qPCR Protocol Research Articles

Detection of Filariid Infections in Mexican Primate Populations Through qPCR.

Filariae are parasitic nematodes of high veterinary and medical importance, responsible for some acute tropical diseases. They are transmitted through the bite of hematophagous vectors such as biting midges and blackflies. Filariae are among the most prevalent vector-borne parasitoses in Neotropical primates in which severe infections can cause inflammatory reactions and tissue damage. Given the location inside the host (peritoneal cavity, bloodstream, and lymphatics), the detection of filariid nematodes is challenging and is mostly postmortem; hence the scarcity of studies on the prevalence of filariae in wild primate populations. Here, we report the prevalence of filariid infections in free-ranging populations of Geoffroy's spider (Ateles geoffroyi) and black howler (Alouatta pigra) monkeys across southern Mexico, using a combination of noninvasive sampling and molecular diagnostic techniques. Fecal samples were screened for filariid DNA by qPCR protocols. A total of 88 samples were examined with an overall prevalence of 26%. Filariae were slightly more common in spider monkeys compared to howler monkeys. This study constitutes the first report of the prevalence of infection of filariid nematodes in populations of wild spider monkey across southern Mexico, and the first reporting of filariae in black howler monkeys, as part of a new era of primate parasitology and the diagnostics of parasite infections in light of the everyday more affordable molecular tools.

Molecular detection and quantification of canine parvovirus 2 using a fast and sensitive SYBR® green-based quantitative polymerase chain reaction assay in dogs affected with gastroenteritis

Background and Aim: Viral gastroenteritis in canines is primarily caused by the canine parvovirus 2 (CPV-2). Infections by this virus can cause severe consequences in dogs, such as fever, vomiting, diarrhea, septicemia, systemic inflammation, and immunosuppression. Therefore, the mortality rate of persistent infections caused by this virus is significantly high. The capsid protein VP2 genome of canine parvovirus has undergone many changes, resulting in the emergence of different genotypes, including CPV-2a, CPV-2b, and CPV-2c. Diagnostic procedures often lack the necessary specificity for early infection diagnosis. Early detection of the infection enhances the likelihood of canine survival because the canine will receive prompt therapy. Hence, this study aimed to develop a quantitative polymerase chain reaction (qPCR)-based diagnostic technique using SYBR Green for the rapid and accurate detection and quantification of CPV-2. Materials and Methods: The assay was specifically designed to identify a portion of the conserved NS gene using primers that amplify a 125-bp fragment. The qPCR method was executed in the fast mode to expedite the process using Power up SYBR Green Master Mix reagent. A standard curve was constructed using the amplified and purified PCR product of the NS gene. Results: The limit of detection and quantification were determined in the one amplified-DNA copy. The standard curve showed an efficiency of 99.5% and inter- and intra-assay coefficients of variation of 0.387%–0.976% and 0.085%–0.430%, respectively. The assay was specific for the amplification of CPV-2, as no amplification was observed for other viral genomes (canine adenovirus II, canine distemper virus, canine coronavirus, and canine astrovirus) or from the negative controls. Inter- and intra-tests for repeatability showed low test variability around the run time. To validate the present assay, 200 samples of fezzes from canines with gastroenteritis and symptoms associated with enteric infection were tested using the qPCR protocol. From the analyzed samples, 136 were positive for CPV-2 by qPCR assay, of which 110 were before diagnostic positive for the virus by endpoint PCR, showing high sensitivity of the current assay. CPV-2 was detected in dogs over 2 weeks old up to dogs 9 years old, where the highest viral concentration found was 16429595 gene copies in dogs aged 2 weeks. Conclusion: In the present study, a rapid, specific, repeatable, and sensitive assay was developed for the detection and quantification of CPV-2. Furthermore, it was demonstrated that in the population of domestic dogs in Ecuador affected with gastrointestinal disease, the virus is presented in dogs of different ages and not only in young dogs. Keywords: canine parvovirus, gastroenteritis, quantitative polymerase chain reaction, SYBR green.

Mosquitoes on a chip-environmental DNA-based detection of invasive mosquito species using high-throughput real-time PCR.

The monitoring of mosquitoes is of great importance due to their vector competence for a variety of pathogens, which have the potential to imperil human and animal health. Until now mosquito occurrence data is mainly obtained with conventional monitoring methods including active and passive approaches, which can be time- and cost-consuming. New monitoring methods based on environmental DNA (eDNA) could serve as a fast and robust complementary detection system for mosquitoes. In this pilot study already existing marker systems targeting the three invasive mosquito species Aedes (Ae.) albopictus, Ae. japonicus and Ae. koreicus were used to detect these species from water samples via microfluidic array technology. We compared the performance of the high-throughput real-time PCR (HT-qPCR) system Biomark HD with real-time PCR (qPCR) and also tested the effect of different filter media (Sterivex® 0.45 µm, Nylon 0.22 µm, PES 1.2 µm) on eDNA detectability. By using a universal qPCR protocol and only 6-FAM-MGB probes we successfully transferred these marker systems on the HT-qPCR platform. All tested marker systems detected the target species at most sites, where their presence was previously confirmed. Filter media properties, the final filtration volume and observed qPCR inhibition did not affect measured Ct values via qPCR or HT-qPCR. The Ct values obtained from HT-qPCR were significantly lower as Ct values measured by qPCR due to the previous preamplification step, still these values were highly correlated. Observed incongruities in eDNA detection probability, as manifested by non-reproducible results and false positive detections, could be the result of methodological aspects, such as sensitivity and specificity issues of the used assays, or ecological factors such as varying eDNA release patterns. In this study, we show the suitability of eDNA-based detection of mosquito species from water samples using a microfluidic HT-qPCR platform. HT-qPCR platforms such as Biomark HD allow for massive upscaling of tested species-specific assays and sampling sites with low time- and cost-effort, thus this methodology could serve as basis for large-scale mosquito monitoring attempts. The main goal in the future is to develop a robust (semi)-quantitative microfluidic-based eDNA mosquito chip targeting all haematophagous culicid species occurring in Western Europe. This chip would enable large-scale eDNA-based screenings to assess mosquito diversity, to monitor species with confirmed or suspected vector competence, to assess the invasion progress of invasive mosquito species and could be used in pathogen surveillance, when disease agents are incorporated.

Extensive low-density Plasmodium falciparum reservoir in the island of Príncipe, an isolated malaria pre-elimination setting

ObjectivesThe isolated Príncipe is at the malaria pre-elimination stage. Autochthonous clinical cases have been reported sporadically on the island, signaling the possibility of a sizable subpatent (i.e., rapid diagnostic test- and microscopy-negative and polymerase chain reaction [PCR]-positive) parasite reservoir. MethodsAsymptomatic low-density infections were detected by quantitative PCR (qPCR) targeting Plasmodium falciparum multicopy genes (pfr364 and varATS). Positivity rates were assayed for samples surveyed by active case detection (n = 112) and reactive case detection (n = 221) in 2022. ResultsqPCR unveiled 70% of low parasitemia carriers, reaching >90% in reactive case detection. The high P. falciparum prevalence was confirmed by the two high-sensitivity qPCR protocols. Higher positivity rates were observed in the localities where most malaria cases were reported in 2022. Most parasitemias were very low (<2 Pf /µl). ConclusionsThese findings suggest that pre-elimination surveillance can benefit from the routine application of highly sensitive tools to unveil otherwise invisible but potentially relevant parasite populations.

Establishment of a Suitable Diagnostic Workflow to Ensure Sensitive Detection of African Swine Fever Virus Genome in Porcine Semen.

The rapid spread of African swine fever virus (ASFV), causing severe and often lethal disease in domestic pigs and Eurasian wild boar, continues to be a threat to pig populations and dependent industries. Despite scientific achievements that have deepened our understanding of ASFV pathogenesis, alternative transmission routes for ASFV remain to be elucidated. We previously demonstrated the efficient transmission of ASFV from infected boars to naïve recipient gilts via artificial insemination, thereby highlighting the importance of surveillance of boar semen prior to its shipment. Since the accurate and reliable detection of even low amounts of ASFV in boar semen is key to disease prevention and control, we established a suitable diagnostic workflow to efficiently detect the ASFV genome in boar semen. Here, we assessed the sensitivity of various routine nucleic acid extraction kits as well as qPCR protocols in detecting the ASFV genome in the blood and semen of infected boars. The feasibility of the respective kits and methods for future use in boar studs was also considered. Variability in sensitivity mostly concerned samples with low to very low amounts of the ASFV genome. Ultimately, we defined a well-suited workflow for precisely detecting the ASFV genome in boar semen as early as 2 days post ASFV infection.

Variant-specific BCR::ABL1 quantification discrepancy in chronic myeloid leukemia.

Accurate quantification of the BCR::ABL1 fusion gene in whole blood is pivotal for the clinical management of chronic myeloid leukemia (CML) patients. The fusion protein encoded by BCR::ABL1 can vary in size, depending on the BCR and/or ABL1 gene breakpoint. The vast majority of CML patients have a p210 BCR::ABL1 fusion gene (M-BCR), which can be attributed to the presence of either e14a2 (b3a2) or e13a2 (b2a2) mRNA transcript junctions. Twenty-five CML samples were analyzed in two different ISO15189-accredited centers that both use an Europe Against Cancer-based quantitative polymerase chain reaction (qPCR) protocol. Reanalysis of the sample set with transcript-specific standard curves and digital droplet PCR (ddPCR) were performed. qPCR quantification revealed a significant (up to 1 log) difference specifically for the e13a2 transcript variant in contrast to e14a2 transcripts (Hodges-Lehman 4.29; p < 0.001). Reanalysis of the sample set with transcript-specific standard curves abolishes the initial transcript-specific difference (Hodges-Lehman 0.003; p = 0.8192). Comparison of transcript-specific qPCR results of both centers with ddPCR, an absolute quantification method, showed a statically significant association, especially in the lower range, indicating the clinical utility of transcript-specific or absolute quantification methods. Our data show that differences between transcript-specific quantification might exist between centers, leading to potential clinical impact on the follow-up of CML patients. The use of transcript-specific standard curves for qPCR quantification, or absolute quantification, can significantly reduce these differences. Specific attention should be applied to the interpretation of quantification differences of CML patients that switch between diagnostic centers.

Assessing methods for detecting Alexandrium catenella (Dinophyceae) and paralytic shellfish toxins in Southeast Alaska.

Blooms of Alexandrium catenella threaten to disrupt subsistence, recreational, and commercial shellfish harvest in Alaska, as the paralytic shellfish toxins (PSTs) produced pose a serious public health risk and can lead to costly shutdowns for shellfish farmers. Current methods of PST detection in the region range from monitoring programs utilizing net tows to detect A. catenella to direct shellfish tissue testing via mouse bioassay (MBA) for commercial aquaculture harvest, as well as various optional testing methods for subsistence and recreational harvesters. The efficacy and feasibility of these methods vary, and they have not been directly compared in Southeast Alaska. In this study, we sought to assess and compare A. catenella and PST early detection methods to determine which can provide the most effective and accurate warning of A. catenella blooms or PST events. We found microscope counts to be variable and prone to missing lower numbers of A. catenella, which may be indicative of bloom formation. However, quantitative polymerase chain reaction (qPCR) significantly correlated with microscope counts and was able to effectively detect even low numbers of A. catenella on all sampling days. Paralytic shellfish toxin concentrations measured by enzyme-linked immunosorbent assay and MBA significantly correlated with each other, qPCR, and some microscope counts. These results show that qPCR is an effective tool for both monitoring A. catenella and serving as a proxy for PSTs. Further work is needed to refine qPCR protocols in this system to provide bloom warnings on an actionable timescale for the aquaculture industry and other shellfish harvesters. Integr Environ Assess Manag 2024;20:2189-2202. © 2024 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

A novel TaqMan qPCR system for the detection of Diaporthe citri, the causative agent of citrus melanose

Citrus melanose, caused by Diaporthe citri, is one of the most economically significant fungal diseases in China. Given that the application of fungicides is the main technique used for the control of citrus melanose, many studies have focused on enhancing the effectiveness and efficiency of the use of fungicides for the control of this economically significant disease. Here, we developed a TaqMan quantitative polymerase chain reaction (qPCR) protocol for monitoring citrus melanose via detection of suspected infected leaves and airborne conidia of D. citri. A primer pair and TaqMan probe targeting D. citri were designed to amplify a region of the fungal β-tubulin gene. The limits of detection and quantification of our TaqMan qPCR method were 159.57 and 683 copies/μL, respectively. The method detected the DNA equivalent to 47 conidia when fungal conidia were cultured in vitro. To evaluate the feasibility of our TaqMan qPCR method, symptomatic and asymptomatic leaf samples, airborne conidia samples were collected from a citrus orchard and tea garden, respectively. D. citri was detected in leaves with citrus melanose symptoms in an orchard; airborne fungal conidia of D. citri were also detected. These findings suggested that this novel TaqMan qPCR method could be used to detect D. citri both in vivo and in vitro. Our methodology could be applied as an alternative method for monitoring the changes in the incidence of D. citri in orchards.

Validation of a Field-Portable, Handheld Real-Time PCR System for Detecting Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome in Bats.

White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans, has decimated bat populations across North America. Despite ongoing management programs, WNS continues to expand into new populations, including in US states previously thought to be free from the pathogen and disease. This expansion highlights a growing need for surveillance tools that can be used to enhance existing monitoring programs and support the early detection of P. destructans in new areas. We evaluated the feasibility of using a handheld, field-portable, real-time (quantitative) PCR (qPCR) thermocycler known as the Biomeme two3 and the associated field-based nucleic acid extraction kit and assay reagents for the detection of P. destructans in little brown bats (Myotis lucifugus). Results from the field-based protocol using the Biomeme platform were compared with those from a commonly used laboratory-based qPCR protocol. When using dilutions of known conidia concentrations, the lowest detectable concentration with the laboratory-based approach was 108.8 conidia/mL, compared with 1,087.5 conidia/mL (10 times higher, i.e., one fewer 10× dilution) using the field-based approach. Further comparisons using field samples suggest a high level of concordance between the two protocols, with positive and negative agreements of 98.2% and 100% respectively. The cycle threshold values were marginally higher for most samples using the field-based protocol. These results are an important step in establishing and validating a rapid, field-assessable detection platform for P. destructans, which is urgently needed to improve the surveillance and monitoring capacity for WNS and support on-the-ground management and response efforts.

New Genetic Determinants for qPCR Identification and the Enumeration of Selected Lactic Acid Bacteria in Raw-Milk Cheese.

Lactic acid bacteria (LAB) play an important role in the ripening of cheeses and contribute to the development of the desired profile of aroma and flavor compounds. Therefore, it is very important to monitor the dynamics of bacterial proliferation in order to obtain an accurate and reliable number of their cells at each stage of cheese ripening. This work aimed to identify and conduct a quantitative assessment of the selected species of autochthonous lactic acid bacteria from raw cow's milk cheese by the development of primers and probe pairs based on the uniqueness of the genetic determinants with which the target microorganisms can be identified. For that purpose, we applied real-time quantitative PCR (qPCR) protocols to quantify Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris cells in cheese directly after production and over three-month and six-month ripening periods. While L. lactis subsp. cremoris shows good acidification ability and the ability to produce antimicrobial compounds, L. delbrueckii subsp. bulgaricus has good proteolytic ability and produces exo-polysaccharides, and S. thermophilus takes part in the formation of the diacetyl flavor compound by metabolizing citrate to develop aroma, they all play an important role in the cheese ripening. The proposed qPCR protocols are very sensitive and reliable methods for a precise enumeration of L. delbrueckii subsp. bulgaricus, S. thermophilus, and L. lactis subsp. cremoris in cheese samples.

Quantitative detection of cassava common mosaic virus for health certification of cassava (Manihot esculenta Crantz) germplasm using qPCR analysis

Cassava (Manihot esculenta Crantz) is a crop of global economic and food safety importance, used for human consumption and in various industrial applications. The genebank of the Genetic Resources Program of the Alliance of Bioversity International and CIAT currently holds the world's largest cassava collection, with 5965 in vitro accessions from 28 countries. Managing this extensive collection involves indexing quarantine pathogens as a phytosanitary certification requirement for safely distributing cassava germplasm. The study therefore aimed to optimize a quantitative diagnostic protocol to detect cassava common mosaic virus (CsCMV) using quantitative PCR (qPCR) as a better alternative to other molecular techniques. This was done through designing primers and a probe in the RdRP region of CsCMV, and optimizing the qPCR conditions of the diagnostic protocol using primer concentration assays, and reaction amplification conditions such as volume and reaction time. We also evaluated the qPCR protocol by comparing the results of 140 cassava accession evaluations using three diagnostic methodologies (DAS-ELISA, end-point PCR, and qPCR) for CsCMV. Our protocol established that qPCR technique analysis is ten-times more sensitive in detecting CsCMV compared to end-point PCR, showing a maximum detection level of 77.97 copies/μL of plasmid, with 76 min of reaction time. The comparison allowed us to verify the level of CsCMV detection through the techniques evaluated, concluding that qPCR was more sensitive and allowed the quantification of viral concentration. The optimized qPCR protocol will be used to accelerate diagnostic screening of cassava germplasm for the presence or absence of CsCMV to ensure safe movement and distribution of disease-free germplasm.

Validation of a real-time polymerase chain reaction for the detection and quantification of the nucleic acid of Histoplasma from equine clinical samples.

Histoplasmosis is a neglected yet major cause of morbidity and mortality in both equids and people in resource-scarce settings. One of the major hindrances to the control of histoplasmosis is a lack of readily available diagnostic tests. Tests are needed to support clinical decision-making and to be applied in population-based research to further understand this disease in situ. This paper reports, for the first time, the validation and application of a qPCR to detect Histoplasma directly from equine clinical samples, bypassing the need to culture this notoriously difficult organism. We report and comment on the performance of the qPCR in comparison with our previously developed nested PCR.

Molecular Diagnosis of Footrot and Contagious Ovine Digital Dermatitis in Small Ruminants in the Iberian Peninsula

Contagious ovine digital dermatitis (CODD) and footrot (FR), a sub-acute or acute necrotic (decaying) infectious disease involving the hoof and underlying tissues, pose economic challenges to herds in Spain and worldwide. The aetiological agent for FR is Dichelobacter nodosus, while CODD is caused by pathogenic Treponema phylogroups. We detail the findings derived from the analysis by qPCR of 105 pooled samples from 100 ovine and five caprine herds in Spain and Portugal, alongside 15 samples from healthy flocks in order to identify Dichelobacter nodosus, Fusobacterium necrophorum, Treponema spp., and three pathogenic Treponema phylogroups (T. phagedenis, T. medium, and T. pedis). Treponema spp. were detected in all 120 pools, including samples from the 15 healthy flocks where only one positive result for F. necrophorum was recorded. Mixed infections by agents different from Treponema spp. were identified in 68.57% of samples. Positive results for F. necrophorum and/or D. nodosus, were obtained for 91.4% of the pools, whereas the presence of the three pathogenic Treponema phylogroups was rare: each of them appeared in isolation in a single pool, while they were found in 18 pools in combination with other agents. While F. necrophorum was the sole finding in 16.2% of samples from affected herds, D. nodosus (the footrot causative agent) was only detected in 61% of affected farms. An improved qPCR protocol was implemented to determine the serogroups of D. nodosus in the samples and found all of them (except the G serogroup), often in combined infections (35.1%). This report concludes with comprehensive proposals for diagnosing, preventing, and treating hoof ailments, remarking the interest of the information about D. nodosus serogroups in order to improve the efficiency of immunization by choosing appropriate vaccine protocols.

Use of saliva-based qPCR diagnostics for the accurate, rapid, and inexpensive detection of strep throat.

Outpatient health care facilities are essential for quickly diagnosing common infectious diseases such as bacterial and viral pharyngitis. The only form of pharyngitis requiring antibiotics is strep throat (ST); however, antibiotic prescription rates are much higher than ST prevalence, suggesting antibiotics are being inappropriately prescribed. Current rapid ST diagnostics may be contributing to this problem due to the low sensitivity and variable specificity of these tests. It is best practice to verify a negative ST diagnosis with a group A Streptococcus (GAS) culture, but many clinics do not perform this test due to the additional cost and 24-72 h required to obtain results. This indicates there is great need for more accurate rapid diagnostic tools in outpatient facilities. We hypothesized that next generation qPCR technology could be adapted to detect GAS DNA from saliva samples (instead of the traditional throat swab) by creating a simple, fast, and inexpensive protocol. Saliva specimens collected from patients at James Madison University Health Center were used to test the effectiveness of our Chelex 100-based rapid DNA extraction method, followed by a fast protocol developed for the Open qPCR machine to accurately detect ST. Our final saliva processing and qPCR protocol required no specialized training to perform and was able to detect ST with 100 % sensitivity and 100 % specificity (n=102) in 22-26 min, costing only $1.12 per sample. Saliva can be rapidly analyzed via qPCR for the accurate and inexpensive detection of ST.

Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.

Onchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. volvulus infection (onchocerciasis) began in 1974 and in recent years, following successful elimination of transmission in much of the Americas, the focus of efforts in Africa has moved from control to the more challenging goal of elimination of transmission in all endemic countries. Mass drug administration (MDA) with ivermectin has reached more than 150 million people and elimination of transmission has been confirmed in four South American countries, with at least two African countries having now stopped MDA as they approach verification of elimination. It is essential that accurate data for active transmission are used to assist in making the critical decision to stop MDA, since missing low levels of transmission and infection can lead to continued spread or recrudescence of the disease. Current World Health Organization guidelines for MDA stopping decisions and post-treatment surveillance include screening pools of the Simulium blackfly vector for the presence of O. volvulus larvae using a PCR-ELISA-based molecular technique. In this study, we address the potential of an updated, practical, standardized molecular diagnostic tool with increased sensitivity and species-specificity by comparing several candidate qPCR assays. When paired with heat-stable reagents, a qPCR assay with a mitochondrial DNA target (OvND5) was found to be more sensitive and species-specific than an O150 qPCR, which targets a non-protein coding repetitive DNA sequence. The OvND5 assay detected 19/20 pools of 100 blackfly heads spiked with a single L3, compared to 16/20 for the O150 qPCR assay. Given the improved sensitivity, species-specificity and resistance to PCR inhibitors, we identified OvND5 as the optimal target for field sample detection. All reagents for this assay can be shipped at room temperature with no loss of activity. The qPCR protocol we propose is also simpler, faster, and more cost-effective than the current end-point molecular assays.

An accurate, reliable, and universal qPCR method to identify homozygous single insert T-DNA with the example of transgenic rice.

Early determination of transgenic plants that are homozygous for a single locus T-DNA insert is highly desirable in most fundamental and applied transgenic research. This study aimed to build on an accurate, rapid, and reliable quantitative real-time PCR (qPCR) method to fast-track the development of multiple homozygous transgenic rice lines in the T1 generation, with low copy number to single T-DNA insert for further analyses. Here, a well-established qPCR protocol, based on the OsSBE4 reference gene and the nos terminator, was optimized in the transgenic Japonica rice cultivar Nipponbare, to distinguish homozygous single-insert plants with 100% accuracy. This method was successfully adapted to transgenic Indica rice plants carrying three different T-DNAs, without any modifications to quickly develop homozygous rice plants in the T1 generation. The accuracy of this qPCR method when applied to transgenic Indica rice approached 100% in 12 putative transgenic lines. Moreover, this protocol also successfully detected homozygous single-locus T-DNA transgenic rice plants with two-transgene T-DNAs, a feature likely to become more popular in future transgenic research. The assay was developed utilizing universal primers targeting common sequence elements of gene cassettes (the nos terminator). This assay could therefore be applied to other transgenic plants carrying the nos terminator. All procedures described here use standardized qPCR reaction conditions and relatively inexpensive dyes, such as SYBR Green, thus the qPCR method could be cost-effective and suitable for lower budget laboratories that are involved in rice transgenic research.

A-299 Clinical Performance Validation of 7 qPCR Kits for the Specific Diagnosis of 5 Enteric Viruses in Clinical Stool Samples

Abstract Background Among the most prevalent enteric viruses associated with gastroenteritis are norovirus (NoV), rotavirus (RV), astrovirus (AstV), sapovirus (SaV) and adenovirus (AdV). The diagnosis of infections associated with gastroenteritis viruses is mainly based on the detection of the genome of these viruses by molecular methods, mainly qPCR and RTqPCR reactions. The aim of this study was to validate the diagnostic performance of 7 qPCR diagnostic kits of the “VIASURE Real Time PCR Detection Kits” series with respect to commercial reference kits used for the diagnosis of these pathogens. Methods A total of 318 clinical stool samples were used, including: 213 stool samples from children from sporadic cases of gastroenteritis collected between 2016–2018; 94 samples from outbreaks of gastroenteritis caused by NoV between 2017–2019; and 11 samples from healthy children with asymptomatic enteric virus infections collected between 2017–2018. Seven VIASURE kits were used for the specific detection of enteric viruses. At the same time, samples were also tested with the following commercial kits used as reference methods: RIDA®GENE Viral Stool Panel II (rotavirus, adenovirus 40/41 and astrovirus) and RIDA®GENE Sapovirus, RIDA®GENE Norovirus I &amp; II. In the case of AdV, the positivity of the samples was confirmed using the qPCR protocol published by Heim et al. (2003). Results The results obtained are summarized in Table 1. Conclusion The results obtained show a high sensitivity and specificity of the VIASURE kits for the detection of rotavirus, adenovirus, sapovirus, astrovirus and norovirus GI and GII (in the latter case using both the monoplex and multiplex product). In addition, it should be noted that all these kits share the same thermal protocol, thus allowing their joint use for the simultaneous diagnosis of these 5 enteric viruses.

Development of a MGB probe based qPCR protocol for detecting Pacific cod Gadus macrocephalus in eDNA samples

Development of a MGB probe based qPCR protocol for detecting Pacific cod Gadus macrocephalus in eDNA samples

Improving the diagnosis of Streptococcus iniae using a novel probe-based qPCR assay combined with an enrichment step.

Streptococcus iniae is a bacterial pathogen that causes streptococcosis, leading to significant losses in fish aquaculture globally. This study reported a newly developed probe-based quantitative polymerase chain reaction (qPCR) method for the detection of S. iniae. The primers and probes were designed to target the lactate oxidase gene. The optimized method demonstrated a detection limit of 20 copies per reaction and was specific to S. iniae, as evidenced by no cross-reactivity when assayed against genetic materials extracted from 23 known aquatic animal pathogens, and fish samples infected with Streptococcus agalactiae or Streptococcus dysgalactiae. To validate the newly developed qPCR protocol with field samples, fish specimens were systematically investigated following the Food and Agriculture Organization of the United Nations & Network of Aquaculture Centres in Asia-Pacific three diagnostic levels approach, which integrated basic and advanced techniques for disease diagnosis, including observation of gross signs (level I), bacterial isolation (level II), qPCR and 16S rDNA sequencing (level III). The result showed that 7/7 affected farms (three Asian seabass farms and four tilapia farms) experiencing clinical signs of streptococcosis were diagnosed positive for S. iniae. qPCR assays using DNA extracted directly from fish tissue detected S. iniae in 11 out of 36 fish samples (30.6%), while 24 out of 36 samples (66.7%) tested positive after an enrichment step, including apparently healthy fish from affected farms. Bacterial isolation of S. iniae was only successful in a proportion of clinically diseased fish but not in healthy-looking fish from the same farm. Overall, the newly developed qPCR protocol combined with enrichment would be a useful tool for the diagnosis and surveillance of S. iniae infections in fish populations, thereby aiding in the disease control and prevention.

Development of a Duplex qPCR System for Detection and Quantification of the Two Canola Blackleg Pathogens Leptosphaeria biglobosa and L. maculans.

Two probe-based qPCR systems, namely P-Lb and P-Lm, specific to the canola blackleg pathogens Leptosphaeria biglobosa and L. maculans, respectively, were developed, and their efficiencies were tested. Each of the two systems targets a single-copy gene exclusively present in the corresponding species. The specificities of the two systems on the species level and their ubiquities on the subspecies level were confirmed by in silico sequence analyses and testing on L. biglobosa (17 strains), L. maculans (10 strains), and other plant pathogens (31 species). For sensitivities, the two systems were tested on synthesized DNA fragments (gBlock) of the targeted regions, from which a standard curve was generated for each system. In addition, standard curves were also generated on gBlocks for duplex qPCR in which the two systems were used in the same reaction. The two systems were further tested in both singleplex and duplex qPCR on DNA samples extracted from fungal spores, inoculated canola cotyledons, and naturally infected canola stubble samples collected from commercial fields. Our data indicated that the two systems are specific to L. biglobosa and L. maculans, respectively, and one reaction could detect as few as 200 spores of either species. When used in duplex qPCR on DNA samples with various origins, the two systems generated similar results as in singleplex qPCR. The duplex qPCR system, along with the sample preparation and DNA extraction specified in this study, constituted a first-reported duplex qPCR protocol for detection and quantification of the two blackleg pathogens from field samples.