses in the poultry industry world-wide. ILT virus (ILTV) belongs to alphaherpesvirinae and the Gallid herpesvirus 1 species. The transmission of ILTV is via respiratory and ocular routes. Clinical and post-mortem signs of ILT can be separated into two forms according to its virulence. The characteristic of the severe form is bloody mucus in the trachea with high mortality. The mild form causes nasal discharge, conjunctivitis, and reduced weight gain and egg production. Conventional polymerase chain reaction (PCR), nested PCR, real-time PCR, and loop-mediated isothermal amplification were developed to detect ILTV samples from natural or experimentally infected birds. The PCR combined with restriction fragment length polymorphism (RFLP) can separate ILTVs into several genetic groups. These groups can separate vaccine from wild type field viruses. Vaccination is a common method to prevent ILT. However, field isolates and vaccine viruses can establish latent infected carriers. According to PCR-RFLP results, virulent field ILTVs can be derived from modified-live vaccines. Therefore, modified-live vaccine reversion provides a source for ILT outbreaks on chicken farms. Two recently licensed commercial recombinant ILT vaccines are also in use. Other recombinant and gene-deficient vaccine candidates are in the developmental stages. They offer additional hope for the control of this disease. However, in ILT endemic regions, improved biosecurity and management practices are critical for improved ILT control.

Rapid, specific and quantitative polymerase chain reaction (PCR) detection of pathogenic protozoa Entamoeba histolytica for drinking water supply

In this study, a quantitative species-specific polymerase chain reaction (PCR) method to rapidly detect E. histolytica in water is developed. First, the specificity of E. histolytica PCR detection was verified by using species-specific primers of 16S-like rRNA genes to clearly differentiate it from the closely related amoebae species E. dispar and E. moshkovskii. The sensitivity of this method was subsequently determined using purified E. histolytica genomic DNA and culture cells as PCR reaction templates. Results indicated that conventional PCR visualized on 1% agarose gel was able to detect as low as 0.02 pg genomic DNA and 5 cells, while real-time PCR could detect 0.01 pg genomic DNA and 2 cells of E. histolytica. The protocols for E. histolytica PCR detection in real water samples were then optimized by spiking E. histolytica cells into tap water and reservoir raw water samples. A two-round centrifugation treatment to concentrate amoeba cells directly as a PCR template was the most effective way to detect E. histolytica in spiked tap water samples, while DNA extraction after concentrating amoeba cells was required for spiked reservoir raw water samples. The detection limit of 50 E. histolytica cells in 100 ml tap water was achieved in 2 h from sample collection to real-time PCR data readout. With these established protocols, 78 tap water samples, 11 reservoir raw water samples and 4 feed water samples from Singapore water supply systems were analyzed by both conventional PCR and real-time PCR methods. No E. histolytica cell was detected in tested samples.

Detection of ‘CandidatusLiberibacter Solanacearum’ in the Potato Psyllid,Bactericera cockerelli(Sulc)1, by Conventional and Real-Time PCR

‘Candidatus Liberibacter solanacearum’ is the putative causal agent of zebra chip disease of potato, Solanum tuberosum L., and is transmitted by the potato psyllid, Bactericera cockerelli (Sulc). Much work has been done on the association of Liberibacter with the potato psyllid and several laboratories currently use polymerase chain reaction (PCR) to test for the bacterium. We have been testing for the bacterium in individual and composite samples of B. cockerelli as part of an ongoing investigation of the incidence of ‘Ca. L. solanacearum’ in potato psyllids collected in various parts of the US. Here we report that the bacterium can be reliably detected by using conventional and quantitative real-time PCR of nucleic acid extracts prepared from composite samples of one psyllid from a Liberibacterinfected colony combined with either one, two, three, four, five, nine, 14, 19, or 29 psyllids from a Liberibacter-free psyllid colony. This ability makes it possible to more easily test large numbers of psyllids for the presence of ‘Ca. Liberibacter solanacearum’. This, in turn, will aid efforts to predict the threat of developing serious zebra chip disease as a result of psyllid infestations in potato fields.

Comparison of a conventional polymerase chain reaction with real-time polymerase chain reaction for the detection of neurotropic viruses in cerebrospinal fluid samples

Purpose: To compare a conventional polymerase chain reaction (PCR) and real-time PCR for the detection of neurotropic DNA viruses. Materials and Methods: A total of 147 cerebrospinal fluid (CSF) samples was collected from patients attending a tertiary care hospital in South India for a period from 2005 to 2008. All these samples were tested using a conventional multiplex/uniplex PCR and a real-time multiplex/uniplex PCR. This technique was used to detect a large number of herpes viruses responsible for central nervous system infections, including HSV-1, HSV-2, VZV, CMV and EBV and the polyoma virus JCV. Results: Overall, in the entire set of samples, the real-time PCR yielded 88 (59.9%) positives and conventional PCR had six (4.1%) positives. Conclusion: Our results suggest that the real-time PCR assay was more sensitive compared with the conventional PCR. The advantage of real-time PCR is that it can be performed much faster than conventional PCR. Real-time PCR is less time-consuming, less labour-intensive and also reduces the chance of contamination as there is no post-amplification procedure. In the entire study population, the major viruses detected using real-time PCR were EBV (34%), HSV-2 (10.8%) and VZV (6.8%).

Detection and Monitoring of Greeneria uvicola and Colletotrichum acutatum Development on Grapevines by Real-Time PCR.

Bitter rot (Greeneria uvicola) and ripe rot (Colletotrichum acutatum, syn. C. simmondsii) occur frequently in subtropical grape-growing regions of Australia, where they cause yield loss and bitter taints in wine. To further advance the epidemiological studies of G. uvicola and C. acutatum and contribute toward their effective management and control, a rapid and reliable species-specific real-time polymerase chain reaction (PCR) method was developed based on the polymorphic portion of the internal transcribed spacer region of the two fungi. It was found that, within 6 to 8 h postinoculation, the assay could detect as little as 20 fg of genomic DNA and 10 conidia for both species. Artificially and naturally infected grape inflorescences and mature berries were analyzed by both conventional plating methods and real-time PCR. Fungal presence was demonstrated on all plant material but development was observed only on mature berries. The results demonstrate that the real-time PCR technique is a highly specific, rapid, and sensitive method that can be used to detect and study the dynamics of G. uvicola and C. acutatum during different stages of infection and on different grape tissues.

DEVELOPMENT OF A DIAGNOSTIC PROTOCOL FOR CUCUMBER MOSAIC VIRUS FOR SCREENING BANANA (MUSA SPP.) PLANTING MATERIAL IN IVORY COAST

Cucumber mosaic virus (CMV) is the primary virus affecting commercial banana (Musa spp.) plantations in Ivory Coast, with the disease also present within nursery systems. However, currently no virus indexing of planting material is performed for banana in the country. This current study attempts to quantify levels of detection of CMV within banana material using flinders technology associates (FTA) cards in order to identify a reliable and reproducible method suitable for local conditions. A suite of PCR primers were developed against CMV coat protein open reading frame (CP ORF) sequence available on GenBank. TaqMan assay generated comparable CT values with either FTA or cetyltrimethyl ammonium bromide (CTAB) derived templates and was able to detect CMV RNA in leaf samples. The conventional real time polymerase chain reaction (RT-PCR) assay, RNeasy and FTA derived RNA templates reliably amplified CMV, whilst CTAB-derived RNA gave variable results. The TaqMan ® reaction was much more sensitive than the conventional RT-PCR for CMV detection in banana leaf samples. The suitability of FTA cards as a matrix for virus nucleic acid capture provides scope for its use in the transportation of banana virus samples to laboratories for diagnosis and characterisation, circumventing constraints of time-related degeneration of sample integrity. Sequence data of the CMV CP ORF region of 16 isolates from Cote d'Ivoire clustered the isolates to CMV Sub-Group IA. This is the first successful application of Real-time PCR and FTA technology for a banana virus in Africa.

Authentication of Atlantic Cod (Gadus morhua) Using Real Time PCR

This work describes the development of a real-time polymerase chain reaction (RT-PCR) system for the detection and identification of Atlantic cod (Gadus morhua). Among the advantages of this technique, it is worth highlighting that this is reliable in terms of specificity and sensitivity. The TaqMan real-time PCR is the simplest, fastest testing process and has the highest potential for automation, therefore representing the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of this species. The method can be applied to all kinds of products, fresh, frozen, and processed products, including those undergoing intensive processes of transformation. The developed methodology using specific primer-probe set was validated and further applied to 40 commercial samples labeled as cod in order to determinate if the species used for their manufacturing corresponded to G. morhua, detecting 20% that were incorrectly labeled. A C(t) value of about 19 was obtained when G. morhua was present. In samples with a species mixture, all samples that had a fluorescence signal were positive (C(t) < 30) for the presence of G. morhua by conventional end-point RT-PCR, and the estimated limit of detection for these type of samples was of 20 pg of DNA. The methodology herein developed is useful to check the fulfilment of labeling regulations for seafood products and verify the correct traceability in commercial trade and for fisheries control.

Microfluidic platform versus conventional real-time polymerase chain reaction for the detection of Mycoplasma pneumoniae in respiratory specimens

Rapid, accurate diagnosis of community-acquired pneumonia (CAP) due to Mycoplasma pneumoniae is compromised by low sensitivity of culture and serology. Polymerase chain reaction (PCR) has emerged as a sensitive method to detect M. pneumoniae DNA in clinical specimens. However, conventional real-time PCR is not cost-effective for routine or outpatient implementation. Here, we evaluate a novel microfluidic real-time PCR platform (Advanced Liquid Logic, Research Triangle Park, NC) that is rapid, portable, and fully automated. We enrolled patients with CAP and extracted DNA from nasopharyngeal wash (NPW) specimens using a biotinylated capture probe and streptavidin-coupled magnetic beads. Each extract was tested for M. pneumoniae-specific DNA by real-time PCR on both conventional and microfluidic platforms using Taqman probe and primers. Three of 59 (5.0%) NPWs were positive, and agreement between the methods was 98%. The microfluidic platform was equally sensitive but 3 times faster and offers an inexpensive and convenient diagnostic test for microbial DNA.

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.

Application of real-time polymerase chain reaction (PCR) coupled with ethidium monoazide treatment for selective quantification of viable bacteria in aquatic environment

Ethidium monoazide (EMA) was used to quantify DNA selectively from viable cells with healthy membrane/cell wall system, but not from dead cells, of a target bacterium in the aquatic environment using real-time PCR. Spiking experiments to determine the EMA treatment conditions showed that EMA treatment with EMA at 10-25 microg/ml and subsequent halogen light exposure for 2 min was suitable for selective quantification of DNA from viable cells in an aquatic sample using real-time PCR coupled with EMA treatment (real-time EMA-PCR). Optimized real-time EMA-PCR was applied in combination with culture-based method and conventional real-time PCR without EMA treatment to elucidate the behavior of an Escherichia coli strain inoculated into a pond water microcosm. Quantification results obtained using real-time EMA-PCR were lower than those by conventional real-time PCR without EMA treatment and higher than those by culture-based method. The results suggest that quantification by real-time EMA-PCR seemed to represent the viable population, which would partly include viable but non-culturable state bacteria. Real-time EMA-PCR optimized here can be a useful tool for selective monitoring of the viable population of a target bacterium in the aquatic environment, and thereby contribute to assessment of potential microbial risks generated from waterborne pathogenic bacteria.

Canine Parvovirus 2c Infection in Central Portugal

Canine parvovirus (CPV) has been evolving, generating new genetic and antigenic variants throughout the world. This study was conducted to determine the types of CPV circulating in dogs in Figueira da Foz, Portugal. Thirty fecal samples, collected between 2006 and 2007 from dogs with clinical signs of CPV infection, were tested for CPV by a rapid, in-clinic, enzyme-linked immunosorbent assay (ELISA)/immunomigration test, by conventional real-time polymerase chain reaction (PCR), and by minor-groove binding TaqMan PCR. Of the 29 PCR-positive samples, 15 were identified as CPV-2b and 14 as CPV-2c. No CPV-2a was detected. The sensitivity of the ELISA test was 82.76% compared with the PCR assays. No significant associations were found between CPV type, clinical outcome, breed, vaccination status, or age.

A prospective study of diagnosis of Toxoplasma gondii infection after bone marrow transplantation

Active infection with Toxoplasma gondii in immunocompromised transplant recipients can lead to toxoplasmosis, which may have a rapid disease course and in some cases be fatal. It is of paramount importance to diagnose toxoplasmosis at an early stage, and to initiate specific treatment to improve the outcome. Polymerase chain reaction (PCR) is today the primary diagnostic tool to diagnose toxoplasmosis in immunocompromised patients. Timely diagnosis may, however, be difficult if toxoplasmosis is at first asymptomatic. To investigate the magnitude of toxoplasmosis after bone marrow transplantation (BMT), we conducted a screening study by PCR where 21 autologous and 12 allogeneic BMT recipients were included. Peripheral blood samples were taken one week prior to BMT; thereafter, blood samples were drawn weekly for the first 6 months, and monthly up to one year after BMT. The samples were analyzed by conventional PCR and real-time PCR. T. gondii DNA was detected in peripheral blood from one patient 5 days post allogeneic BMT. There were no clinical signs of toxoplasmosis. Medical records were reviewed and showed a previously undiagnosed eye infection in another allogeneic BMT recipient. These two patients were seropositive for T. gondii. We concluded that monitoring for T. gondii DNA in peripheral blood samples using PCR might be a valuable method for identifying toxoplasma-seropositive stem cell transplant recipients.