Sensitive Real-time PCR Assay Research Articles

TET2–BCLAF1 transcription repression complex epigenetically regulates the expression of colorectal cancer gene Ascl2 via methylation of its promoter

Ascl2 has been shown to be involved in tumorigenesis in colorectal cancer (CRC), although its epigenetic regulatory mechanism is largely unknown. Here, we found that methylation of the Ascl2 promoter (bp -1670 ∼ -1139) was significantly increased compared to the other regions of the Ascl2 locus in CRC cells and was associated with elevated Ascl2 mRNA expression. Furthermore, we found that promoter methylation was predictive of CRC patient survival after analyzing DNA methylation data, RNA-Seq data, and clinical data of 410 CRC patient samples from the MethHC database, the MEXPRESS database, and the Cbioportal website. Using the established TET methylcytosine dioxygenase 2 (TET2) knockdown and ectopic TET2 catalytic domain–expression cell models, we performed glucosylated hydroxymethyl–sensitive quatitative PCR (qPCR), real-time PCR, and Western blot assays to further confirm that hypermethylation of the Ascl2 promoter, and elevated Ascl2 expression in CRC cells was partly due to the decreased expression of TET2. Furthermore, BCLAF1 was identified as a TET2 interactor in CRC cells by LC-MS/MS, coimmunoprecipitation, immunofluorescence colocalization, and proximity ligation assays. Subsequently, we found the TET2–BCLAF1 complex bound to multiple elements around CCGG sites at the Ascl2 promoter and further restrained its hypermethylation by inducing its hydroxymethylation using chromatin immunoprecipitation-qPCR and glucosylated hydroxymethyl-qPCR assays. Finally, we demonstrate that TET2-modulated Ascl2-targeted stem gene expression in CRC cells was independent of Wnt signaling. Taken together, our data suggest an additional option for inhibiting Ascl2 expression in CRC cells through TET2–BCLAF1–mediated promoter methylation, Ascl2-dependent self-renewal of CRC progenitor cells, and TET2–BCLAF1–related CRC progression.

DNA methylation analysis of tumor suppressor genes in liquid biopsy components of early stage NSCLC: a promising tool for early detection

PurposeCirculating tumor cells (CTCs) and circulating tumor DNA (ctDNA) analysis represents a liquid biopsy approach for real-time monitoring of tumor evolution. DNA methylation is considered to be an early event in the process of cancer development and progression. The aim of the present study was to evaluate whether detection of DNA methylation of selected tumor suppressor genes in CTC and matched ctDNA provides prognostic information in early stage NSCLC.Experimental designThe methylation status of five selected gene promoters (APC, RASSFIA1, FOXA1, SLFN11, SHOX2) was examined by highly specific and sensitive real-time methylation specific PCR assays in: (a) a training group of 35 primary tumors and their corresponding adjacent non-cancerous tissues of early stage NSCLC patients, (b) a validation group of 22 primary tumor tissues (FFPEs) and 42 peripheral blood samples of early stage NSCLC patients. gDNA was isolated from FFPEs, CTCs (size-based enriched by Parsortix; Angle and plasma, and (c) a control group of healthy blood donors (n = 12).ResultsAll five gene promoters tested were highly methylated in the training group; methylation of SHOX2 promoter in primary tumors was associated with unfavorable outcome. RASSFIA and APC were found methylated in plasma-cfDNA samples at 14.3% and 11.9%, respectively, whereas in the corresponding CTCs SLFN11 and APC promoters were methylated in 7.1%. The incidence of relapses was higher in patients with a) promoter methylation of APC and SLFN11 in plasma-cfDNA (P = 0.037 and P = 0.042 respectively) and b) at least one detected methylated gene promoter in CTC or plasma-cfDNA (P = 0.015).ConclusionsDNA methylation of these five gene promoters was significantly lower in CTCs and plasma-cfDNA than in the primary tumors. Combination of DNA methylation analysis in CTC and plasma-cfDNA was associated with worse DFI of NSCLC patients. Additional studies are required to validate our findings in a large cohort of early stage NSCLC patients.

Components of apoptotic pathways modulate HIV-1 latency in Jurkat cells

The ability of the human immunodeficiency virus type 1 (HIV-1) to establish latent infections serves as a major barrier for its cure. This process could occur when its host cells undergo apoptosis, but it is uncertain whether the components of the apoptotic pathways affect viral latency. Using the susceptible Jurkat cell line, we investigated the relationship of apoptosis-associated components with HIV-1 DNA levels using the sensitive real-time PCR assay. Here, we found that the expression of proapoptotic proteins, including Fas ligand (FasL), FADD, and p53, significantly decreased HIV-1 viral DNA in cells. In contrast, the expression of antiapoptotic molecules, such as FLIP, Bcl2, and XIAP, increased the levels of viral DNA. Furthermore, promoting cellular antiapoptotic state via the knockdown of Bax with siRNA and FADD with antisense mRNA or the treatment with the Caspase-3 inhibitor, Z-DEVD, also raised viral DNA. We also simultaneously measured viral RNA from supernatants of these cell cultures and found that HIV-1 latency is inversely proportional to viral replication. Furthermore, we demonstrated that HIV-1-infected cells that underwent the transient expression of FLIP- or XIAP-induced viral latency would then produce an increased level of viral RNA upon the reversal of these antiapoptotic effects via PMA treatment compared to LacZ control cells. Taken together, these data suggest that HIV-1 infection could be adapted to employ or even manipulate the cellular apoptotic pathway to its advantage: when the host cell remains in a pro-apoptotic state, HIV-1 favors active replication, while when the host cell prefers an anti-apoptotic state, the virus establishes viral latency and promotes latent reservoir seeding in a way which would enhance viral replication and cytopathogenesis when the cellular conditions shift to encourage the productive infection phase.

Real-Time PCR Assay for Rapid and Simultaneous Detection of vanA and vanB Genes in Clinical Strains.

Here, we develop a robust and sensitive real-time PCR assay which allows the simultaneous detection of vanA and vanB genes using common primers. The system was designed using the Primer3 online software. The specificity of primers and probes was first checked by in silico PCR and by BlastN analysis. The genomic DNA of 255 bacterial isolates, including Enterococcus spp., Gram-negative, and Gram-positive strains, as well as a collection of 50 stool and 50 rectal swab samples, were tested to evaluate the specificity of the new real-time PCR (RT-PCR) system. The results of the designed RT-PCR were 100% specific and 100% positive on tested vancomycin resistant isolates harboring either the vanA or vanB gene. RT-PCR assays were negative for all other bacterial species tested including vancomycin-sensitive Enterococci and Enterococcus strains harboring vanC genes. The limit of detection of vanA and vanB genes by RT-PCR assay was 47 CFU/mL and 32 CFU/mL, respectively. The rapid and accurate detection of vancomycin-resistant Enterococci is the cornerstone for minimizing the risk of nosocomial transmissions and outbreaks. We believe that this assay will strengthen routine diagnostics and surveillance programs.

Development of a new real-time PCR for the detection of pilchard orthomyxovirus (POMV) in apparently healthy fish

Pilchard orthomyxovirus (POMV) is a virus of concern to the Atlantic salmon aquaculture industry in Tasmania. First isolated from wild pilchards in southern Australia in 1998, the virus is now a recognised pathogen of farmed Atlantic salmon (Salmo salar) in Tasmania. While the current real-time PCR for POMV targets segment 5 of the viral genome, recent viral gene expression data suggests that other segments of the POMV genome presented higher transcription levels and thus may be better candidates for the early detection of the virus. This study aimed to design and begin validating a more sensitive reverse transcriptase real-time PCR (RT-qPCR) assay to detect POMV. Primers and probes were developed targeting two independent viral genes derived from segments 7 and 8, which presented higher transcription levels than segment 5 in both cell culture and infected fish. These were compared with the current POMV RT-qPCR. The POMV segment 8 assay had a higher analytical sensitivity than segment 7, detecting at least 1 plasmid copy μl−1, and was 10-fold more sensitive than both POMV segment 7 and 5 assays when analysing nucleic acid from a positive field sample. Both new assays also had high analytical specificity, detecting the 11 POMV isolates tested (inclusivity testing) and not amplifying nucleic acids from other viruses, including ISAV, a related orthomyxovirus. In the latent class model analysis, the diagnostic sensitivity of the segment 8 and 7 assays were higher than segment 5 in 93% and 92% of simulations, respectively. Seven samples (18.4%), all from subclinical fish infected with POMV, returned a positive result only with the segment 8 assay. Both new assays showed reproducible results when applied to aliquots of the same samples tested in three different laboratories. The new POMV segment 8 assay shows promising results as a surveillance tool for detecting POMV in fish without any symptoms.

Development and validation of real-time PCR assays for the detection of Ehrlichia species and E. chaffeensis in clinical specimens

Ehrlichiosis, caused by Gram-negative bacteria of the genus Ehrlichia, is considered an emerging infectious disease due to the increasing number of reported cases. Symptoms are non-specific and occur within 1 to 2 weeks following the bite of an infected tick. Confirmatory laboratory diagnostic methods vary in sensitivity and specimen requirements, which can lead to delayed diagnosis. PCR testing serves as an efficient approach to Ehrlichia confirmation in the acute stage of illness. Published assays have been effectively used to detect human ehrlichiosis at limit of detections ranging from 10 to 50 genomic copies (GC) of Ehrlichia DNA. With the discovery of new species capable of human infection, we wanted to develop assays that are sensitive and encompass a wide range of Ehrlichia. Here we developed and validated two sensitive and specific real-time PCR assays (PanE1 and PanE2) for the detection of Ehrlichia species, as well as two real-time PCR assays (ECh2 and ECh4) for the detection of Ehrlichia chaffeensis, specifically. The limit of detection was determined to be 10 GC per reaction with 100% confidence, and as little as 1 GC with lower efficiencies. Accuracy was assessed at 100% correlation. Specificity from exclusivity testing demonstrated that neither the Ehrlichia species assays (n = 60), nor the E. chaffeensis specific assays (n = 64) had cross reactivity with near neighbors or environmental bacteria. A positive predictive value of 100% and a negative predictive value of ≥93% was determined by evaluating banked clinical specimens from 62 patients with the assays. These real-time PCR assays are effective tools to detect human Ehrlichia species during the acute stage of illness. Early detection of Ehrlichia infection by these real-time PCR assays can facilitate diagnosis and treatment.

Development of a Duplex Real-Time PCR Assay for the Differentiation of Blastomyces dermatitidis and Blastomyces gilchristii and a Retrospective Analysis of Culture and Primary Specimens from Blastomycosis Cases from New York (2005 to 2019).

Blastomycosis due to Blastomyces dermatitidis and Blastomyces gilchristii is a significant cause of respiratory mycoses in North America with occasional reported outbreaks. We developed a highly sensitive, specific, and reproducible TaqMan duplex real-time PCR assay for the differentiation of B. dermatitidis and B. gilchristii The new assay permitted retrospective analysis of Blastomyces cultures (2005 to 2019) and primary clinical specimens from blastomycosis cases (2013 to 2019) from New York patients. We identified B. dermatitidis as the predominant pathogen in 38 cases of blastomycosis, while B. gilchristii was a minor pathogen involved in five cases; these findings expand understanding of blastomycosis in New York. The duplex real-time PCR assay could be implemented in reference and public health laboratories to further understand the ecology and epidemiology of blastomycosis due to B. dermatitidis and B. gilchristii.

Detection of meat from horse, donkey and their hybrids (mule/hinny) by duplex real-time fluorescent PCR.

Meat adulteration is currently a common practice worldwide. In China, adulteration of donkey meat products with the similar species (horse and mule/hinny) meat and mislabeling are becoming widespread concerns. In this study, a sensitive and species-specific duplex real-time PCR assay based on the simultaneous amplification of fragments of the creatine kinase muscle gene family, was developed and optimized for the identification of horse, donkey and mule /hinny species in raw and heat-processed meat products. Duplex real-time PCR results showed different fluorescence amplification curves for horse and donkey. Both kinds of fluorescence amplification curves appeared simultaneously for mule/hinny. The limit of detection (LOD) of the method was up to 0.01 ng /μL. The method and strategy developed in this study could be applied to detect the presence of adulterants from horse and mule /hinny meat in raw donkey meat and meat products.

KMT2C promoter methylation in plasma-circulating tumor DNA is a prognostic biomarker in non-small cell lung cancer.

MLL3 histone methyltransferase, encoded by the KMT2C gene, is a tumor suppressor that has an essential role in cell-type-specific gene expression. We evaluated the prognostic significance of KMT2C promoter methylation as a circulating epigenetic biomarker in plasma cell-free DNA (cfDNA) in non-small cell lung cancer (NSCLC). We examined the methylation status of KMT2C promoter using a novel highly specific and sensitive real-time methylation-specific PCR (MSP) assay in (a) operable NSCLC: 48 fresh-frozen NSCLC tissues, their corresponding adjacent non-neoplastic tissues, and 48 matched plasma samples; (b) metastatic NSCLC: 91 plasma samples; and (c) 60 plasma samples from healthy donors (HD). KMT2C promoter methylation in plasma cfDNA was detected in 7/48 (14.6%) patients with operable and in 18/91 (19.8%) patients with advanced NSCLC but in none (0/60, 0%) of the plasma samples from HD. In operable NSCLC, in corresponding adjacent non-neoplastic tissue samples, KMT2C promoter methylation was detected in 3/48 (6.3%) cases. Moreover, in operable NSCLC, KMT2C promoter methylation in plasma cfDNA was related to reduced disease-free survival (ΗR=0.239; P=0.001) and worse overall survival (OS; HR=0.342, P=0.023). In metastatic NSCLC, KMT2C promoter methylation in plasma cfDNA was related to worse progression-free survival (PFS; HR=0.431; P=0.005) and worse OS (HR=0.306; P<0.001). Our data strongly suggest that the detection of KMT2C promoter methylation in plasma cfDNA predicts poor prognosis in patients with both operable and metastatic NSCLCs. KMT2C promoter methylation in plasma cfDNA therefore merits further evaluation and validation as a noninvasive circulating epigenetic biomarker.

Multicenter Comparative Study of Six Cryptosporidium parvum DNA Extraction Protocols Including Mechanical Pretreatment from Stool Samples.

Background: Nowadays, many commercial kits allow the detection of Cryptosporidium sp. in stool samples after deoxyribonucleic acid (DNA) extraction. Protocols of stool pretreatment have been proposed to optimize oocysts’ DNA extraction. Among them, mechanical grinding was reported to improve the performance of Cryptosporidium oocysts’ DNA extraction. Methods: A multicenter comparative study was conducted within the framework of the French National Reference Center-Expert Laboratory for Cryptosporidiosis. Six extraction systems (i.e., manual or automated) associated with various mechanical pretreatment protocols, were compared for the Cryptosporidium parvum oocyst’ DNA extraction, before amplification using the same real-time PCR method targeting. Results: The sensitivity of real-time PCR assay was unequally impacted by the pretreatment/extraction protocol. We observed significant differences for the lowest concentrations of C. parvum oocysts (i.e., 0–94.4% and 33.3–100% respectively for 10 and 50 oocysts/mL). All in all, the protocol using Quick DNA Fecal/Soil Microbe-Miniprep® manual kit showed the best performances. In addition, optimal performances of mechanical pretreatment were obtained by combining a grinding duration of 60 s with a speed of 4 m/s using Fastprep24® with Lysing Matrix E®. Conclusions: Sample pretreatment, as well as the extraction method, needs to be properly adapted to improve the diagnostic performances of the C. parvum DNA amplification methods.

Rapid and highly sensitive portable detection of African swine fever virus.

African swine fever (ASF) continues to spread across Asia, devastating pig populations. The disease is nearly 100% fatal in pigs, and currently, there is no effective vaccine available. Therefore, early detection of ASF is critical for effective disease control. The testing process usually requires samples to be shipped to a central laboratory, which may take many hours of travel or shipping time, delaying the results needed for a rapid response. The ability to confirm ASFV-infected animals on-site or in a regional laboratory that has limited technical capacity and/or infrastructure should eliminate these issues. This study describes the successful transfer of a highly sensitive and specific laboratory-validated real-time PCR assay to a portable pen-side thermocycler, which can be operated in the field for rapid detection of ASFV following a quick manual nucleic acid extraction from a wide array of clinical samples including aggregate samples such as oral fluids. The performance of the portable assay was comparable to the laboratory-based assay. The true portability of the assay was evaluated in seven ASF-suspected farms in Vietnam by testing eighty-nine freshly collected whole blood samples on-site. The results obtained on-site were in agreement with the laboratory data obtained the following day. Availability of this field-deployable molecular assay would eliminate the need to ship samples to a central laboratory, when rapid laboratory results are required, ultimately improving the response time.

Features of Cytomegalovirus DNAemia Blips in Allogeneic Hematopoietic Stem Cell Transplant Recipients: Implications for Optimization of Preemptive Antiviral Therapy Strategies.

Cytomegalovirus (CMV) DNAemia occurs frequently in allogeneic hematopoietic stem cell transplant recipients (allo-HSCT). There is limited information about the incidence, features, and clinical impact of CMV DNAemia blips (episodes defined by an isolated positive PCR result) in this setting. In this retrospective study, 225 consecutive adult patients undergoing any modality of allo-HSCT at our center between May 2012 and July 2019 were included. Plasma CMV DNA load was monitored using a highly sensitive real-time PCR assay. In all, 187 of 225 patients had CMV DNAemia through day 365 after allo-HSCT (total number of episodes, n=379). Eighty-three of the 187 patients had 1 or more blips (n=104). Blips occurred as a first episode of CMV DNAemia as opposed to prolonged CMV DNAemia (≥2 consecutive positive PCR results) in 47 patients; in 20 of these patients, blips represented the only documented episode throughout the study period, and in 27 patients, blips preceded a prolonged CMV DNAemia episode. In the remaining 36 patients, blips developed as recurrences. Blips presenting as initial episodes occurred more frequently (P < .001) in patients receiving an allograft from a CMV-seropositive donor. The cumulative incidence of recurrent CMV DNAemia following initial blips, self-resolving prolonged CMV DNAemia episodes, or CMV DNAemia episodes treated preemptively with antivirals was not significantly different (P=.34). Receiver operating characteristic curve analysis indicated that a CMV DNA load cutoff of 48 IU/mL yielded the highest combined sensitivity (66%) and specificity (70.2%) for predicting a prolonged CMV DNAemia episode. The practical implications of our data in the optimization of preemptive antiviral therapy strategies are discussed.

A real-time PCR assay based on a specific mutation of PstS1 gene for detection of M. bovis strains

The Mycobacterium tuberculosis complex (MTBC) is composed of several genetically related and pathogenic mycobacterial species, including M. tuberculosis, M. bovis and M.africanum et al. In our previous study, we found that M. bovis strains had a unique SNP located in position 1055 in the sequence of the pstS1 gene in which a T was substituted by a C. In this study, specific primers and MGB probes were designed according to the mutation in PstS1 gene, and a sensitive, specific and rapid real-time PCR assay for M. bovis was established. Then the assay was used to detect M. bovis in simulation samples. The minimum detectable concentration is 101 copies for M. bovis DNA. The standard curve showed correlation coefficient between threshold cycle and PstS1 gene fragment copy number was 0.997 and slope is −3.144. The minimum detectable concentration is 101 cells/ml for simulation sample. In addition, M.bovis strain 93006, 14 clinical BCG stains and 7 clinical M.bovis strain showed positive while the other strains showed negative results, which proved good specificity. This assay had high sensitivity and specificity for identification of M. bovis from the simulation specimens. The assay can be applied for epidemiological and ecological surveillance of M. bovis strains.

A highly sensitive internally-controlled real-time PCR assay for mycoplasma detection in cell cultures

Mycoplasma contamination of cell lines is a common occurrence and may affect the cell line behaviour in a variety of ways. Contamination with mycoplasma is usually not obvious so cell lines should be frequently tested. Several commercially available kits for mycoplasma detection exist, however the Ph. Eur. culture method which can take several weeks to yield results is still considered to be the ‘gold standard’ method. There is therefore a need for rapid alternative methods with comparable sensitivity, specificity and species range. Here, we describe an internally-controlled Taqman-based real-time PCR assay for cell culture medium without the need for DNA extraction. The assay can detect less than 10 CFU of the most frequently encountered mycoplasma contaminants in mammalian cell cultures. The validated assay has the potential to be used as a routine test in the production of cell culture-based Biologicals.

Novel multiplex RT-PCR assay to detect BCR/ABL mRNA variants

The detection of the t(9;22) translocation, which results in the formation of the BCR/ABL oncoprotein, in patients diagnosed with chronic and acute leukaemias, allows for the administration of highly effective tyrosine kinase inhibitors such as imatinib and nilotinib. For the effective management of these patients it is important to monitor for minimal residual disease, allowing early therapy intervention prior to haematological relapse. Currently this is achieved through very sensitive quantitative real-time PCR assays. Unfortunately, these assays are highly specific to the BCR/ABL variant expressed: p210, p190 or p230 and require identification of the transcript type prior to selection of the correct monitoring assay. We have developed a novel multiplex BCR/ABL variant PCR assay that can identify the variant in a single qualitative assay, creating both a cost and time effective way to identify the most appropriate monitoring methodology for each patient.

DNA Extraction for Sensitive Detection of Shiga Toxin-Producing Escherichia coli in Food by Real-Time PCR Assays

Alkali-heat DNA extraction, a rapid and economical method, was evaluated for use in the detection of Shiga toxin-producing Escherichia coli in food using real-time PCR assays. Alkali-heat DNA extracts led to highly sensitive detection (102-104 CFU/mL) of stx and O-antigen genes in beef liver, ground beef, sliced pork, cheese, lettuce, radish sprouts, tomato, and spinach, equivalent to the sensitivity obtained using a commercial DNA extraction kit that utilizes proteinase K lysis, and silica membrane purification. Although there were differences in DNA concentration and purity between DNA extraction methods, the sensitivity of real-time PCR assays was similar. These results indicate that alkali-heat DNA extraction is a viable method when testing food products with real-time PCR assays for the presence of stx and O-antigen genes.

Duplex TaqMan real-time PCR assay for simultaneous detection and quantification of Anaplasma capra and Anaplasma phagocytophilum infection

Anaplasma capra and A. phagocytophilum, two species of the family Anaplasmataceae, are zoonotic tick-borne obligate intracellular bacteria affecting wild and domestic ruminants, dogs, cats, horses and humans. A. capra and A. phagocytophilum infections have been steadily increasing in both number and geographic distribution, and the accurate diagnosis of these infections is challenging. This study aimed to develop a rapid, sensitive and reliable duplex real-time PCR assay for the specific detection and differentiation of these Anaplasma species.We designed primers and probes against the conserved regions of A. capra groEL and A. phagocytophilum 16S rRNA genes. A range of PCR-related parameters were evaluated such as the dosage of primers and probes, and annealing temperature. The specificity, sensitivity and repeatability of this assay were evaluated. Assay performance was further evaluated using samples collected from 124 goats in four regions of Henan, China. This set of samples was also tested using conventional PCR under conditions previously described.The developed duplex real-time PCR assay allowed the simultaneous detection of A. capra and A. phagocytophilum in a reasonably short time at levels as small as 102 copies/μL, respectively, with optimal specificity and reproducibility. In addition, this duplex real-time PCR assay is the first DNA-based method designed to detect A. capra and A. phagocytophilum, and will be valuable for timely diagnosis and treatment of these infections.

Specific detection and differentiation of classic goose parvovirus and novel goose parvovirus by TaqMan real-time PCR assay, coupled with host specificity

BackgroundClassic goose parvovirus (cGPV) causes high mortality and morbidity in goslings and Muscovy ducklings. Novel GPV (N-GPV) causes short beak and dwarfism syndrome (SBDS) in Cherry Valley ducks, Pekin ducks and Mule ducks. Both cGPV and N-GPV have relatively strict host specificity, with obvious differences in pathogenicity. Specific detection of cGPV and N-GPV may result in false positives due to high nucleotide similarity with Muscovy duck parvovirus (MDPV). The aim of this study was to develop a highly specific, sensitive, and reliable TaqMan real-time PCR (TaqMan qPCR) assay for facilitating the molecular detection of cGPV and N-GPV.ResultsAfter genetic comparison, the specific conserved region (located on the NS gene) of cGPV and N-GPV was selected for primer and probe design. The selected regions were significantly different from MDPV. Through a series of optimization experiments, the limit of detection was 50.2 copies/μl. The assay was highly specific for the detection of cGPV and N-GPV and no cross-reactivity was observed with E. coli., P.M., R.A., S.S., MDPV, N-MDPV, DAdV-A, DEV, GHPV, DHAV-1, DHAV-3, ATmV, AIV, MDRV and N-DRV. The assay was reproducible with an intra-assay and inter-assay variability of less than 2.37%. Combined with host specificity, the developed TaqMan qPCR can be used for cGPV and N-GPV in differential diagnoses. The frequency of cGPV in Muscovy duckling and goslings was determined to be 12 to 44%, while N-GPV frequency in Mule ducks and Cherry Valley ducks was 36 to 56%. Additionally, fluorescence-positive signals can be found in Mule duck embryos and newly hatched Mule ducklings. These findings provide evidence of possible vertical transmission of N-GPV from breeding Mule ducks to ducklings.ConclusionsWe established a quantitative platform for epidemiological investigations and pathogenesis studies of cGPV and N-GPV DNA that was highly sensitive, specific, and reproducible. N-GPV and cGPV infections can be distinguished based on host specificity.

Detection and Quantification of Spongospora subterranea Sporosori in Soil by Quantitative Real-Time PCR.

Powdery scab on potato tubers is caused by the obligate soilborne biotroph Spongospora subterranea and is known to cause substantial losses in potato production. The pathogen also infects roots of susceptible hosts, forming galls which can negatively affect root function. S. subterranea is also the vector of Potato mop-top virus, which causes a tuber necrosis disease that can, depending on temperature and cultivar, render potato tubers unmarketable. In this study, we adapted a published protocol to develop a sensitive and robust quantitative real-time PCR (qPCR) assay using specific primers and probes for detecting and quantifying S. subterranea sporosori in soil types that differ in physical properties, including organic matter content and soil pH. For the first time, an external control was utilized and applied directly to the soil prior to DNA extraction, which facilitated normalization of S. subterranea sporosori soil levels from sample to sample. The duplex qPCR protocol was demonstrated to be highly sensitive, capable of detecting and quantifying as few as 1 sporosorus/g of soil, with consistently high qPCR efficiency and the coefficient of determination (R2) values ranging from 94 to 99% and 0.98 to 0.99, respectively. The protocol was successfully implemented in enumerating S. subterranea sporosori in naturally infested field soil collected from several states and in artificial potting mixes with high organic matter content ranging from 64 to 71%. The qPCR method developed can be useful for potato growers to avoid agricultural soils highly infested with S. subterranea and in the development of risk assessment models in the future that incorporate cultivar susceptibility to powdery scab and soil infestation levels.

Genome Mining–Based Identification of Identical Multirepeat Sequences in Plasmodium falciparum Genome for Highly Sensitive Real-Time Quantitative PCR Assay and Its Application in Malaria Diagnosis

Developing ultrasensitive methods capable of detecting submicroscopic parasitemia-a challenge that persists in low transmission areas, asymptomatic carriers, and patients showing recrudescence-is vital to achieving malaria eradication. Nucleic acid amplification techniques offer improved analytical sensitivity but are limited by the number of copies of the amplification targets. Herein, we perform a novel genome mining approach to identify a pair of identical multirepeat sequences (IMRSs) that constitute 170 and 123 copies in the Plasmodium falciparum genome and explore their potential as primers for PCR. Real-time quantitative PCR analyses have shown the ability of P. falciparum IMRSs to amplify as low as 2.54 fg of P. falciparum genomic DNA (approximately 0.1 parasite), with a striking 100-fold increase in detection limit when compared with P. falciparum 18S rRNA (251.4 fg; approximately 10 parasites). Validation with clinical samples from malaria-endemic regions has shown 6.70±1.66 cycle better detection threshold in terms of Ct value for P. falciparum IMRSs, with approximately 100% sensitivity and specificity. Plasmodium falciparum IMRS assays are also capable of detecting submicroscopic infections in asymptomatic samples. To summarize, this approach of initiating amplification at multiple loci across the genome and generating more products with increased analytical sensitivity is different from classic approaches amplifying multicopy genes or tandem repeats. This can serve as a platform technology to develop advanced diagnostics for various pathogens.