Sensitive Real-time PCR Assay Research Articles

Campylobacter hepaticus, the cause of spotty liver disease in chickens, is present throughout the small intestine and caeca of infected birds

Spotty liver disease (SLD) causes significant egg production losses and mortality in chickens and is therefore a disease of concern for some sectors of the poultry industry. Although the first reports of the disease came from the United States in the 1950s it is only recently that the organism that causes the disease was identified, isolated, and characterised as a new bacterial species, Campylobacter hepaticus. The first isolations of C. hepaticus were from the livers and bile of SLD affected birds. Isolates could only be recovered from samples that had a monoculture of C. hepaticus in the tissues, as a selective culturing method has not yet been developed. In non-selective growth conditions the slow growing C. hepaticus is quickly outgrown by many other members of the chicken microbiota. Therefore, it is currently not possible to use a culturing approach to evaluate C. hepaticus carriage in tissues, such as the gastrointestinal tract (GIT), that also carry complex microbial populations. As it is suspected that birds become infected via the faecal-oral route it is important that pathogen carriage in the GIT is investigated. In the present study, a specific and sensitive quantitative real-time PCR assay, based on the glycerol kinase gene of C. hepaticus, was developed. The assay facilitated the detection and quantification of C. hepaticus in tissue samples from clinical cases of SLD. It was shown that in infected birds C. hepaticus colonises the small intestine, increasing in abundance from duodenum to ileum, and is at highest levels within the ceaca. C. hepaticus was also readily detected in cloacal swabs, indicating that thecl-oral infection.

Simultaneous identification of Haemophilus influenzae and Haemophilus haemolyticus using real-time PCR.

To design a highly specific and sensitive multiplex real-time PCR assay for the differentiation of the pathogen Haemophilus influenzae from its nonpathogenic near-neighbor Haemophilus haemolyticus. A comparison of 380 Haemophilus spp. genomes was used to identify loci specific for each species. Novel PCR assays targeting H. haemolyticus (hypD) and H. influenzae (siaT) were designed. PCR screening across 143 isolates demonstrated 100% specificity for hypD and siaT. These two assays were multiplexed with the recently described fucP assay for further differentiation among H. influenzae. The triplex assay provides rapid, unambiguous, sensitive and highly specific genotyping results for the simultaneous detection of hypD and siaT, including fucose-positive H. influenzae (fucP), in a single PCR.

Pneumocystis colonization in asthmatic patients not receiving oral corticosteroid therapy

Pneumocystis jirovecii can colonize patients with chronic obstructive pulmonary disease. To determine if colonization occurs in asthma patients, sputum samples from 10 patients with mild asthma, who were not receiving...

Possible chromosomal and germline integration of human herpesvirus 7.

Human herpesvirus 7 (HHV-7) is a betaherpesvirus, and is phylogenetically related to both HHV-6A and HHV-6B. The presence of telomeric repeat sequences at both ends of its genome should make it equally likely to integrate into the human telomere as HHV-6. However, numerous studies have failed to detect germline integration of HHV-7, suggesting an important difference between the HHV-6A/-6B and HHV-7 genomes. In search of possible germline integrated HHV-7, we developed a sensitive and quantitative real-time PCR assay and discovered that primers designed against some parts of the HHV-7 genome can frequently miss HHV-7 positive clinical samples even though they work efficiently in cell-culture-derived HHV-7 positive materials. Using a primer pair against the U90 ORF of HHV-7, we identified a possible case of germline integration of HHV-7 with one copy of viral genome per cell in both peripheral blood cells and hair follicles. Chromosomal integration of HHV-7 in these individuals was confirmed by fluorescence in situ hybridization analysis. Germline integration of HHV-7 was further confirmed by detection of ~2.6 copies of HHV-7 in the hair follicles of one of the parents. Our results shed light on the complex nature of the HHV-7 genome in human-derived materials in comparison to cell-culture-derived materials and show the need for stringent criteria in the selection of primers for epidemiological HHV-7 studies.

Development and validation of a highly sensitive real-time PCR assay for rapid detection of parapoxviruses.

Parapoxviruses (PaPVs) cause widespread infections in ruminants worldwide. All PaPVs are zoonotic and may infect humans after direct or indirect contact with infected animals. Herein we report the development and validation of a highly sensitive real-time PCR assay for rapid detection of PaPVs. The new assay (referred to as the RVSS assay) was specific for PaPVs only and had no cross-reactivity against other pox viruses. Using a recombinant plasmid as positive control, the analytical sensitivity of the assay was determined to be 16 genome copies of PaPV per assay. The amplification efficiency estimate (91-99%), the intra- and interassay variability estimate (standard deviation [SD]: 0.28-1.06 and 0.01-0.14, respectively), and the operator variability estimate (SD: 0.78 between laboratories and 0.28 between operators within a laboratory) were within the acceptable range. The diagnostic specificity was assessed on 100 specimens from healthy normal animals and all but 1 tested negative (99%). The diagnostic sensitivity (DSe) was assessed on 77 clinical specimens (skin/scab) from infected sheep, goats, and cattle, and all tested positive (100%). The assay was multiplexed with beta-actin as an internal positive control, and the multiplex assay exhibited the same DSe as the singleplex assay. Further characterization of the PaPV specimens by species-specific real-time PCR and nucleotide sequencing of the PCR products following conventional PCR showed the presence of Orf virus not only in sheep and goats but also in 1 bovid. The validated RVSS assay demonstrated high specificity, sensitivity, reproducibility, and ruggedness, which are critical for laboratory detection of PaPVs.

Development of a Recombinase Polymerase Amplification Assay for Rapid Detection of the Mycobacterium avium subsp. paratuberculosis.

BackgroundThe detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in ruminants is crucial to control spread among animals and to humans. Cultivation of MAP is seen as the gold standard for detection, although it is very time consuming and labour intensive. In addition, several PCR assays have been developed to detect MAP in around 90 minutes, but these assays required highly sophisticated equipment as well as lengthy and complicated procedure.Methodology/Principal FindingsIn this study, we have developed a rapid assay for the detection of MAP based on the recombinase polymerase amplification (RPA) assay targeting a MAP specific region, the IS900 gene. The detection limit was 16 DNA molecules in 15 minutes as determined by the probit analysis on eight runs of the plasmid standard. Cross reactivity with other mycobacterial and environmentally associated bacterial strains was not observed. The clinical performance of the MAP RPA assay was tested using 48 MAP-positive and 20 MAP-negative blood, sperm, faecal and tissue samples. All results were compared with reads of a highly sensitive real-time PCR assay. The specificity of the MAP RPA assay was 100%, while the sensitivity was 89.5%.Conclusions/SignificanceThe RPA assay is quicker and much easier to handle than real-time PCR. All RPA reagents were cold-chain independent. Moreover, combining RPA assay with a simple extraction protocol will maximize its use at point of need for rapid detection of MAP.

A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences.

Background Campylobacter fetus is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testudinum. Campylobacter fetus fetus affects a broad range of hosts and induces abortion in sheep and cows. Campylobacter fetus venerealis is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus testudinum has been proposed recently based on genetically divergent strains isolated from reptiles and humans. Both C. fetus fetus and C. fetus testudinum are opportunistic pathogens for immune-compromised humans. Biochemical tests remain as the gold standard for identifying C. fetus but the fastidious growing requirements and the lack of reliability and reproducibility of some biochemical tests motivated the development of molecular diagnostic tools. These methods have been successfully tested on bovine isolates but fail to detect some genetically divergent strains isolated from other hosts. The aim of the present study was to develop a highly specific molecular assay to identify and quantify C. fetus strains.ResultsWe developed a highly sensitive real-time PCR assay that targets a unique region of the 16S rRNA gene. This assay successfully detected all C. fetus strains, including those that were negative for the cstA gene-based assay used as a standard for molecular C. fetus identification. The assay showed high specificity and absence of cross-reactivity with other bacterial species. The analytical testing of the assay was determined using a standard curve. The assay demonstrated a wide dynamic range between 102 and 107 genome copies per reaction, and a good reproducibility with small intra- and inter-assay variability.ConclusionsThe possibility to characterize samples in a rapid, sensitive and reproducible way makes this assay a good option to establish a new standard in molecular identification and quantification of C. fetus species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0913-3) contains supplementary material, which is available to authorized users.

Leishmania-FAST15: A rapid, sensitive and low-cost real-time PCR assay for the detection of Leishmania infantum and Leishmania braziliensis kinetoplast DNA in canine blood samples

We describe an improved real-time PCR assay (designated as “Leishmania-FAST15”) for the detection and quantification of Leishmania infantum and Leishmania braziliensis kinetoplast DNA minicircles in canine blood samples. The analytical sensitivity of this technique is 0.1 fg of DNA, which is equivalent to 0.002 parasite per reaction. This assay uses a small reaction volume (15 μl) and is rapid to perform, with the results being available in less than 34 min. This improved assay might also be suitable for detecting and quantifying L. infantum and L. braziliensis DNA in other tissues, such as bone marrow and lymph nodes.

Real time PCR assay for detection of all known lineages of West Nile virus

West Nile virus (WNV) is one of the most widespread arbovirus and a large variety of WNV strains and lineages have been described. The molecular methods for the diagnosis of WNV target mainly lineages 1 and 2, which have caused outbreaks in humans, equines and birds. But the last few years new and putative WNV lineages of unknown pathogenicity have been described. Here we describe a new sensitive and specific real-time PCR assay for the detection and quantification of all the WNV lineages described until now. Primers and probe were designed in the 3′-untranslated region (3′-UTR) of the WNV genome and were designed to match all sequenced WNV strains perfectly. The sensitivity of the assay ranged from 1,5 to 15 copies per reaction depending on the WNV lineage tested. The method was validated for WNV diagnosis using different viral strains, human samples (cerebrospinal fluid, biopsies, serum and plasma) and mosquito pools. The assay did not amplify any other phylogenetically or symptomatically related viruses. All of the above make it a very suitable tool for the diagnosis of WNV and for surveillance studies.

Development and evaluation of a simple and effective real time PCR assay for mitochondrial quantification in racing camels

Camel racing is a popular sport in the Middle East region, where the demand is high for racing camels with higher stamina and endurance. Devising a technique to measure oxidative capacity and endurance in camels should be useful. Mitochondria are highly specialized organelles involved in metabolism in all higher organisms for sustaining life and providing energy for physical functions. The ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) is often used as an estimate for the metabolic status of the tissue. A greater quantity of mitochondria per unit of tissue translates into greater oxidative capacity and endurance. In this report, we describe a simple, sensitive and efficient real-time PCR assay for the quantification of blood mitochondria in racing camels. The primer sequences selected for the SYBR green-based PCR assay included mitochondrial D-loop region, mitochondrial ATP6ase gene and the nuclear β-actin gene. The assay was validated using two groups of camels comprising racing and dairy camels. The racing camels demonstrated a higher mtDNA/nDNA ratio compared with dairy camels based on the ΔΔCt values, with a higher variability among racing camels. The mean ΔΔCt values of adult and young racing camels did not vary considerably. The findings show that the present assay can be used as an evaluative tool for racing camels.

High detection rate of dog circovirus in diarrheal dogs.

BackgroundDiarrhea is one of the most common clinical symptoms reported in companion animal clinics. Dog circovirus (DogCV) is a new mammalian circovirus that is considered to be a cause of alimentary syndromes such as diarrhea, vomiting and hemorrhagic enteritis. DogCV has previously only been identified in the United States, Italy, Germany (GeneBank accession number: KF887949) and China (GeneBank accession number: KT946839). Therefore, the aims of this study were to determine the prevalence of DogCV in Taiwan and to explore the correlation between diarrhea and DogCV infection. Clinical specimens were collected between 2012 and 2014 from 207 dogs suffering from diarrhea and 160 healthy dogs.ResultsIn this study, we developed a sensitive and specific SYBR Green-based real-time PCR assays to detected DogCV in naturally infected animals. Of the analyzed fecal samples from diarrheal dogs and health dogs, 58 (28.0 %) and 19 (11.9 %), respectively, were DogCV positive. The difference in DogCV prevalence was highly significant (P = 0.0002755) in diarrheal dogs.ConclusionsThis is the first study to reveal that DogCV is currently circulating in domestic dogs in Taiwan and to demonstrate its high detection rate in dogs with diarrhea.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0722-8) contains supplementary material, which is available to authorized users.

A robust and specific real-time PCR tool for the detection of Phytophthora lateralis in plant tissues

Phytophthora lateralis is an oomycete responsible for Port-Orford Cedar (Chamaecyparis lawsoniana) dieback and mortality since the 1920s in western North America. It is recommended for quarantine regulation by EPPO (A2 list) and has been recently detected in Europe. In order to implement efficient control of the disease, a sensitive real-time PCR assay was developed to detect the presence of P. lateralis in plant tissues. In this study, we used the species-specific polymorphisms within the RAS-related protein gene Ypt1 to design a primer pair and a hydrolysis probe targeting the pathogen. The tool proved to be specific and inclusive, based on in silico and in vitro assessments, and could detect as few as 47 copies of target DNA. In addition, the test demonstrated its robustness and remained highly sensitive and specific under deliberately modified experimental conditions such as the hybridization temperature, or the reaction and DNA template volumes. The DNA extraction step from diseased tissues was also optimized, and the reliability of the results was ensured by a set of controls and a test targeting plant DNA that enabled the assessment of the quality of the extracts.

Development of conventional and real time PCR assay for detection and quantification of Rhizoctonia solani infecting pulse crops

AbstractA specific and sensitive conventional and real-time PCR assays were developed for the detection and quantification of

Application of real-time PCR for evaluation of distribution of equine herpesvirus type 1 in tissues of aborted fetuses.

A highly sensitive and specific real-time PCR assay was used for detection and quantitation of equine herpesvirus type 1 (EHV-1) in the different internal organs of aborted fetuses. Tissue samples from 23 aborted fetuses submitted to the Department of Virology of the National Veterinary Research Institute in Pulawy between 2012 and 2013 were used for testing. Total DNA was extracted using a phenol-chloroform-isoamyl alcohol standard protocol. A real-time PCR with forward and reverse primers encompassing a highly conserved region encoding viral glycoprotein B was adapted for diagnosis of EHV-1 infection. The detection limit of the assay was shown to be 6.0 × 10⁰ of viral DNA copies and the obtained standard curve exhibited a linear range from 10⁰ to 10⁷ molecules. Sixteen out of twenty three aborted fetuses (69.5%) were positive for EHV-1 in real-time PCR. The highest EHV-1 DNA load was obtained for liver (mean Ct value: 15.7) and lung (18.2) samples, while the lowest was in the thymus (29.6) and placenta (28.4).

Biological control of Botrytis cinerea on tomato plants using Streptomyces ahygroscopicus strain CK-15.

We developed a real-time PCR assay to specifically detect and quantify the efficacy of a biological fungicide from Streptomyces ahygroscopicus var. wuyiensis on tomato leaves. This fungicide, the natural secondary metabolite wuyiencin, is an antifungal agent against Botrytis cinerea. Specific primers were designed based on the β-actin gene sequences, which were used to detect a 303 bp fragment from B. cinerea isolates. Our assay is highly sensitive and can be used to reliably detect and quantify as little as 1·75 pg of B. cinerea DNA. We used this detection method to monitor the progression of B. cinerea infection in inoculated plant material under preventive (wuyiencin) and nonpreventive treatment. After 5 days, plants under preventive treatment exhibited a sharp decrease in fungal biomass and no symptoms, whereas plants under nonpreventive treatment displayed severe disease symptoms. The results demonstrate that wuyiencin has significant effects on B. cinerea in tomato plants and that real-time PCR is a reliable method for evaluating the effects of Streptomyces wuyiensis CK-15 on B. cinerea. Botrytis cinerea commonly produces latent or nonsymptomatic infection on and within plant tissues, which can develop into symptomatic infection when triggered by changes in environmental conditions or host plant physiology. In this study, we develop a specific, sensitive real-time PCR assay for detecting and quantifying B. cinerea on tomato leaves to determine the control efficacy of Streptomyces ahygroscopicus var. wuyiensis as a biological fungicide. Our findings demonstrate that wuyiencin has significant effects on B. cinerea in tomato plants and that real-time PCR is a reliable method for evaluating the effects of biological fungicides on plant pathogens.

Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes.

BackgroundClostridium perfringens is a widespread pathogen, but the precise quantification of this subdominant gut microbe remains difficult due to its low fecal count (particularly in asymptomatic subjects) and also due to the presence of abundant polymerase-inhibitory substances in human feces. Also, information on the intestinal carriage of toxigenic C. perfringens strains in healthy subjects is sparse. Therefore, we developed a sensitive quantitative real-time PCR assays for quantification of C. perfringens in human feces by targeting its α-toxin and enterotoxin genes. To validate the assays, we finally observed the occurrence of α-toxigenic and enterotoxigenic C. perfringens in the fecal microbiota of healthy Japanese infants and young adults.MethodsThe plc-specific qPCR assay was newly validated, while primers for 16S rRNA and cpe genes were retrieved from literature. The assays were validated for specificity and sensitivity in pre-inoculated fecal samples, and were finally applied to quantify C. perfringens in stool samples from apparently healthy infants (n 124) and young adults (n 221).ResultsThe qPCR assays were highly specific and sensitive, with a minimum detection limit of 103 bacterial cells/g feces. Alpha-toxigenic C. perfringens was detected in 36 % infants and 33 % adults, with counts ranging widely (103-107 bacterial cells/g). Intriguingly, the mean count of α-toxigenic C. perfringens was significantly higher in infants (6.0 ± 1.5 log10 bacterial cells/g), as compared to that in adults (4.8 ± 1.2). Moreover, the prevalence of enterotoxigenic C. perfringens was also found to be significantly higher in infants, as compared to that in adults. The mean enterotoxigenic C. perfringens count was 5.9 ± 1.9 and 4.8 ± 0.8 log10 bacterial cells/g in infants and adults, respectively.ConclusionsThese data indicate that some healthy infants and young adults carry α-toxigenic and enterotoxigenic C. perfringens at significant levels, and may be predisposed to related diseases. Thus, high fecal carriage of toxigenic C. perfringens in healthy children warrants further investigation on its potential sources and clinical significance in these subjects. In summary, we present a novel qPCR assay for sensitive and accurate quantification of α-toxigenic and enterotoxigenic C. perfringens in human feces, which should facilitate prospective studies of the gut microbiota.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0561-y) contains supplementary material, which is available to authorized users.

Tropheryma whipplei, a Potential Commensal Detected in Individuals Undergoing Routine Colonoscopy.

Mucosal biopsy samples from individuals not suspected of having Whipple's disease were tested for the presence of Tropheryma whipplei. A sensitive and specific real-time PCR assay targeting a sequence present seven times in the T. whipplei genome was used. T. whipplei DNA was detected in 2.0 and 3.8% of the patients undergoing gastroduodenoscopy and colonoscopy, respectively, who were tested.

Development of real-time PCR assays for the detection of Moraxella macacae associated with bloody nose syndrome in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques.

Moraxella macacae is a recently described bacterial pathogen that causes epistaxis or so-called bloody nose syndrome in captive macaques. The aim of this study was to develop specific molecular diagnostic assays for M. macacae and to determine their performance characteristics. We developed six real-time PCR assays on the Roche LightCycler. The accuracy, precision, selectivity, and limit of detection (LOD) were determined for each assay, in addition to further validation by testing nasal swabs from macaques presenting with epistaxis at the Tulane National Primate Research Center. All assays exhibited 100% specificity and were highly sensitive with an LOD of 10 fg for chromosomal assays and 1 fg for the plasmid assay. Testing of nasal swabs from 10 symptomatic macaques confirmed the presence of M. macacae in these animals. We developed several accurate, sensitive, and species-specific real-time PCR assays for the detection of M. macacae in captive macaques.

Establishment and preliminary application of an assay for the detection of porcine parvovirus in cells used for production

Objective To establish an assay for the detection of porcine parvovirus (PPV) and to verify its application for monitoring cells used for production. Methods A pair of primers and one probe were designed according to the conserved sequence encoding non-structural protein 1 (NS1). Based on the designed primers, a real-time fluorescent quantitative PCR assay for the detection of PPV was developed. Several parameters including the linearity, precision, minimum detection limit and anti-interference of the established assay were evaluated. A stock of PPV strains was prepared by infecting swine testicle (ST) cells with PPV strains. An assay for the detection of PPV infection was developed by using ST cells as sensitive cells. A combined ST cell infection-PCR test was developed by combining the ST cell infection assay with the real-time fluorescent quantitative PCR assay. The sensitivity of ST cell infection-PCR test was analyzed. The cell samples used for production of biological products were detected by using the established assay. Results The real-time fluorescent quantitative PCR assay was specific for the detection of PPV without cross-reaction to other species of parvovirus virus, SV40 virus and other porcine viruses. The linear range of the assay was 1×109-1×104 copies/μl with a R2 value more than 0.98. The sensitivity of the real-time quantitative PCR assay was 1×104 copies/μl. Both of the intra- and inter-coefficient of variation (CV) were less than 5% in Ct values. The intra- and inter-CV in copies of detection were 5%-15% and 30%-40% respectively. The minimum detection limit of the real-time fluorescent quantitative PCR assay was 1CCID50/ml. The PPV strains were detected in cell samples with no interference. The sensitivity of ST cells infection-PCR test was 0.01CCID50/ml. All of the 22 cell samples were negative for PPV by using the real-time fluorescent quantitative PCR assay. Conclusion The real-time fluorescent quantitative PCR and the ST cell infection-PCR test for the detection of PPV in cells were established successfully. The application of the two assays was conducive to further enhance the safety of using cells for production and therapy. Key words: Porcine parvovirus (PPV); Real-time fluorescent quantitative PCR assay; Swine testicle (ST) cells; Infection assay; Infection-PCR assay

Dynamics of HIV DNA and Residual Viremia in Patients Treated With a Raltegravir-Containing Regimen

Dynamics of HIV DNA and Residual Viremia in Patients Treated With a Raltegravir-Containing Regimen