Triplex Quantitative Real-time PCR Research Articles

Triplex real-time qPCR for the simultaneous detection of Botryosphaeriaceae species in woody crops and environmental samples.

Species of Botryosphaeriaceae fungi are relevant pathogens of almond causing trunk cankers, extensive gumming, necrosis of internal tissues and plant dieback and dead, threatening almond productivity. A novel triplex quantitative real-time PCR (qPCR) assay was designed for the simultaneous detection and quantification of Neofusicoccum parvum, Botryosphaeria dothidea and the Botryosphaeriaceae family. The method was validated in symptomatic and asymptomatic almond, avocado, blueberry and grapevine plants and in environmental samples, such as cropping soil and rainwater and in artificially inoculated trapped spores, demonstrating the same performance on several matrices. The limit of detection of the triplex qPCR was 10 fg of genomic DNA for the three fungal targets, with high correlation coefficients (R2) and amplification efficiencies between 90 and 120%. Although the triplex qPCR demonstrated to be more sensitive and accurate than the traditional plate culturing and further sequencing method, a substantial agreement (kappa index = 0.8052 ± 0.0512) was found between the two detection methods. The highly sensitive qPCR assay allows for accurate diagnosis of symptomatic plants and early detection of Botryosphaeriaceae fungi in asymptomatic plants (rootstocks and grafting scions from almond nurseries). Furthermore, the triplex qPCR successfully detected Botryosphaeriaceae fungi in environmental samples, such as cropping soils and rainwater. It was also capable of detecting as few as 10 conidia in artificially inoculated tapes. Therefore, the triplex qPCR is a valuable tool for accurate diagnosis, aiding in the implementation of suitable control measures. It enables preventive detection in asymptomatic samples, helping to avoid the introduction and spread of these pathogens in production fields. Moreover, it assists in identifying inoculum sources and quantifying inoculum levels in crop environments, contributing to a precise phytosanitary application schedule, thereby reducing production costs and preserving the environment.

Development and evaluation of recombinase-aided amplification assays incorporating competitive internal controls for detection of human adenovirus serotypes 3 and 7

BackgroundHuman adenoviruses are a common group of viruses that cause acute infectious diseases. Human adenovirus (HAdV) 3 and HAdV 7 cause major outbreaks of severe pneumonia. A reliable and practical method for HAdV typing in clinical laboratories is lacking. A simple, rapid and accurate molecular typing method for HAdV may facilitate clinical diagnosis and epidemiological control.MethodsWe developed and evaluated duplex real-time recombinase-aided amplification (RAA) assays incorporating competitive internal controls for detection of HAdV 3 and HAdV 7, respectively. The assays were performed in a one-step in a single tube reaction at 39° for 20 min.ResultsThe analytical sensitivities of the duplex RAA assays for HAdV 3 and HAdV 7 were 5.0 and 14.8 copies per reaction, respectively (at 95% probability by probit regression analysis). No cross-reaction was observed with other types of HAdV or other common respiratory viruses. The duplex RAA assays were used to detect 152 previously-defined HAdV-positive samples. These results agreed with those obtained using a published triplex quantitative real-time PCR protocol.ConclusionsWe provide the first report of internally-controlled duplex RAA assays for the detection of HAdV 3 and HAdV 7. These assays effectively reduce the rate of false negative results and may be valuable for detection of HAdV 3 and HAdV 7 in clinical laboratories, especially in resource-poor settings.

Newborn screening using TREC/KREC assay for severe T and B cell lymphopenia in Iran.

T-cell receptor excision circles (TRECs) and κ-deleting recombination excision circles (KRECs) are recently used for detection of T or B cell lymphopenia in neonates based on region-specific cutoff levels. Here, we report cutoffs for TREC and KREC copies useful for newborn screening and/or diagnosis of primary immunodeficiency diseases (PID) in Iran. DNA was extracted from a single 3.2mm punch of dried blood spots collected from 2160 anonymized newborns referred to two major referral health centres between 2014 and 2016. For refinement of the cutoffs, 51 patients with a definite diagnosis of severe combined immunodeficiency, X-linked agammaglobulinaemia and combined immunodeficiency, including ataxia telangiectasia, human phosphoglucomutase 3 and Janus kinase-3 deficiency, as well as 47 healthy controls were included. Samples from patients with an X-linked hyper-IgM-syndrome, Wiskott-Aldrich syndrome and DNA ligase 4 deficiency were considered as disease controls. Triplex-quantitative real-time PCR was used. Cutoffs were calculated as TRECs<11 and KRECs<6 copies with an ACTB > 700 copies with sensitivity of 100% for TREC and 97% for KREC. Among thirty anonymized newborn samples (1.5%) with abnormal results for TREC and/or KREC, only twenty-one available cases were retested and shown to be in the normal range except for three samples (0.15%). All of the patients with a definitive diagnosis were correctly identified based on our established TREC/KREC copy numbers. Determining cutoffs for TREC/KREC is essential for correctly identifying children with PID in newborn screening. Early diagnosis of PID patients enables appropriate measures and therapies like stem cell transplantation.

A triplex quantitative real-time PCR assay for differential detection of human adenovirus serotypes 2, 3 and 7

BackgroundHuman adenovirus (HAdV) serotypes 2, 3 and 7 are more prevalent than other serotypes and have been associated with severe pneumonia in pediatric children. Molecular typing of HAdV is not routinely performed in clinical diagnostic laboratories as it is time-consuming and labor-intensive.MethodsIn the present study, we developed a triplex quantitative real-time PCR assay (tq-PCR) in a single closed tube for differential detection and quantitative analysis of HAdV serotypes 2, 3 and 7. The sensitivity, specificity, reproducibility and clinical performance of tq-PCR were evaluated.ResultsThe analytical sensitivity of the tq-PCR was 100 copies/reaction for each of HAdV serotypes 2, 3 and 7, and no cross-reaction with other common respiratory viruses or HAdV serotypes 1,4,5,6,31,55 and 57 was observed. The coefficients of variation (CV) of intra-assay and inter-assay were between 0.6% to 3.6%. Of 138 previously-defined HAdV-positive nasopharyngeal aspirates samples tested, the detection agreement between tq-PCR and nested PCR was 96.38% (133/138).ConclusionThe proposed tq-PCR assay is a sensitive, specific and reproducible method and has the potential for clinical use in the rapid and differential detection and quantitation of HAdV serotypes 2, 3 and 7.

Development and validation of a real-time quantitative PCR assay to detect Xanthomonas axonopodis pv. allii from onion seed

Bacterial blight of onion is an emerging disease threatening world onion production. The causal agent Xanthomonas axonopodis pv. allii is seed transmitted and a reliable and sensitive tool is needed to monitor seed exchanges. A triplex quantitative real-time PCR assay was developed targeting two X. axonopodis pv. allii-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Amplification of at least one marker indicates the presence of the bacterium in seed extracts. This real-time PCR assay detected all the 79 X. axonopodis pv. allii strains tested and excluded 85.2% of the 135 non-target strains and particularly all 39 saprophytic and pathogenic bacteria associated with onion. Cross-reactions were mainly obtained for strains assigned to nine phylogenetically related X. axonopodis pathovars. The cycle cut-off was estimated statistically at 36.3 considering a risk of false positive of 1%. The limit of detection obtained in at least 95% of the time (LOD 95%) was 5×103CFU/g (colony forming unit/g). The sensitivity threshold was found to be 1 infected seed in 32,790 seeds. This real-time PCR assay should be useful for preventing the long-distance spread of X. axonopodis pv. allii via contaminated seed lots and determining the epidemiology of the bacterium.

Mycobacterium avium subsp. avium in Lymph Nodes and Diaphragms of Pigs from One Infected Herd in the Czech Republic

This study was performed on 40 finished pigs from one herd naturally infected with Mycobacterium avium subsp. avium. The aim was to investigate the presence and amount of M. a. avium in samples of lymph nodes and diaphragm tissues collected during routine postmortem inspection using the triplex quantitative real time PCR (qPCR) method. We collected, in total, 107 samples: various lymph nodes affected by gross tuberculosis (TB)–like lesions from 17 pig carcasses, as well as samples of head and mesenteric lymph nodes from 23 carcasses without TB-like lesions. Samples of diaphragm tissues were collected from all carcasses. M. a. avium was detected in one or more tissue samples collected from half of the slaughtered pigs tested. Samples of diaphragm tissues of three pigs with detected TB-like lesions contained M. a. avium (102 to 103 cells per g of sample); the organism was not detected in diaphragm tissues from pigs without TB-like lesions. The qPCR method may be useful for quantification of M. a. avium in pigs for the purposes of foodborne risk assessment.

Mycobacterium avium subsp. avium Found in Raptors Exposed to Infected Domestic Fowl

We report a case of a falcon breeding facility, where raptors (both diurnal and nocturnal) were raised in contact with domestic fowl (Gallus gallus f. domesticus) infected by Mycobacterium avium subsp. avium. Fecal and environmental samples from 20 raptors and four common ravens (Corvus corax) were collected. Mycobacterium a. avium DNA was detected in feces of four raptors (bald eagle [Haliaeetus leucocephalus], eagle owl [Bubo bubo], barn owl [Tyto alba], and little owl [Athene noctua]) using triplex quantitative real-time PCR. As both the flock of domestic fowl and one of the infected raptors had the same origin (zoological collection), they might have had a common source of colonization/infection. However, the detection of M. a. avium in feces of three other raptors may point at transmission of the agent between the birds in the facility. Contact of raptors with domestic fowl infected by M. a. avium may pose a risk for transmission of the infection for them; however, raptors from the falcon breeding facility seemed to be relatively resistant to the infection.

Optimisation of a triplex real time RT-PCR for detection of hepatitis E virus RNA and validation on biological samples

The aim of this study was to optimise a two-tube reverse transcription triplex quantitative real time PCR (qRT-PCR) combining amplification of two loci with an internal amplification control (IAC) for detection and quantitation of hepatitis E virus (HEV) RNA and to validate its performance on a pool of biological samples. Optimisation was performed on serially diluted "home-made" RNA standards. The limit of detection was determined experimentally as 10 copies/μl of the RNA standard for both amplification targets. The qRT-PCR was validated on a cohort of samples originating from 48 wild boars (Sus scrofa), 17 fallow deer (Dama dama) and 28 mouflons (Ovis musimon), with nested RT-PCR used as a reference method. qRT-PCR was found to be more specific for the detection of HEV RNA in examined samples. HEV RNA was detected in samples of five more animals (one wild boar and four mouflons) in comparison with nested RT-PCR.

Spinal muscular atrophy: Clinical validation of a single-tube multiplex real time PCR assay for determination of SMN1 and SMN2 copy numbers

ObjectivesTo describe and validate a new protocol for molecular diagnosis of spinal muscular atrophy (SMA), a frequent neuromuscular disease of childhood. Design and methodsSMA is caused in most cases by homozygous deletion of the SMN1 gene. We describe a triplex quantitative real-time PCR method in which fragments of SMN1, SMN2 (a nearly-identical neighboring gene with markedly reduced function) and of a control gene, CFTR, are amplified in the same tube. ResultsWe validated this method in three ways. First, testing the same samples ten times yielded CV values <4.6%. Second, in 104 previously-genotyped individuals, SMN copy numbers identical to those of the previously-determined genotype was unambiguously obtained in all cases. Finally, results using the technique in practice are described and analyzed for reproducibility of amplification efficiency and for inter-run variability. ConclusionsIn over 1200 samples, this technique has proven accurate, fast, economical and reproducible.

Outbreak of Mycobacterium avium subsp. avium Infection in One Flock of Domestic Pigeons

An outbreak of Mycobacterium avium subsp. avium infection was diagnosed in one breed of domestic pigeons (Columba livia f. domestica) in the Czech Republic. Nodular granulomatous lesions were found in 42 (9.7%) pigeons of the 435 examined; histopathologic examination of livers with gross lesions of mycobacteriosis from 15 randomly selected pigeons revealed granulomatous inflammation typical for avian mycobacteriosis in all samples. Direct Ziehl-Neelsen (ZN) microscopy and conventional culture were performed for a total of 117 liver samples (42 pigeons with nodular lesions, 55 randomly selected pigeons without nodular lesions, and 20 randomly selected squabs). Acid-fast bacilli were observed in 19 (16.2%), and conventional culture yielded growth of M. a. avium in 40 (34.2%) liver samples. A triplex quantitative real-time PCR assay based on the IS901 detection system was performed successfully in 115 liver samples and revealed M. a. avium in 63 (54.8%) of them. Mycobacterium a. avium was also detected in two squabs. Eight domestic rabbits (Oryctolagus cuniculus f. domestica) living in the breeding facility were also examined. Pyogranulomatous lesions were only found in one adult male rabbit. At necropsy, both direct ZN microscopy and culture gave negative results for mycobacteria in all examined rabbit tissues. Mycobacterium a. avium was diagnosed in a liver sample of one juvenile rabbit using triplex qPCR, suggesting that M. a. avium infection can occur as early as juvenile animals.

Mycobacterium avium Subsp. avium and Salmonella enterica Serotype Typhimurium Var. Copenhagen Phage Type DT2 in Pigeons

We report on a coinfection of Mycobacterium avium subsp. avium and Salmonella enterica serotype Typhimurium var. Copenhagen phage type DT2 in pigeons from one flock, from which squabs were occasionally consumed by humans. Triplex quantitative real-time PCR and culture methods were used for M. a. avium detection in livers and culture method was used for the detection of Salmonella sp. in samples of liver and caecum of 33 examined birds. M. a. avium was detected in a total of 31 (93.9%) and Salmonella Typhimurium in a total of 11 (33.3%) pigeons. Coinfection with both pathogens was found in 10 (30.3%), infection with Salmonella Typhimurium alone in 1 (3.0%), and infection with M. a. avium alone in 21 (63.7%) pigeons. Neither pathogen was detected in one pigeon. There was no difference in clinical symptoms exhibited by pigeons infected by M. a. avium and/or Salmonella Typhimurium. All Salmonella Typhimurium isolates were sensitive to all 15 antimicrobials tested. According to these results we emphasize good heat treatment of consumed squabs.

“ Mycobacterium avium subsp. hominissuis ” in Neck Lymph Nodes of Children and their Environment Examined by Culture and Triplex Quantitative Real-Time PCR

"Mycobacterium avium subsp. hominissuis" often causes cervical lymphadenitis in children; its prompt and accurate identification enables adequate therapy, tracing, and prevention. The aims of this study were to determine the causative agent of lymphadenitis using culture, PCR, and triplex quantitative real-time PCR (qPCR) methods with DNA directly isolated from tissue, as well as to identify possible sources of infection from the environment. We confirmed the diagnoses by detecting M. avium subsp. hominissuis using qPCR with DNA directly isolated from lymph node biopsy specimens of two patients. In order to trace the source of infection from the environment, a method of DNA isolation from soil and other environmental samples, such as dust, cobwebs, and compost, was developed. The triplex qPCR examination revealed the presence of M. avium subsp. hominissuis in a high proportion of the environmental samples (42.8% in the first patient's house and 47.6% in the second patient's house). Both patients were also exposed to M. avium subsp. avium, which was present due to the breeding of infected domestic hens. The high infectious dose of M. avium subsp. hominissuis or the increased susceptibility of humans to M. avium subsp. hominissuis compared to M. avium subsp. avium could be the reason why the children were infected with M. avium subsp. hominissuis.

Examination of Mycobacterium avium subsp. avium distribution in naturally infected hens by culture and triplex quantitative real time PCR

Mycobacterium avium subsp. avium (MAA) is the etiologic agent of avian tuberculosis, a chronic contagious disease described in a wide variety of domestic and wild bird species. The aims of this study were to assess the advantages of triplex quantitative real time PCR (qPCR) in comparison with culture testing for distribution of MAA in the organs of hens displaying varying degrees of clinical symptoms of the disease. From one small flock of ten hens and one cock with a history of weight loss, 98 tissue samples were examined in total. Pathological lesions were observed in six hens from which two were clinically ill. A total of 12 samples were positive by culture and 16 were positive by IS901 and IS1245 qPCR, confirming MAA infection. In conclusion, qPCR was a faster and more reliable alternative method in comparison with conventional culture analysis. Due to the detection of MAA in the muscle tissue of one hen, consumption of under cooked meat originating from infected fowl could pose a threat to immunosuppressed individuals.