scholarly journals Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping of Viable Cryptosporidium Oocysts

2009 ◽  
Vol 75 (21) ◽  
pp. 6856-6863 ◽  
Author(s):  
Cristin C. Brescia ◽  
Shannon M. Griffin ◽  
Michael W. Ware ◽  
Eunice A. Varughese ◽  
Andrey I. Egorov ◽  
...  
Keyword(s):  
Human Health Risk ◽  
Protozoan Parasite ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Pcr Analysis ◽  
Propidium Monoazide ◽  
Pcr Assay ◽  
Severe Diarrhea ◽  
Cryptosporidium Oocysts ◽  
Surface Water Samples

ABSTRACT Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.

A new real-time PCR assay for detecting fungi in genus Ceratocystis

Plant Disease ◽  
2021 ◽  
Author(s):  
Karthikeyan Dharmaraj ◽  
Alice Merrall ◽  
Julie A. Pattemore ◽  
Joanne Mackie ◽  
Brett J.R Alexander ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Stem Tissue ◽  
Natural Ecosystems ◽  
Pcr Assay ◽  
Practical Applications ◽  
Wide Range

The genus Ceratocystis contains several significant plant pathogens, causing wilt and canker disease on a wide range of plants species. Currently, there are over 40 known species of Ceratocystis, some of which are becoming increasingly important in agricultural or natural ecosystems. The diagnostics for most Ceratocystis species currently relies on time consuming and labour-intensive culturing approaches. To provide more time efficient and sensitive molecular diagnostic tools for Ceratocystis, a generic Taq-Man real-time PCR assay was developed using the ITS gene. This novel two-probe Taq-man assay amplified DNA from all tested Ceratocystis species. Some non-specific amplification of a few species from closely related genera was observed under certain conditions; however, these false positive detections could be ruled out using the additional PCR primers developed for further sequence based identification of the detected species. The assay was highly sensitive as it detected 0.2 pg/µl of Ceratocystis DNA in water as well as in host DNA matrix. Further validation with artificially inoculated fig stem tissue demonstrated that the assay was also able to effectively detect the pathogen in infected asymptomatic stem tissue. This newly developed real-time PCR assay has practical applications in biosecurity, conservation, and agriculture, enabling to detect Ceratocystis species directly from plant material, to facilitate more sensitive screening of imported plant germplasm, and allow rapid tracking of pathogens in case of disease outbreaks.

scholarly journals New Subgenotyping and Consensus Real-Time Reverse Transcription-PCR Assays for Hepatitis A Outbreak Surveillance

2019 ◽  
Vol 57 (9) ◽  
Author(s):  
William S. Probert ◽  
Jill K. Hacker
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Outbreak Detection ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Laboratory Surveillance

ABSTRACTLaboratory surveillance plays an important role in the detection and control of hepatitis A outbreaks and requires the application of rapid and accurate molecular diagnostic tools for hepatitis A virus (HAV) RNA detection, subgenotype identification, and sequence-based genotyping. We describe the development and validation of a triplex real-time, reverse transcription-PCR (triplex rRT-PCR) assay for the identification and discrimination of HAV subgenotypes IA, IB, and IIIA and a singleplex rRT-PCR assay designed to detect all HAV genotypes infecting humans. Overall, the accuracy, sensitivity, and specificity of the new assays were >97% for serum and plasma specimens collected during unrelated outbreaks of HAV in California and Michigan compared to a nested RT-PCR genotyping assay and the ISO 15216-1 rRT-PCR method for HAV detection. The new assays will permit the rapid detection of HAV RNA and discrimination among subgenotypes IA, IB, and IIIA in serum and plasma specimens, which will strengthen public health surveillance efforts for HAV outbreak detection and response.

scholarly journals microFLOQ® Direct: a helpful tool for the coronavirus SARS-CoV-2 rapid detection without RNA purification

2020 ◽  
Author(s):  
Mathilde Recipon ◽  
Amaury Pussiau ◽  
Sébastien Follot ◽  
Noussair Latifa ◽  
Jean-Louis Herrmann ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Virus Disease ◽  
Sampling Procedure ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Pcr Analysis ◽  
Infected People ◽  
Bacterial Diseases ◽  
Transport Medium ◽  
Rna Purification

Abstract In the context of SARS-Cov-2 virus disease (COVID-19) pandemic, molecular diagnostic tools were rapidly developed as there are fundamental for a rapid detection of infected people. In this context, and in order to optimize the manipulations and reduce the time to get results, we report the successful use of a sampling tool for COVID-19 diagnosis named microFLOQ® Direct (MFD). Hundred upper respiratory specimens sampled from patients with potential COVID-19 were evaluated using MFD, and results were compared to the results obtained by standard sampling procedure using dry swabs and physiologic serum as the transport medium. MFD results compared to results issued from the classic RNA purification and amplification steps from transport medium showed that MFD can be directly used for RT-PCR analysis without the preliminary inactivation and extraction steps. So, MFD could limit handling errors compared to the different treatment steps with dry swabs and transport medium. It therefore limits the risk of contamination, simplify the analytical process and enables to get results in less than 2 hours. We also show that the MFD kit is operational as a screening tool in the field of molecular detection of viral and bacterial diseases during an outbreak and then it can be used for public health or agro-veterinary purposes.

scholarly journals Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass

3 Biotech ◽  
2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Domenico Rizzo ◽  
Nicola Luchi ◽  
Daniele Da Lio ◽  
Linda Bartolini ◽  
Francesco Nugnes ◽  
...  
Keyword(s):  
Lamp Assay ◽  
Isothermal Amplification ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Loop Mediated Isothermal Amplification ◽  
Larval Stages ◽  
Longhorn Beetle ◽  
Rapid Monitoring ◽  
Major Pest ◽  
Wood Borer

AbstractThe red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.

scholarly journals The new Internal Transcribed Spacer 2 diagnostic tool clarifies the taxonomic position and geographic distribution of the North American malaria vector Anopheles punctipennis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
James M. Hodge ◽  
Andrey A. Yurchenko ◽  
Dmitriy A. Karagodin ◽  
Reem A. Masri ◽  
Ryan C. Smith ◽  
...  
Keyword(s):  
Malaria Vector ◽  
Internal Transcribed Spacer ◽  
Single Species ◽  
Its2 Sequence ◽  
Pathogen Transmission ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Sequence Length ◽  
Phylogenetic Position ◽  
Internal Transcribed Spacer 2

Abstract Background The malaria mosquito Anopheles punctipennis, a widely distributed species in North America, is capable of transmitting human malaria and is actively involved in the transmission of the ungulate malaria parasite Plasmodium odocoilei. However, molecular diagnostic tools based on Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA are lacking for this species. Anopheles punctipennis is a former member of the Anopheles maculipennis complex but its systematic position remains unclear. Methods In this study, ITS2 sequences were obtained from 276 An. punctipennis specimens collected in the eastern and midwestern United States and a simple and robust Restriction Fragment Length Polymorphism approach for species identification was developed. The maximum-likelihood phylogenetic tree was constructed based on ITS2 sequences available through this study and from GenBank for 20 species of Anopheles. Results The analysis demonstrated a consistent ITS2 sequence length and showed no indications of intragenomic variation among the samples based on ITS2, suggesting that An. punctipennis represents a single species in the studied geographic locations. In this study, An. punctipennis was found in urban, rural, and forest settings, suggesting its potential broad role in pathogen transmission. Phylogeny based on ITS2 sequence comparison demonstrated the close relationship of this species with other members of the Maculipennis group. Conclusions This study developed molecular tools based on ITS2 sequences for the malaria vector An. punctipennis and clarified the phylogenetic position of the species within the Maculipennis group.

scholarly journals LAMP in Neglected Tropical Diseases: A Focus on Parasites

Diagnostics ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 521
Author(s):  
Juan García-Bernalt Diego ◽  
Pedro Fernández-Soto ◽  
Antonio Muro
Keyword(s):  
Neglected Tropical Diseases ◽  
Treatment Success ◽  
Public Health Problem ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
World Population ◽  
Major Public Health Problem ◽  
Tropical Diseases ◽  
Early 21St Century

Neglected Tropical Diseases (NTDs), particularly those caused by parasites, remain a major Public Health problem in tropical and subtropical regions, with 10% of the world population being infected. Their management and control have been traditionally hampered, among other factors, by the difficulty to deploy rapid, specific, and affordable diagnostic tools in low resource settings. This is especially true for complex PCR-based methods. Isothermal nucleic acid amplification techniques, particularly loop-mediated isothermal amplification (LAMP), appeared in the early 21st century as an alternative to PCR, allowing for a much more affordable molecular diagnostic. Here, we present the status of LAMP assays development in parasite-caused NTDs. We address the progress made in different research applications of the technique: xenomonitoring, epidemiological studies, work in animal models and clinical application both for diagnosis and evaluation of treatment success. Finally, we try to shed a light on the improvements needed to achieve a true point-of-care test and the future perspectives in this field.

scholarly journals A 2.8-Angstrom-Resolution Cryo-Electron Microscopy Structure of Human Parechovirus 3 in Complex with Fab from a Neutralizing Antibody

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Aušra Domanska ◽  
Justin W. Flatt ◽  
Joonas J. J. Jukonen ◽  
James A. Geraets ◽  
Sarah J. Butcher
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
High Resolution ◽  
Cultured Cells ◽  
Human Monoclonal Antibody ◽  
Neutralizing Antibody ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Human Parechovirus ◽  
Cryo Electron Microscopy

ABSTRACTHuman parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward the latter, we present a 2.8-Å-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody, AT12-015, using cryo-electron microscopy (cryo-EM) and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally, at high resolution, it was possible to model a stretch of RNA inside the virion and, from this, identify the key features that drive and stabilize protein-RNA association during assembly.IMPORTANCEHuman parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for which, despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Toward this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high resolution, it was possible to precisely define the epitope that, when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.

scholarly journals The burden of congenital Chagas disease and implementation of molecular diagnostic tools in Latin America

2018 ◽  
Vol 3 (5) ◽  
pp. e001069 ◽  
Author(s):  
Albert Picado ◽  
Israel Cruz ◽  
Maël Redard-Jacot ◽  
Alejandro G Schijman ◽  
Faustino Torrico ◽  
...  
Keyword(s):  
Latin America ◽  
Chagas Disease ◽  
Diagnostic Algorithm ◽  
Diagnostic Methods ◽  
Diagnosis And Treatment ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Molecular Tests ◽  
Epidemiological Impact ◽  
And Performance

It is estimated that between 8000 and 15 000 Trypanosoma cruzi infected babies are born every year to infected mothers in Chagas disease endemic countries. Currently, poor access to and performance of the current diagnostic algorithm, based on microscopy at birth and serology at 8–12 months after delivery, is one of the barriers to congenital Chagas disease (CCD) control. Detection of parasite DNA using molecular diagnostic tools could be an alternative or complement to current diagnostic methods, but its implementation in endemic regions remains limited. Prompt diagnosis and treatment of CCD cases would have a positive clinical and epidemiological impact. In this paper, we analysed the burden of CCD in Latin America, and the potential use of molecular tests to improve access to early diagnosis and treatment of T. cruzi infected newborns.

scholarly journals Frequency of benzimidazole resistance inHaemonchus contortus populations isolated from buffalo, goat and sheep herds

2013 ◽  
Vol 22 (4) ◽  
pp. 548-553 ◽  
Author(s):  
Ronaldo Luiz Nunes ◽  
Livia Loiola dos Santos ◽  
Eduardo Bastianetto ◽  
Denise Aparecida Andrade de Oliveira ◽  
Bruno Santos Alves Figueiredo Brasil
Keyword(s):  
Haemonchus Contortus ◽  
Significant Variation ◽  
Genetic Basis ◽  
Anthelmintic Resistance ◽  
Allelic Frequency ◽  
Molecular Diagnostic ◽  
Economic Losses ◽  
Diagnostic Tools ◽  
Ruminant Species ◽  
Benzimidazole Resistance

Anthelmintic resistance is an increasing problem that threatens livestock production worldwide. Understanding of the genetic basis of benzimidazole resistance recently allowed the development of promising molecular diagnostic tools. In this study, isolates of Haemonchus contortus obtained from goats, sheep and buffaloes raised in Brazil were screened for presence of the polymorphism Phe200Tyr in the β-tubulin 1 gene, which confers resistance to benzimidazole. The allelic frequency of the mutation conferring resistance ranged from 7% to 43%, and indicated that resistance to benzimidazole could be found in nematodes isolated from all the ruminant species surveyed. Although significant variation in the frequency of the F200Y mutation was observed between different herds or host species, no significant variation could be found in populations isolated from animals within the same herd. These findings suggest that screening of samples from a few animals has the potential to provide information about the benzimidazole resistance status of the entire herd, which would enable a considerable reduction in the costs of diagnosis for the producer. Molecular diagnosis has practical advantages, since it can guide the choice of anthelmintic drug that will be used, before its application in the herd, thus reducing the economic losses driven by anthelmintic resistance.

scholarly journals Identification of pathogenic Leptospira species and serovars in New Zealand using metabarcoding

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257971
Author(s):  
David A. Wilkinson ◽  
Matthew Edwards ◽  
Jackie Benschop ◽  
Shahista Nisa
Keyword(s):  
New Zealand ◽  
Diagnostic Techniques ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Pathogenic Leptospira ◽  
Molecular Barcoding ◽  
Sequencing Platform ◽  
Oxford Nanopore ◽  
Culture Independent ◽  
Variant Identification

Leptospirosis is a zoonotic disease of global importance. The breadth of Leptospira diversity associated with both human and animal disease poses major logistical challenges to the use of classical diagnostic techniques, and increasingly molecular diagnostic tools are used for their detection. In New Zealand, this has resulted in an increase in positive cases reported nationally that have not been attributed to the infecting serovar or genomospecies. In this study, we used data from all pathogenic Leptospira genomes to identify a partial region of the glmU gene as a suitable locus for the discrimination of the infecting species and serovars of New Zealand-endemic Leptospira. This method can be used in culture and culture-independent scenarios making it flexible for diagnostics in humans, animals, and environmental samples. We explored the use of this locus as a molecular barcoding tool via the Oxford Nanopore Technology (ONT) sequencing platform MinION. Sequences obtained by this method allowed specific identification of Leptospira species in mixed and enriched environmental cultures, however read error inherent in the MinION sequencing system reduced the accuracy of strain/variant identification. Using this approach to characterise Leptospira in enriched environmental cultures, we detected the likely presence of Leptospira genomospecies that have not been reported in New Zealand to date. This included a strain of L. borgpetersenii that has recently been identified in dairy cattle and sequences similar to those of L. mayottensis. L. tipperaryensis, L. dzianensis and L. alstonii.

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