Real-time PCR Assay For Detection Research Articles

Novel peptide nucleic acid-based real-time PCR assay for detection and genotyping of Megalocytivirus

Megalocytivirus, the causative agent for Red Sea Bream Iridoviral Disease (RSIVD), is classified into three genotypes based on its phylogeny: Red Sea Bream Iridovirus (RSIV), Infectious Spleen and Kidney Necrosis Virus (ISKNV), and Turbot Reddish Body Iridovirus (TRBIV). To overcome drawbacks such as limitation of genotypic variant type discrimination and unexpected PCR reaction from an SYBR green-based genotyping method by high-resolution melting analysis, peptide nucleic acid (PNA)-based real-time PCR assay for detecting and genotyping Megalocytivirus was developed. Compared with DNA hybridization, PNA has more suitable hybridization traits that allowed the difference in melting temperature (Tm) even by a single nucleotide mismatch. Based on the consensus sequences of the viral major capsid protein gene, four PNA probes labeled with respective fluorescence at their 3′ ends were designed as reporter molecules. PNA-based real-time PCR assay allowed quantification of Megalocytivirus copies using a synthetic DNA as standard (detection limit of 103 copies) and discrimination of the genotypes based on Tm in a single-tube reaction. Furthermore, the results of this new method were consistent with those of sequencing analysis of Megalocytivirus-infected field and artificial samples, proving its accuracy and efficiency. The novel PNA-based real-time PCR assay could contribute to the detection as well as discrimination of the genotypes of Megalocytivirus in a single simple assay, and could be used as a highly robust diagnostic tool in the field of aquatic animal diseases.

Development of a real-time PCR assay for detection of Cryptosporidium canis in dog fecal samples.

Cryptosporidiosis is an emerging zoonotic disease caused by the worldwide distributed parasitic protozoa Cryptosporidium spp. The host-adapted species Cryptosporidium canis is most frequently found in dogs, although human infections with this species have been described. This study aimed to develop a real-time PCR targeting the HSP70 protein gene for C. canis DNA detection in dog fecal samples collected from two municipalities in the state of São Paulo, Brazil. Furthermore, the occurrence of Cryptosporidium spp. and. C. canis was also determined by nested PCR. Fecal samples from 367 dogs (21 puppies and 346 adults) were purified by water-ether sedimentation. A real-time PCR protocol targeting the HSP70 gene for the species-specific detection of C. canis was developed and compared with nested PCR results. Real-time PCR identified C. canis in 15.3% (58/367) samples. Nested PCR revealed that 10.4% (38/367) of samples were positive for Cryptosporidium spp. All sequenced 18S rRNA amplicons were C. canis. There was a higher prevalence of Cryptosporidium spp. and C. canis in puppies compared to adult dogs. No non-specific amplification was observed in C. canis specific real-time PCR assay.

252. Development and Evaluation of a Novel MultiCode Real-Time PCR Assay for the Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid and Induced Sputum

Background Pneumocystis jirovecii is a medically important fungal pathogen responsible for opportunistic infections in immunocompromised hosts with high morbidity and mortality. Compared with standard microscopy based assays, home-brew nucleic acid amplification tests (NAAT) have emerged as sensitive tools for the diagnosis of P. jirovecii pneumonia, but their sensitivities vary depending upon selected genetic targets. Recent studies suggest that the mitochondrial small subunit (mtSSU) is a better NAAT target given its higher copy number and stable expression in the disease process. We aimed to develop and evaluate a mtSSU-targeted MultiCode real-time PCR assay that incorporates a sample processing control (SPC) and enables detection of P. jirovecii in bronchoalveolar lavage fluid (BALF) and induced sputum.MethodsFirstly, we compared manual DNA extraction using Zymo Quick DNA kit with automated extraction using the NucliSENS easyMAG system after sample pretreatment with either FastPrep mechanical grinding or vortex-based bead beating. We then determined the mouse hepatitis virus SPC (Luminex) spike-in amount, and optimized the PCR conditions on the ABI 7500 PCR system. A new Pneumocystis mtSSU run control was generated by cloning and transforming mtSSU gene into a genetically engineered E. coli strain, and quantified with a home-brew quantitative TaqMan PCR. Lastly, the performance characteristics of the MultiCode PCR assay were determined.ResultsMechanical grinding of BALF or sputum before the easyMAG based extraction was better than the other extraction protocols as evidenced by lower CT of mtSSU or SPC. Diluted SPC added to samples before DNA extraction made its CT within 31–34. With the mtSSU run control, the limit of detection of the new assay was 80 copies/mL. No cross-reactivity was found with 9 respiratory viruses, 8 bacteria or 11 fungi. The assay has high reproducibility for three-day detection of the same sample aliquots for mtSSU (CT: 30.0–30.3; CV%: 0.5–1.6) and SPC (CT: 32.1–32.2; CV%: 0.8–2.4).ConclusionWe developed a novel MultiCode real-time PCR assay for detection of P. jirovecii in BALF and sputum, which demonstrated high analytical sensitivity, specificity and reproducibility and warrants further clinical validation.Disclosures All authors: No reported disclosures.

Development of an EvaGreen-based real-time PCR assay for detection of Aleutian mink disease virus

The objective of this study was to develop a rapid, sensitive and specific EvaGreen (EG)-based real-time PCR assay capable of detecting Aleutian mink disease virus (AMDV) and to evaluate the reliability of the assay for analysis of blood or tissue samples. For this assay, a pair of primers was designed based on a nonstructural protein (NS)-encoding gene of AMDV, and the identity of PCR products was identified based on a melting temperature of 82.8°C. The EG-based real-time PCR assay did not detect canine distemper virus or mink enteritis virus, and the assay could be used to detect Chinese and American AMDV strains, in contrast to a commercial TaqMan kit that could only detect American AMDV strains. The amplification efficiencies of the EG assay were 104.8% for the Chinese strain and 94.4% for the American strain, and the detection limit was 1 copy/μL of AMDV plasmid or 3 pg/μL of viral DNA (Chinese strain). The intra- and inter-assay variation coefficients of melting temperature were all lower than 0.15%, confirming the high reproducibility of the assay. Forty-five clinical blood samples were simultaneously analyzed using the EG real-time PCR, TaqMan kit and conventional PCR, and the detection rates were 91.1%, 0.0% and 86.7%, respectively. Serum samples were also collected from the corresponding blood samples and tested using the counterimmunoelectrophoresis (CIEP) assay, where positive samples accounted for 24.4% of the 45 samples. In conclusion, EG-based real-time PCR is a rapid, sensitive, universal assay that can be effectively utilized as a reliable and specific tool for detection and quantitation of AMDV.

Validation of a real-time PCR assay for detection of swinepox virus.

Swine are the only known hosts of swinepox virus (SWPV), the sole member of the genus Suipoxvirus, family Poxviridae. Rapid diagnosis is recommended for appropriate interventions because of the high morbidity associated with this virus. This study describes a real-time quantitative PCR (qPCR) assay for rapid detection and quantification of SWPV. The detection limit, repeatability, reproducibility, and specificity of this assay were determined. The efficiency was 96%, and the R2 value was 0.996. The detection limit was 1 fg or 10-0.5 TCID50/50 μL. Tests showed that the greatest source of error in the SWPV qPCR assay was variation between analysts rather than different qPCR kits or equipment. All nucleic acids from other viruses or samples collected from swine were negative in the specificity test. qPCR for SWPV is a new method with tested variables that allows main sources of error in laboratory diagnosis and viral quantification to be identified.

Development and use of a triplex real-time PCR assay for detection of three DNA viruses in psittacine birds.

Aves polyomavirus 1, psittacine beak and feather disease virus, and psittacid herpesvirus 1 are important pathogens of psittacine birds with the potential to cause substantial morbidity and mortality. Using publically available nucleotide sequences, we developed and validated a triplex real-time PCR (rtPCR) assay to rapidly detect these 3 viruses. The assay had high analytical sensitivity, detecting <6 copies of viral DNA per reaction, and 100% analytical specificity, showing no cross-reactivity with 59 other animal pathogens. Archived formalin-fixed, paraffin-embedded tissues from psittacine birds diagnosed at postmortem as infected with each of the viruses as well as virus-negative birds were used to validate the utility of the assay. Birds were selected for the positive cohort if they showed histologic evidence of infection (i.e., characteristic inclusion bodies in tissues); birds in the negative cohort had final diagnoses unrelated to the pathogens of interest. The triplex rtPCR assay confirmed 98% of histopathology-positive cases, and also identified subclinical infections that were not observed by histologic examination, including coinfections. Birds that tested positive only by rtPCR had significantly higher cycle threshold values compared to those with histologic evidence of infection. Positive, negative, and overall percentage agreements as well as the kappa statistic between the results of the assay and histopathology were high, demonstrating the usefulness of the assay as a tool to confirm disease diagnoses, and to improve detection of subclinical infections.

A real-time PCR assay for detection of light leaf spot on Brassica crops in the Pacific Northwest of the USA

Outbreaks of light leaf spot (Pyrenopeziza brassicae), a disease new to North America, occurred along with the diseases blackleg (Leptosphaeria spp.) and white leaf spot (Mycosphaerella capsellae) on Brassica crops in western Oregon. Disease symptoms in seed fields planted in the fall of 2013 were observed in the spring of 2014. Field surveys were conducted for disease during 2014 and 2015 to determine the geographic range of light leaf spot, blackleg, and white leaf spot within the Willamette Valley of western Oregon. Infected seed crops included turnip and oilseed rape in Benton, Lane, Linn, Marion, Polk, and Yamhill counties. New protocols that are more sensitive for detection of light leaf spot were developed for the pathogen population in the Pacific Northwest of the USA. Pyrenopeziza brassicae was detected when samples also contained L. maculans or M. capsellae. The test did not react with a range of other fungi evaluated. Sensitivity of the PCR protocol reported is robust at 10 pg of genomic DNA. Standard and real-time PCR screenings developed in this research increase sensitivity for detection of P. brassicae in affected Brassica hosts, which is critical for timely diagnosis and management of this newly-introduced disease in Brassica crops in the Pacific Northwestern region of the USA.

Development and Validation of a Clinical Laboratory Improvement Amendments-Compliant Multiplex Real-Time PCR Assay for Detection of mcr Genes.

Increased use of colistin in both human and veterinary medicine has led to the emergence of plasmid-mediated colistin resistance (mcr genes). In this study, we report the development of a real-time PCR assay using TaqMan probe-based chemistry for detection of mcr genes from bacterial isolates. Positive control isolates harboring mcr-1 and mcr-2 yielded exponential amplification curves with the assay, and the amplification efficiency was 98% and 96% for mcr-1 and mcr-2, respectively. Each target gene could be reproducibly detected from a sample containing 103 cfu/mL of mcr-harboring bacteria, and there was no cross-reactivity with DNA extracted from several multidrug-resistant bacteria harboring other resistance genes, but lacking mcr genes. Both sensitivity and specificity of the mcr real-time PCR assay were 100% in a method validation performed with a set of 25 previously well-characterized bacterial isolates containing mcr-positive and -negative bacteria. This newly developed assay is a rapid and sensitive tool for detecting emerging mcr genes in cultured bacterial isolates. The assay was successfully validated according to quality standards of the Clinical Laboratory Improvement Amendments (CLIA).

EvaGreen-based real-time PCR assay for sensitive detection of enzootic nasal tumor virus 2

Enzootic nasal tumor virus 2 (ENTV-2), the aetiological agent of enzootic nasal adenocarcinoma in goats, is prevalent in China; resulting in substantial economic losses to the goat-breeding industry. Therefore, it is necessary to establish an efficient detection method for the diagnosis and prevention of ENTV-2 infection. More recently, EvaGreen is emerging as a novel alternative fluorescent dye for quantitative real-time PCR because of its low cost, specific amplification and high resolution. In this study, we developed a specific, sensitive, and cost-effective detection method-an EvaGreen-based real-time PCR assay for the detection of ENTV-2. This assay exhibited high specificity and sensitivity and was able to detect ENTV-2 at concentrations as low as 3.0 × 101 copies, which was more sensitive than the conventional PCR method (detection limit, 3.0 × 102 copies). In addition, the reproducibility test indicated that EvaGreen dye in our assay had a good reproducibility. In conclusion, we report that a highly sensitive, specific, and cost-effective EvaGreen-based real-time PCR assay is successful for the rapid detection of ENTV-2.

Rapid detection of human mastadenovirus species B by recombinase polymerase amplification assay

BackgroundAs an important component of the causative agent of respiratory tract infections, enteric and eye infections, Human mastadenoviruses (HAdVs) species B spread easily in the crowd. In this study, we developed a recombinase polymerase amplification (RPA) assay for rapidly detecting HAdVs species B which was comprised of two different formats (real-time and lateral-flow device).ResultsThis assay was confirmed to be able to detect 5 different HAdVs species B subtypes (HAdV-B3, HAdV-B7, HAdV-B11, HAdV-B14 and HAdV-B55) without cross-reactions with other subtypes and other respiratory tract pathogens. This RPA assay has not only highly sensitivity with low detection limit of 50 copies per reaction but also short reaction time (< 15 min per detection). Furthermore, the real-time RPA assay has excellent correlation with real-time PCR assay for detection of HAdVs species B presented in clinical samples.ConclusionsThus, the RPA assay developed in this study provides an effective and portable approach for the rapid detection of HAdVs species B.

Conventional and real-time pcr assays for detection and identification of rhizoctonia solani AG-2-2, the causal agent of root rot of sugar beet

Soil-borne fungi belonging to the genus Rhizoctonia are considered to be among the most destructive sugar beet pathogens. Although multinucleate R. solani AG-2-2 is frequently detected as the main causal agent of root rot of sugar beet worldwide, several binucleate (AG-A, AG-E and AG-K) and multinucleate Rhizoctonia (R. solani AG-4, AG-5 and AG-8) have also been included in the disease complex. Due to their soil-borne nature and wide host range, the management of Rhizoctonia root rot of sugar beet is highly demanding. Identification of Rhizoctonia AG associated with root rot of sugar beet is the essential first step in determining a successful disease management strategy. In this paper we report a highly specific and sensitive real-time PCR protocol for detection of R. solani AG-2-2 which showed a high level of specificity after testing against 10 different anastomosis groups and subgroups, including AG-2-1 as the most closely related. Moreover, a similar conventional PCR assay showed the same specificity but proved to be at least a 100 times less sensitive. Future research will include further testing and adaptation of this protocol for direct detection and quantification of R. solani AG-2-2 in different substrates, including plant tissue and soil samples.

Development of a Taqman-based real-time PCR assay for detection of porcine sapelovirus infection in pigs

The objective of the present study was to develop a rapid, simple, specific and sensitive Taqman-based real-time PCR assay for porcine sapelovirus (PSV) detection. Specific primers and probe were designed from the five untranslated regions (UTRs) of the viral genome. The detection limit of the real-time PCR was 102 copies. The specificity of the Taqman real-time PCR assay was evaluated using other animal viruses and nuclease free water as a negative control. Strong fluorescent signals were obtained only in the detection of PSV real-time PCR and conventional RT-PCR were preformed simultaneously on 90 faecal samples. Based on conventional RT-PCR study 17.7% (16/90) of the faecal samples were positive for PSV. Whereas 21 of 90 samples (23.3%) were positive by real-time RT-PCR. The results showed that real-time PCR was more sensitive than the conventional RT-PCR assay. In conclusion, the Taqman real-time PCR assay for detection of PSV developed, herein, is sensitive, specific, and reliable. This assay will be useful for clinical diagnosis, epidemiological, and pathogenesis studies.

Real-time PCR assays for detection of melanogenesis-related genes from Chinese traditional medicine components

IntroductionMany Chinese traditional medicines provide good therapeutic effects. To screen their pharmacological activities and to discover new bioactive constituents, a SYBR® Green real-time PCR method was established based on information of the melanogenesis-related genes sequences. MethodsSpecific primers were designed according to the sequences of melanogenesis-related genes TYR, TRP-1, TRP-2 and MITF, to establish and optimize a quantitative real-time PCR reaction system based on treatment with 8-methoxypsoralen. Using this system, the effects of Vernonia anthelmintica (L.) Willd injections, Muniziqi granules, Suziafu tablet, Atirilali tablet, Yiti 'erfeili Aikemuaili Migao and Bairesi Migao on the expression of TYR, TRP-1, TRP-2 and MITF in mouse melanoma B16 cells were assessed. ResultsPrimers were designed to be specific to melanogenesis-related genes, and standard curve was constructed using recombinant plasmids containing gene fragments. Cycle thresholds and the template concentrations showed a good linear relationship, the absorption peak of the dissociation curves was single, correlation coefficients were between 0.983 and 0.999, and amplification efficiencies was between 100.95% and 103.05%. A significant positive correlation was observed between the drug concentration (from 25 to 100 μM) and gene expression after 24 h treatment, although Muniziqi granules and Yiti 'erfeili Aikemuaili Migao had not passed this route. ConclusionReal-time PCR was shown to verify compounds with bioactivities that promote melanin production. It also validated that 8-methoxypsoralen and some traditional medicines increased melanin production by increasing the expression of TYR, TRP-1 and TRP-2 and MITF in B16 cells.

BlaNDM-1-producing Vibrio parahaemolyticus and V. vulnificus isolated from recreational beaches in Lagos, Nigeria.

Twenty-six strains of Vibrio parahaemolyticus and 14 strains of V. vulnificus isolated from selected beaches in Lagos State, Nigeria, were examined for virulence and antimicrobial resistance genes. The V. parahaemolyticus isolates were further serotyped and subjected to pulsed field gel electrophoresis (PFGE). Five strains of V. vulnificus and one of V. parahaemolyticus carried the New Delhi-metallo-beta-lactamase gene blaNDM-1, seven strains carried blaTEM, and four strains of V. vulnificus and one of V. parahaemolyticus carried blaCMY. Real-time PCR assay for detection of virulence genes tdh and trh in the V. parahaemolyticus isolates showed that five isolates were positive for tdh, two for trh, and one isolate carried both genes. Ten V. parahaemolyticus serogroups and 23 pulsotypes were identified from 26 isolates based on O and K antigens typing and PFGE. Five of the isolates belong to the pandemic strains O1:Kut and O3:K6, and three belonged to the highly virulent O4:Kut serotype. Nineteen of the isolates showed distinct PFGE banding patterns. These results highlighted the importance of Nigerian recreational beaches as reservoirs of antimicrobial resistance genes of global public health interest, such as blaNDM-1.

Development of a real-time assay to determine the frequency of qac genes in methicillin resistant Staphylococcus aureus

PurposeThe emergence of antiseptic resistance and/or antiseptic-resistance genes in methicillin-resistant Staphylococcus aureus (MRSA) may result in failure of decolonization treatments. Plasmid-encoded efflux pump genes qacA/B and qacC (smr) confer tolerance to chlorhexidine and quaternary ammonium compounds. The objective of this study was to develop and validate a multiplex real time-PCR assay for detection of antiseptic-resistance genes, apply the assay on 200 MRSA isolates and explore if carriage of these genes was associated with resistance to topical antibiotics. MethodologyA SYBR-Green based multiplex real time-PCR assay was developed to detect qacA/B, qacC, and mecA (internal control) simultaneously. The multiplex assay was compared against conventional single-plex PCR followed by agarose gel electrophoresis, using DNA from the first 73 MRSA isolates, followed by multiplex testing of the remaining 127 MRSA isolates. All 200 MRSA isolates were tested for susceptibility to mupirocin, retapamulin, neomycin, bacitracin and octenidine. The genetic diversity of the isolates was investigated by spa-typing. ResultsThe concordance between multiplex and conventional PCR, in assignments of qacA/B and qacC status were 99%(72/73) and 100%(73/73) respectively. Among 200 MRSA isolates, 48(24%) and 44(23%) were found to harbour qacA/B and qacC genes, respectively. These isolates remained susceptible to many common decolonization agents, except mupirocin. The predominant spa-types were t020 and t1081 (41 and 32 isolates respectively). ConclusionThe real-time assay performed acceptably for the detection of qac genes. A high prevalence of antiseptic-resistance genes were detected in the MRSA isolates in our population and appeared to be associated with spa-type t1081.

Development and evaluation of a multiplex reverse-transcription real-time PCR assay for detection of equine respiratory disease viruses

We developed a multiplex reverse-transcription real-time PCR (RT-rtPCR) assay for the simultaneous detection of the main equine respiratory viruses: equid alphaherpesviruses 1 and 4 (EHV-1, -4) and equine influenza virus (EIV; species Influenza A virus). The primers and probes amplified only the targeted viruses, and there were no inter-assay cross-amplifications or nonspecific interactions. The multiplex assay efficiencies were 92.5%, 97%, and 90% for EHV-1, EHV-4, and EIV, respectively. The R2 values of the monoplex and multiplex assays were ⩾0.990, and the slopes were -3.37 to -3.59. The performance of the assay was evaluated by analyzing 152 samples from clinically infected horses. EHV-1 DNA was detected in 12 samples, EHV-4 DNA in 9 samples, and both EHV-1 and EHV-4 in 4 samples. The accuracy of the assay was confirmed by comparing these results using commercial rtPCR and RT-rtPCR kits. Our multiplex RT-rtPCR was a sensitive, specific, accurate, and cost-effective method for the detection of the target viruses whether they occur alone or as part of coinfections.

A real-time PCR assay for detection and absolute quantitation of Citrus exocortis viroid in two sensitive and tolerant rootstocks

Citrus exocortis viroid (CEVd) is the causal agent of exocortis disease, the economically important viroid disease of citrus. In this research, CEVd copy number was quantified in sour orange (Citrus aurantium L.) and citrange (Poncirus trifoliata L. Raf. × Citrus sinensis L.) seedlings by absolute real-time PCR. A full-length in vitro transcript of a single-sequence CEVd-S1 variant was synthesized and mechanically inoculated into sour orange and Troyer citrange seedlings as tolerant and sensitive hosts, respectively. CEVd copy number was subsequently monitored at different times after inoculation in the green bark of infected seedlings for up to 16 weeks post inoculation (wpi). CEVd was detected at 3 wpi and totally increased up to 16 wpi. The copy number of CEVd per milligram of bark tissue in both host species was in the range of 103 to 104 at the third and sixth weeks post inoculation, without any significant differences. Although the viroid titer was remarkably higher in citrange (sensitive host) than in sour orange (tolerant host) at 10–16 wpi, we demonstrated that CEVd titer differed in citrus species depending on the time of sampling and host plant species.

PCR assays for detection of human astroviruses: In silico evaluation and design, and in vitro application to samples collected from patients in the Netherlands

BackgroundHuman astroviruses (HAstV) comprise three phylogenetically compact and non-adjacent groups of species including classical HAstV (HAstV-C) and the novel ones (HAstV-VA/HMO and HAstV-MLB). Of these, HAstV-C is known to be responsible for gastroenteritis while the novel HAstV are associated with cases of neurological disorders. Accurate detection of all known variants by (real-time) PCR is challenging because of the high intra- and intergroup genetic divergence of HAstV. ObjectivesTo evaluate published HAstV PCR assays in silico, design de novo real-time PCR assays that can detect and discriminate three groups of HAstV, and apply those to patient samples to analyse the prevalence of HAstV in stool and cerebrospinal fluid (CSF) specimens. Study designIn silico evaluation of published PCR assays and design of real-time PCR assays for detection of different subsets of HAstV was conducted within a common computational framework that used all astrovirus full genome sequences from GenBank. The newly designed real-time PCR assays were evaluated in vitro and applied to faecal samples (collected in January–May 2016) and cerebrospinal fluid specimens (2010–2016) from patients in the Netherlands. ResultsQuantitative in silico evaluation of published PCRs is provided. The newly designed real-time PCR assays can reliably assign all available HAstV genome sequences to one of the three phylogenetic groups in silico, and differentiate among HAstV-specific controls in vitro. A total of 556 samples were tested using these PCR assays. Fourteen fecal samples (2.5%) tested positive for HAstV, 3 of which could be identified as the novel HAstV-MLB variants. No novel HAstV were found in CSF specimens. ConclusionNewly designed real-time PCR assays with improved detection of all known HAstV allowed the first-time identification of novel astroviruses from stool samples in the Netherlands.

One-step real-time PCR assay for detection and quantification of RNA HCV to monitor patients under treatment in Brazil

The Brazilian Public Health Service provides freely αPEG-IFN to treat patients infected with HCV. The primary goal of HCV therapy is the long-term elimination of HCV from the blood to reduce the risk of HCV associated complications and death. Patient viremia affects the treatment duration and response, thus influencing clinical decisions. We developed a high-throughput method to perform the quantification of RNA hepatitis C virus (HCV) virus load in plasma samples to monitor patients under treatment. The method is based on a duplex detection, in a one-step real-time RT-PCR assay and it has been validated according to the rules established by the official Brazilian regulatory agency (ANVISA). This new method was compared to a commercial kit (Cobas/Taqman HCV Test v2.0 - Roche), showing virus load results with significant correlation between them (p = 0,012) using commercial and clinical panels. In addition, 611 samples from patients treated with peguilated alfa-interferon (αPEG-IFN) from different regions of Brazil were analyzed. Our one-step real-time RT-PCR assay demonstrated good performance in viral load measurement and in treatment course monitoring, with acceptable sensitivity and specificity values

Enhanced sensitivity and fast turnaround time in laboratory diagnosis for bovine paratuberculosis in faecal samples

Bovine paratuberculosis (Johne's disease in cattle) is caused by the pathogen Mycobacterium avium subsp. paratuberculosis (MAP) and is a widespread chronic bacterial infectious disease in cattle. Due to the peculiarities of the pathogen, detection of MAP in faeces remains difficult.DNA extraction and real-time PCR for detection of MAP in bovine faeces (direct PCR) have been refined and feasible procedures for rapid, sensitive and automatable detection of the pathogen agent have been developed. Accordingly, in a first step we tested 20 faecal samples using two MAP complete kits (DNA extraction kits based on magnetic beads combined with real-time PCR assays) and six other DNA extraction kits for faeces. MAP-specific DNA was detected by real-time PCR assays. Cultivation of MAP on the solid medium HEYM and in the liquid medium M7H9C served as reference standards.The two complete kits detected significantly more MAP-DNA positive samples than the other procedures applied (p < 0.04). Ct values of 37 and 38 served as cut-off for the respective real-time PCR assays calculated on the basis of standard curves and droplet digital PCR (ddPCR).In a second step, the two MAP complete kits were employed for a comprehensive study including 107 positive and 50 negative faecal samples which had been previously tested on HEYM cultivation. The MAP complete kits yielded sensitivity values of 86% and 89% and specificity values of 100% compared to cultivation of MAP in the liquid medium M7H9C. In detail, cultivation of MAP in M7H9C detected the pathogen in 97% and 100% of the samples tested after an incubation period of six and twelve weeks, respectively. However, the cultivation of MAP on HEYM succeeded in only 74% after twelve weeks of incubation. In all these solid culture positive samples, MAP was also detected using the two complete kits.Additionally, the impact of repeated freezing and thawing of samples on re-cultivation of MAP was tested using 20 faecal samples and resulted in a reduction to 75% and 25% of bacterial growth when using liquid medium M7H9C and solid medium HEYM, respectively.The results of this study show that complete kits with refined automatable protocols for DNA extraction in combination with real-time PCR assays for detection of MAP can compete with sensitive cultivation of the pathogen in liquid medium.