Probe-based Real-time PCR Research Articles

Blood culture-negative endocarditis caused by Bartonella quintana in Iran.

Blood culture-negative endocarditis (BCNE) is a challenging disease because of the significant impact of delayed diagnosis on patients. In this study, excised heart valves and blood serum samples were collected from 50 BCNE patients in two central hospitals in Tehran, Iran. Sera were tested by IFA for the presence of IgG and IgM antibodies against Bartonella quintana and B. henselae. Genomic DNA extracted from the heart valves was examined for Bartonella-specific ssrA gene in a probe-based method real-time PCR assay. Any positive sample was Sanger sequenced. IgG titer higher than 1024 was observed in only one patient and all 50 patients tested negative for Bartonella IgM. By real-time PCR, the ssrA gene was detected in the valve of one patient which was further confirmed to be B. quintana. Bartonella-like structures were observed in transmission electron microscopy images of that patient. We present for the first time the involvement of Bartonella in BCNE in Iran. Future research on at-risk populations, as well as domestic and wild mammals as potential reservoirs, is recommended.

Quantitative real time PCR for distinction between Pneumocystis jirovecii infection/colonization in hospitalized patients.

Identification of the opportunistic fungus Pneumocystis jirovecii in respiratory specimens presents challenges, particularly in differentiating between colonization and active infection. The present study assessed a probe-based real time PCR (qPCR) diagnostic effectiveness in patients with diverse underlying conditions, particularly those with COVID-19 and pulmonary insufficiency. To set up the qPCR, clinical samples from 281 patients with respiratory ailments were tested. Subsequently, a descriptive study was conducted on 112 patients with pulmonary insufficiency with and without COVID-19 suspected of P. jirovecii infection. All specimens were subjected to DNA extraction followed by nested PCR and qPCR targeting the mitochondrial large subunit (mtLSU)-rRNA gene. Based on nested PCR and qPCR, P. jirovecii was identified in 40 out of 281 patients, with slight variations in positive samples observed across dilutions. Three patients who tested positive in nested PCR yielded negative results in probe-based qPCR. Conversely, three patients who tested positive in probe-based qPCR yielded negative results in nested PCR. Considering nested PCR as the golden standard, probe-based qPCR demonstrated good diagnostic performance, with 92.5% sensitivity and 98.7% specificity. Based on cycle threshold (Ct) values, the positive cases were categorized: ≤32 as infection, >35 as colonization, and a grey zone between these values (32 < X ≤ 35). The analysis of 112 PCP-suspected patients revealed a prevalence ranging from 6.25% (nested PCR) to 7% (probe-based qPCR). This study suggested Ct values to differentiate Pneumocystis pneumonia/colonization in immunocompromised patients. To further augment the diagnostic sensitivity, it is recommended to integrate qPCR results with clinical parameters and biomarkers to offer a more precise understanding of Pneumocystis-related conditions.

A Quantitative Real-Time PCR Method to Detect the Quinoa Downy Mildew Pathogen, Peronospora variabilis.

Quinoa downy mildew, caused by Peronospora variabilis, is the most devastating disease of quinoa globally. Rapid, sensitive diagnostic methods are needed to detect and quantify this pathogen in seeds and plant tissue. A hydrolysis probe-based quantitative real-time PCR (qPCR) assay including a competitive internal control was developed for P. variabilis detection. This assay could detect as low as 20 ag of DNA or approximately 25 internal transcribed spacer (ITS) copies per reaction with efficiencies ranging from 93.9 to 98.2%. No nontarget amplification was observed when tested against DNA from other downy mildew pathogens and related oomycetes. P. variabilis strains from multiple countries were detected using this assay. The assay was successfully applied to quantify the pathogen in quinoa seeds from a field trial conducted in the state of Washington. Downy mildew disease was recorded on all 14 genotypes, with the genotypes 104.88 and 106.49 recording the highest area under the disease progress curve values (mean ± SE; 3,236 ± 303 and 2,851 ± 198, respectively) and J6 and Dutchess recording the lowest (441 ± 107 and 409 ± 129, respectively). Seed washes obtained from field samples were subjected to the qPCR assay, and the pathogen was detected in all samples. The highest pathogen ITS copy number was recorded with 106.49 (194,934 ± 38,171), and the lowest was observed in Pasto (5,971 ± 1,435) and Riobamba (9,954 ± 4,243). This qPCR assay could lead to improved detection and quantification of P. variabilis as well as increased understanding of quinoa-P. variabilis interactions and epidemiology.

Development of a TaqMan probe-based multiplex real-time PCR for the simultaneous detection of four clinically important filamentous fungi.

World Health Organization developed the first fungal priority pathogens list (WHO FPPL) in 2022. Aspergillus fumigatus, Mucorales, Fusarium spp., and Histoplasma spp. are the four types of pathogenic fungi with filamentous morphology in the critical priority group and high priority group of WHO FPPL. These four filamentous fungal infections have become more common and severe in immunocompromised patients with the increase in susceptible populations in recent decades, which resulted in a substantial burden on the public health system. However, prompt and precise identification of them during infection remains challenging. Our study established successfully a TaqMan probe-based multiplex real-time qPCR assay for four clinically important filamentous fungi, A. fumigatus, Fusarium spp., Mucorales, and Histoplasma capsulatum, with high sensitivity and specificity, which shows promising potential for prompt and precise diagnosis against fungal infection.

Evaluating the efficiency of ELISA, monoplex and multiplex probe-based real-time reverse-transcription PCR assays in the detection of SARS-CoV-2 (COVID-19) and influenza A and B viruses: A cross-sectional study.

The current study aimed to evaluate the efficiency of Enzyme-linked immunosorbent assay (ELISA) assay and monoplex and multiplex real-time reverse-transcription PCR (rRT-PCR) in the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A and B viruses (Flu A and Flu B). The SARS-CoV-2 -specific IgG and IgM antibodies, as well as, Flu A (H1N1 and H3N2 serotypes) and Flu B virus antibodies were determined by ELISA assay. The one-step qRT-PCR method was used to detect the SARS-CoV-2 in nasopharyngeal swab samples. Furthermore, the presence of Flu A and B viruses was evaluated using probe-based RT-PCR. Simultaneous detection of SARS-CoV-2, Flu A and B viruses was performed by multiplex rRT-PCR assay. SARS CoV-2 IgM and IgG antibodies were detected in 33.3% and 58.3% of patients, respectively. In contrast, the SARS CoV-2 genome was detected in 50% of patients using the one-step monoplex RT-PCR assay. Flu A serotypes H1N1 and H3N2 were found in 16.7% and 8.3% of patients. Probe-based RT-PCR revealed that 39.3% of patients were positive for the Flu A virus. Multiplex rRT-PCR detect the SARS-CoV-2, Flu A, and Flu B in 50%, 39.3%, and 19% of samples, respectively. The sensitivity and specificity of multiplex rRT-PCR assay in comparison to monoplex RT-PCR were 100% and 55%, respectively. Coinfection with SARS-CoV-2, Flu A, and Flu B viruses was found in 9.5% of patients. Multiplex rRT-PCR can be used as a repaid, cost-effective and suitable tool for molecular surveillance of SARS-CoV-2 and Flu A/B viruses.

Rapid diagnosis of canine respiratory coronavirus, canine influenza virus, canine distemper virus and canine parainfluenza virus with a Taqman probe-based multiplex real-time PCR

Canine Infectious Respiratory Disease Complex (CIRDC) is a highly infectious diseases. Canine respiratory coronavirus (CRCoV), Canine influenza virus (CIV), Canine distemper virus (CDV), and Canine parainfluenza virus (CPiV) are crucial pathogens causing CIRDC. Due to the similar clinical symptoms induced by these viruses, differential diagnosis based solely on symptoms can be challenging. In this study, a multiplex real-time PCR assay was developed for detecting the four RNA viruses of CIRDC. Specific primers and probes were designed to target M gene of CRCoV, M gene of CIV, N gene of CDV and NP gene of CPiV. The detection limit is 10 copies/μL for CIV or CRCoV, while the detection limit of CDV or CPiV is 100 copies/μL. Intra-group and inter-group repeatability coefficient of variation (CV) were both less than 2 %. A total of 341 clinical canine samples were analyzed, and the results indicated that the method developed in our study owns a good consistency and better specificity compared with the conventional reverse transcription PCR. This study provides a new method to enable the simultaneous detection of all four pathogens in a single reaction, improving the efficiency for monitoring the prevalence of four viruses in CIRDC, which benefits the control of CIRDC.

Development of Rapid Detection Methods for Fusarium oysporum f. sp. melonis in Melon Seeds.

Melon (Cucumis melo L.) is a global commercial crop that is sensitive to seed-borne wilt infections caused by Fusarium oxysporum f. sp. melonis (Fom). To address the challenge of detecting Fom contamination, we designed a probe-based real-time PCR method, TDCP2, in combination with rapid or column-based DNA extraction protocols to develop reliable molecular detection methods. Utilizing TDCP2, the detection rate reached 100% for both artificially Fom-inoculated (0.25-25%) and pod-inoculated melon seeds in conjunction with DNA samples from either the rapid or column-based extraction protocol. We performed analyses of precision, recall, and F1 scores, achieving a maximum F1 score of 1 with TDCP2, which highlights the robustness of the method. Additionally, intraday and interday assays were performed, which revealed the high reproducibility and stability of column-based DNA extraction protocols combined with TDCP2. These metrics confirm the reliability of our developed protocols, setting a foundation for future enhancements in seed pathology diagnostics and potentially broadening their applicability across various Fom infection levels. In the future, we hope that these methods will reduce food loss by improving the control and management of melon diseases.

Characterization and Performance Evaluation of Anaerobic Night Soil Biodegrading Inoculum

The efficiency of anaerobic night soil (human excreta) biodegradation depends on the characteristics of the inoculum used. This anaerobic microbial inoculum (AMI) contains a microbial consortium, including rate-limiting methanogens whose growth depends on various physicochemical and biological parameters necessitating the inoculum characterisation essential for optimum night soil (NS) biodegradation. The inoculum contained 24.567 g/L (±0.07) total solid, 5.022 (±0.57) g/L total dissolved solid, 18.148 (±0.0.058) g/L volatile solid (VS), and 7.0±0.2 pH. The Gas chromatography analysis confirmed the presence of a small amount of volatile fatty acid (VFA), (≤ 4 mM except propionate) showing rapid conversion of VFA to methane, and 55% methane. Further, the presence of methanosaetaceae group of methanogens was detected through Taqman probe-based real-time PCR. The inoculum performance was evaluated for NS biodegradation in semicontinuous mode of feeding in four 2 L anaerobic biodigesters (2D HRT, 4D HRT, 5D HRT, and 10D HRT) with varying hydraulic retention time (HRT) (2, 5, 8, and 10 days, respectively) at mesophilic temperature (35°C). After 5 HRT, VS reduction for 2D HRT and 5D HRT was &lt;40% while for 8D HRT and 10D HRT was &gt;45-50% and &gt;45-60%, respectively. The methane content was &lt;20% in biogas for 2 and 5 days HRT while for 8 and 10 days &gt;45-50% and 50-65% methane, respectively. The % VS reduction and methane content showed the overfeeding of NS in biodigesters 2D HRT and 5D HRT while 8D HRT showed towards optimum and 10D HRT optimum with improved VS reduction and methane content.

Risk assessment of wild fish as environmental sources of red sea bream iridovirus (RSIV) outbreaks in aquaculture.

Red sea bream iridovirus (RSIV) causes substantial economic damage to aquaculture. In the present study, RSIV in wild fish near aquaculture installations was surveyed to evaluate the risk of wild fish being an infection source for RSIV outbreaks in cultured fish. In total, 1102 wild fish, consisting of 44 species, were captured from 2 aquaculture areas in western Japan using fishing, gill nets, and fishing baskets between 2019 and 2022. Eleven fish from 7 species were confirmed to harbor the RSIV genome using a probe-based real-time PCR assay. The mean viral load of the RSIV-positive wild fish was 101.1 ± 0.4 copies mg-1 DNA, which was significantly lower than that of seemingly healthy red sea bream Pagrus major in a net pen during an RSIV outbreak (103.3 ± 1.5 copies mg-1 DNA) that occurred in 2021. Sequencing analysis of a partial region of the major capsid protein gene demonstrated that the RSIV genome detected in the wild fish was identical to that of the diseased fish in a fish farm located in the same area in which the wild fish were captured. Based on the diagnostic records of RSIV in the sampled area, the RSIV-infected wild fish appeared during or after the RSIV outbreak in cultured fish, suggesting that RSIV detected in wild fish was derived from the RSIV outbreak in cultured fish. Therefore, wild fish populations near aquaculture installations may not be a significant risk factor for RSIV outbreaks in cultured fish.

Establishment and application of TaqMan real-time PCR method for detection of Theileria annulata resistant to buparvaquone

Tropical theileriosis is a tick-borne disease that caused by Theileria annulata, and leads to substantial economic impact in endemic area. Distinguishes to other piroplasms, Theileria is the only eukaryotic parasite could transform mammalian leukocytes. At present, buparvaquone is the most effective drug used for treatment of Theileria infection. However, frequently reported of failure treatment with buparvaquone for some T. annulata isolates. Mutation of TaPIN1 was reported to be the direct reason for failure of buparvaquone treatment. Through in vitro culture, a T. annulata isolate with a TaPIN1 mutation that is similar to the reported strain was recently identified in China. In order to understand the distribution of Theileria with mutation of TaPIN1 in China, here we developed a TaqMan probe-based real-time PCR technology to detect the mutated TaPIN1 gene. The specificity, sensitivity and reproducibility of the established TaqMan Real-time PCR method were evaluated, and field cattle blood samples collected from Xinjiang Uyghur Autonomous Region were used to test its application. Among 1683 samples, 335 samples were confirmed positive for T. annulata by traditional PCR method and 34 samples were positive for buparvaquone-resistant. The TaPIN1 gene of those 34 samples was sequenced and analyzed with the published gene sequences from NCBI database. The results showed that the sequence obtained from the present study has good consistency with those published sequences. In conclusion, the TaqMan probe-based real-time PCR targeting T. annulata mutated TaPIN1 gene was successfully established and can be used to detect clinical samples to investigation of buparvaquone-resistant parasites in Xinjiang region quickly and accurately, which will be useful for guiding clinical medicine application.

Diagnostic utility of intranodular saline lavage in TaqMan probe-based real-time PCR diagnosis of lumpy skin disease

Lumpy skin disease (LSD), caused by the poxvirus lumpy skin disease virus has entered India, causing serious economic consequences for the Indian dairy sector. To evaluate the diagnostic value of intranodular saline lavage in the identification of LSD-infected animals, skin nodule tissue biopsy, intranodular saline lavage, whole blood, and nasal swab specimens were collected simultaneously from LSD-symptomatic animals. TaqMan probe-based real-time PCR was performed targeting the EEV glycoprotein gene (LSDV126) and the results were compared. qPCR analysis could detect LSDV in all the skin nodule tissue biopsy and intranodular saline lavage. The mean cycle threshold (Cq) values of skin nodule tissue biopsy samples (23.94 ± 2.97) were also comparable with intranodular saline lavage (25.23 ± 2.41) samples. Whole blood and nasal swabs have an extremely poor detection rate for PCR-based viral detection.Our findings demonstrate that intranodular saline lavage is an excellent alternative specimen for the detection of LSDV. Taking into consideration, the simplicity of specimen collection, intranodular saline lavage could be adopted in field conditions for accurate LSDV surveillance testing.

Emergence of Ancylostoma caninum parasites with the benzimidazole resistance F167Y polymorphism in the US dog population

BackgroundAnthelmintic resistance to benzimidazole has been detected in the canine hookworm, Ancylostoma caninum. Benzimidazole resistance is believed to have developed originally in greyhounds, but has also been detected in non-greyhound pet dogs. The aim of this study was to validate a probe-based allele-specific real-time PCR tests for the F167Y polymorphism on the β-tubulin isotype-1 gene and to determine the geographic distribution. MethodsAllele-specific real-time PCR tests were established and validated to detect the codon 167 polymorphism in the Ancylostoma caninum β-tubulin isotype-1gene. Additionally, real-time PCR tests were validated for Ancylostoma spp. and Uncinaria stenocephala. Two nucleic acid extraction protocols were validated including mechanical disruption of parasite structures in stool. The frequency of the F167Y single nucleotide polymorphism (SNP) was determined in hookworm confirmed stool samples. Samples with the resistant 167Y genotype were confirmed by β-tubulin gene sequencing and allele frequencies were determined. ResultsThe Ancylostoma spp. and A. caninum F167Y allele-specific real-time PCR tests were highly sensitive and specific when tested against synthetic DNA, spiked samples, and characterized parasites. Using an optimized total nucleic acid extraction protocol, 54 of 511 (10.6%) were found to contain the benzimidazole resistance allele. All 55 samples containing hookworms with the resistance mutation were confirmed by β-tubulin gene sequencing. The majority of resistant hookworms (44 resistant, 183 tested; 24.4%) originated from Florida, five from California (103 tested, 4.9%), three from Idaho (40 tested, 7.5%), two from Nevada (22 tested, 9.1%), and one sample from Hawaii (13 tested, 7.7%). Resistant genotypes were found in 14 different dog breeds including eight in Greyhounds. Allele-frequency determination revealed resistance allele frequencies between 1 and 100% with 58% above 50%. ConclusionsThis data strongly supports recent findings of benzimidazole resistant canine hookworms present throughout the general US pet dog population.

DNA extraction procedures and validation parameters of a real-time PCR method to control milk containing only A2 β-casein

Bovine milk mainly contains two types of β-casein: A1 and A2 variants. In recent years, a new variety of cows’ milk has emerged in dairy sector called “A2 milk”. This novel product is characterised by the absence of A1 β-casein, which has been associated with possible gastrointestinal discomfort due to β-casomorphin-7 (BCM-7) release during gastrointestinal digestion. In this context, methods to verify A1 allele absence in A2 milk are required as a quality control in the A2 milk commercialisation. Therefore, the aim of the present study was to develop a locked nucleic acid (LNA) probe-based duplex real-time PCR (qPCR) assay for A1 allele detection in A2 milk samples. Firstly, four DNA isolation methods from milk somatic cells were optimised and evaluated. Results suggests that the commercial kit NucleoSpin Tissue was the most suitable method in terms of DNA quality and amplificability for downstream applications. Then, optimisation and validation of the qPCR assay were carried out. For both A1 and A2 alleles, the absolute limits of detection of this qPCR assay were 7.3 DNA copies/reaction (2 x 10 −5 ng DNA) and 30.4 DNA copies/reaction (0.1 ng DNA) at a 95% confidence level with synthetic reference DNA samples and heterozygous genotyped DNA sample, respectively. The relative limits of detection were 2% (15 copies) and 5% (152 copies) for A1 allele in A2 samples at a 95% confidence with synthetic reference and genotyped DNA samples, respectively. The qPCR assay was robust, with intra- and inter-assay variability below 4.3%, and specific, differentiating between A1 and A2 alleles with 100% genotyping accuracy. In conclusion, this cost-effective and fast method could be used to discriminate A1 allele in A2 samples and, consequently, to verify A1 allele absence in “A2 milk” by screening commercial product on the market. • The LNA duplex qPCR assay developed could be used to verify A1 allele absence in “A2 milk”. • LNA probes reliably discriminated between A1 and A2 alleles. • The relative limits of detection reach up to 2% (15 copies) for A1 allele in A2 samples at a 95% confidence. • Milk is a challenging matrix for DNA isolation due to the presence of PCR inhibitors that leads to low DNA yield.

Rapid Detection of Fusarium oxysporum Using Insulated Isothermal PCR and a Rapid, Simple DNA Preparation Protocol.

We developed an insulated isothermal PCR (iiPCR) method for the efficient and rapid detection of Fusarium oxysporum (Fo), which is a fungus that infects various hosts and causes severe crop losses. The Fo iiPCR method was sensitive enough to detect up to 100 copies of standard DNA template and 10 fg of Fo genomic DNA. In addition, it could directly detect 1 pg of mycelium and 10 spores of Fo without DNA extraction. Our study compared the performance of Fo iiPCR to that of three published in planta molecular detection methods-conventional PCR, SYBR green-based real-time PCR, and hydrolysis probe-based real-time PCR-in field detection of Fo. All diseased field samples yielded positive detection results with high reproducibility when subjected to an Fo iiPCR test combined with a rapid DNA extraction protocol compared to Fo iiPCR with an automated magnetic bead-based DNA extraction protocol. Intraday and interday assays were performed to ensure the stability of this new rapid detection method. The results of detection of Fo in diseased banana pseudostem samples demonstrated that this new rapid detection method was suitable for field diagnosis of Fusarium wilt and had high F1 scores for detection (the harmonic mean of precision and recall of detection) for all asymptomatic and symptomatic Fo-infected banana samples. In addition, banana samples at four growth stages (seedling, vegetative, flowering and fruiting, and harvesting) with mild symptoms also showed positive detection results. These results indicate that this new rapid detection method is a potentially efficient procedure for on-site detection of Fo.

Single Nucleotide Polymorphism-Based Real-Time PCR Screening Assay for Rapid Tracking of Bacterial Infection Clusters To Complement Whole-Genome Sequencing Efforts during Outbreak Investigations.

Infection clusters of multidrug-resistant bacteria increase mortality and entail expensive infection control measures. Whereas whole-genome sequencing (WGS) is the current gold standard to confirm infection clusters, PCR-based assays targeting cluster-specific signatures, such as single nucleotide polymorphisms (SNPs) derived from WGS data, are more suitable to initially screen for cluster isolates within large sample sizes. Here, we evaluated four software tools (SeqSphere+, RUCS, Gegenees, and Find Differential Primers) regarding their efficiency to find SNPs within WGS data sets that were specific for two bacterial monospecies infection clusters but were absent from a WGS reference data set comprising several hundred diverse genotypes of the same bacterial species. Cluster-specific SNPs were subsequently used to establish a probe-based real-time PCR screening assay for in vitro differentiation between cluster and noncluster isolates. SeqSphere+ and RUCS found 2 and 24 SNPs for clusters 1 and 14 and 24 SNPs for cluster 2, respectively. However, some signatures detected by RUCS were not cluster specific. Interestingly, all SNPs identified by SeqSphere+ were also detected by RUCS. In contrast, analyses with the remaining tools either resulted in no SNPs (with Find Differential Primers) or failed (Gegenees). Design of six cluster-specific real-time PCR assays enabled reliable cluster screening in vitro. Our evaluation revealed that SeqSphere+ and RUCS identified cluster-specific SNPs that could be used for large-scale screening in surveillance samples via real-time PCR, thereby complementing WGS efforts. This faster and simplified approach for the surveillance of bacterial clusters will improve infection control measures and will enhance protection of patients and physicians. IMPORTANCE Infection clusters of multidrug-resistant bacteria threaten medical facilities worldwide and cause immense health care costs. In recent years, whole-genome sequencing (WGS) has been increasingly applied to detect and to further control bacterial clusters. However, as WGS is still expensive and time-consuming, its exclusive application for screening and confirmation of bacterial infection clusters contributes to high costs and enhanced turnaround times, which many hospitals cannot afford. Therefore, there is need for alternative methods that can enable further surveillance of bacterial clusters that are initially detected by WGS in a faster and more cost-efficient way. Here, we established a system based on real-time PCR that enables rapid large-scale sample screening for bacterial cluster isolates within 7 days after the initial detection of an infection cluster, thereby complementing WGS efforts. This faster and simplified surveillance of bacterial clusters will improve infection control measures and will enhance protection of patients and physicians.

Specific detection and quantification of three microsporidia infecting bees, Nosema apis, Nosema ceranae, and Nosema bombi, using probe-based real-time PCR

Among stressors affecting bee health, Nosema microsporidia are prevalent intracellular parasites. Nosema apis and Nosema ceranae have been described in honey bees (Apis spp.), while Nosema bombi has been described in bumble bees (Bombus spp.). Although available molecular methods serve as a complement to microscopic diagnosis of nosemosis, they do not enable accurate quantification of these three Nosema species. We developed three quantitative real-time PCRs (qPCRs) starting from in silico design of specific primers, probes, and recombinant plasmids, to target the RNA polymerase II subunit B1 (RPB1) gene in the three species. The complete methods, including bee grinding, DNA purification, and qPCR, were validated in honey bee (Apis mellifera) homogenate. Specificity was assessed in silico and in vitro with several types of bee samples. The limit of detection was estimated at 4 log10 copies/honey bee. A small, systematic method bias was corrected for accurate quantification up to 10 log10 copies/honey bee. Method accuracy was also verified in bumble bee (Bombus terrestris) and mason bee (Osmia bicornis) homogenates in the range of 5 to 10 log10 copies/bee. These validated qPCR methods open perspectives in nosemosis diagnosis and in the study of the parasite’s eco-dynamics in managed and wild bees.

A Probe-Based qPCR Method, Targeting 16S rRNA Gene, for the Quantification of Paenibacillus larvae Spores in Powdered Sugar Samples

Paenibacillus larvae (P. larvae) is responsible for American foulbrood (AFB), the most severe bacterial disease of honeybees. The enumeration of P. larvae spores in substrates taken from hives allows for the identification of the contamination levels of the colonies, mostly in those with atypical symptoms or with asymptomatic infections; in these cases, it is essential for the effective control of American foulbrood (AFB). In this work we described a new quantitative TaqMan® probe-based real-time PCR (qPCR) assay, targeting the 16S rRNA gene of P. larvae, used for the quantification of P. larvae spores in powdered sugar samples collected from hives, in comparison to the culture. A total of 105 colonies were selected, belonging to 10 apiaries with different levels of infection, located in northern Italy. The proportions of positive colonies was 54% (57/105) with the culture and 66% (69/105) with qPCR. A significant difference between the two methods was found with McNemar’s test (p = 0.02). Out of the 51 positive samples by both methods, 45 showed higher infection by qPCR compared to the culture. A close concordance with the clinical–epidemiological status of the hives was observed by both methods, with higher infection levels found by qPCR.

Molecular diagnosis and direct quantification of cereal cyst nematode (Heterodera filipjevi) from field soil using TaqMan real-time PCR

Heterodera filipjevi continues to be a major threat to wheat production worldwide. Rapid detection and quantification of cyst nematodes are essential for more effective control against this nematode disease. In the present study, a TaqMan-minor groove binder (TaqMan-MGB) probe-based fluorescence quantitative real-time PCR (qPCR) was successfully developed and used for quantifying H. filipjevi from DNA extracts of soil. The primers and probe designed from the obtained RAPD-SCAR marker fragments of H. filipjevi showed high specificity to H. filipjevi using DNA from isolates-confirmed species of 23 Heterodera spp., 1 Globodera spp. and 3 Pratylenchus spp. The qPCR assay is highly sensitive and provides improved H. filipjevi detection sensitivity of as low as 4–3 single second-stage juvenile (J2) DNAs, 10–3 female DNAs, and 0.01 μg μL–1 genomic DNAs. A standard curve relating to the threshold cycle and log values of nematode numbers was generated and validated from artificially infested soils and was used to quantify H. filipjevi in naturally infested field soils. There was a high correlation between the H. filipjevi numbers estimated from 32 naturally infested field soils by both conventional methods and the numbers quantified using the qPCR assay. qPCR potentially provides a useful platform for the efficient detection and quantification of H. filipjevi directly from field soils and to quantify this species directly from DNA extracts of field soils.

A TaqMan Probe-Based Multiplex Real-Time PCR for Simultaneous Detection of Porcine Epidemic Diarrhea Virus Subtypes G1 and G2, and Porcine Rotavirus Groups A and C.

Porcine viral diarrhea diseases affect the swine industry, resulting in significant economic losses. Porcine epidemic diarrhea virus (PEDV) genotypes G1 and G2, and groups A and C of the porcine rotavirus, are major etiological agents of severe gastroenteritis and profuse diarrhea, particularly among piglets, with mortality rates of up to 100%. Based on the high prevalence rate and frequent co-infection of PEDV, RVA, and RVC, close monitoring is necessary to avoid greater economic losses. We have developed a multiplex TaqMan probe-based real-time PCR for the rapid simultaneous detection and differentiation of PEDV subtypes G1 and G2, RVA, and RVC. This test is highly sensitive, as the detection limits were 20 and 100 copies/μL for the G1 and G2 subtypes of PEDV, respectively, and 50 copies/μL for RVA and RVC, respectively. Eighty-eight swine clinical samples were used to evaluate this new test. The results were 100% in concordance with the standard methods. Since reassortment between porcine and human rotaviruses has been reported, this multiplex test not only provides a basis for the management of swine diarrheal viruses, but also has the potential to impact public health as well.

Development, Validation, and Utility of Species-Specific Diagnostic Markers for Detection of Peronospora belbahrii

Peronospora belbahrii is an oomycete and the cause of basil downy mildew, one of the most destructive diseases affecting basil production worldwide. Disease management is challenging due to wind-dispersed sporangia and contaminated seed; therefore, identifying P. belbahrii in seed lots before sale or planting or in the field before symptoms develop could allow for timely deployment of disease management strategies. In this study, a draft genome assembly and next-generation sequencing reads for P. belbahrii, as well as publicly available DNA-seq and RNA-seq reads of several other downy mildew pathogens, were incorporated into a bioinformatics pipeline to predict P. belbahrii-specific diagnostic markers. The specificity of each candidate marker was validated against a diverse DNA collection of P. belbahrii, host tissue, and related oomycetes using PCR. Two species-specific markers were identified and used as templates to develop a highly sensitive probe-based real-time quantitative PCR (qPCR) assay that could detect P. belbahrii in leaf tissue and seed samples. Both markers were capable of reliably detecting as low as 500 fg/µl of P. belbahrii genomic DNA and as few as 10 sporangia. The qPCR assay was then validated with seed samples collected from a basil cultivar experiment. In total, 48 seed samples were collected and tested; P. belbahrii was detected in samples of all cultivars at estimated concentrations of 600 fg/µl up to 250 pg/µl and at as few as 10 sporangia up to &gt;1,000 sporangia. The markers and assays are valuable for diagnostics and identifying P. belbahrii-contaminated seed lots to mitigate the effects of future basil downy mildew epidemics.