Determination of the viability of Toxoplasma gondii oocysts by PCR real-time after treatment with propidium monoazide

November 12, 2020
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RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells

 

Immunization-based antibody discovery platforms require robust and effective protocols for the amplification, cloning, expression, and screening of antibodies from large numbers of B-cells in order to effectively capture the diversity of an experienced Ig-repertoire. Multiplex PCR using a series of forward and reverse primers designed to recover antibodies from a range of different germline sequences is challenging because primer design requires the recovery of full length antibody sequences, low starting template concentrations, and the need for all the primers to function under the same PCR conditions.
Here we demonstrate several advantages to incorporating RNase H2-dependent PCR (rh-PCR) into a high-throughput, antibody-discovery platform. Firstly, rh-PCR eliminated primer dimer synthesis to below detectable levels, thereby eliminating clones with a false positive antibody titer. Secondly, by increasing the specificity of PCR, the rh-PCR primers increased the recovery of cognate antibody variable regions from single B-cells, as well as downstream recombinant antibody titers.
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hsa-mir-521 Real-time RT-PCR Detection Kit
20-abx097600
  • EUR 551.00
  • EUR 787.00
  • EUR 398.00
  • 100 rxns
  • 200 rxns
  • 50 rxns

Novel Coronavirus COVID-19 (2019-nCoV) Real Time RT-PCR Kit
RR-0478-02 25 tests/kit
EUR 991
Description: Novel Coronavirus (2019-nCoV) Real Time RT-PCR Kit is used for the qualitative detection of a novel coronavirus, which was identified in 2019 at Wuhan City, Hubei Province, China, in upper respiratory tract specimens (nasopharyngeal extracts, deep cough sputum, etc.) and lower respiratory tract specimens (alveoli irrigation fluid, etc.) by real time PCR systems.

0,1ML THIN WALL TUBES 8 PER STRIP. CLEAR & REAL TIME STRIP CAPS
PCR-0108-LP-RT-C 125/pk
EUR 709
Description: PCR Plates & Tubes; PCR Strip Tubes and Strip Caps - Axygen

0,1ML THIN WALL TUBES 8 PER STRIP. WHITE & REAL TIME STRIP CAPS
PCR-0108-LP-RT-W 125/pk
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Description: PCR Plates & Tubes; PCR Strip Tubes and Strip Caps - Axygen

Rubella virus One-Step PCR kit
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hsa-mir-521 Real-Time RT-PCR Detection and U6 Calibration Kit
20-abx097601
  • EUR 732.00
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  • EUR 523.00
  • 100 rxns
  • 200 rxns
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Virus Zika RT PCR kit
RTq-H434-100R 100T
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Virus Zika RT PCR kit
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Virus Zika RT PCR kit
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EUR 963

Dengue virus RT PCR kit
RTq-H451-100R 100T
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Dengue virus RT PCR kit
RTq-H451-150R 150T
EUR 1787

Dengue virus RT PCR kit
RTq-H451-50R 50T
EUR 963

Chikungunya virus RT PCR kit
RTq-H525-100R 100T
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Chikungunya virus RT PCR kit
RTq-H525-150R 150T
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Chikungunya virus RT PCR kit
RTq-H525-50R 50T
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Measles Virus RT PCR kit
RTq-H639-100R 100T
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Measles Virus RT PCR kit
RTq-H639-150R 150T
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Measles Virus RT PCR kit
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EUR 963

Mumps Virus RT PCR kit
RTq-H640-100R 100T
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Mumps Virus RT PCR kit
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Mumps Virus RT PCR kit
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EUR 963

Uukuniemi virus RT PCR kit
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Uukuniemi virus RT PCR kit
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Reticuloendotheliosis virus RT PCR kit
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Reticuloendotheliosis virus RT PCR kit
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Reticuloendotheliosis virus RT PCR kit
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Distemper virus RT PCR kit
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Pseudorabies Virus RT PCR kit
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Finally, we demonstrate that rh-PCR primers provide a more homogeneous sample pool and greater sequence quality in a Next Generation Sequencing-based approach to obtaining DNA sequence information from large numbers of cloned antibody cognate pairs. Furthermore, the higher specificity of the rh-PCR primers allowed for a better match between native antibody germline sequences and the VL/VH fragments amplified from single B-cells.

Determination of the viability of Toxoplasma gondii oocysts by PCR real-time after treatment with propidium monoazide

 

This study aimed to investigate a methodology for discriminating viable and non-viable T. gondii oocysts in water. Analyses included two steps: (i) microscopic investigation with vital dyes; (ii) molecular investigation, using a real time PCR (qPCR), after parasite treatment (or not) with propidium monoazide (PMA).
The method was called qPCR-PMA. Oocyst aliquots were incubated (15 min) at 25 ºC or 100 ºC and analyzed by microscopy, after trypan blue and neutral red staining. Microscopic investigation determined viable and non-viable oocysts. For the molecular investigation, both aliquots of oocysts were treated with PMA. Non-viable oocysts, after PMA treatment, exhibited an inhibition of DNA amplification by qPCR. Although analyses were carried out with oocysts treated experimentally, these results suggest that qPCR-PMA can be a useful strategy to distinguish viable and non-viable T. gondii oocysts in water safety testing, showing if water is safe to drink.

Diagnostic Utility of a Custom 34-Gene Anchored Multiplex PCR-Based Next-Generation Sequencing Fusion Panel for the Diagnosis of Bone and Soft Tissue Neoplasms With Identification of Novel USP6 Fusion Partners in Aneurysmal Bone Cysts

 

Context.—: Bone and soft tissue tumors are heterogeneous, diagnostically challenging, and often defined by gene fusions.
Objective.—: To present our experience using a custom 34-gene targeted sequencing fusion panel.
Design.—: Total nucleic acid extracted from formalin-fixed, paraffin-embedded (FFPE) tumor specimens was subjected to open-ended, nested anchored multiplex polymerase chain reaction and enrichment of 34 gene targets, thus enabling detection of known and novel fusion partners.
Results.—: During a 12-month period, 145 patients were tested as part of routine clinical care. Tumor percentage ranged from 10% to 100% and turnaround time ranged from 3 to 15 (median, 7.9) days. The most common diagnostic groups were small round blue cell tumors, tumors of uncertain differentiation, fibroblastic/myofibroblastic tumors, and adipocytic tumors.
In-frame fusion transcripts were identified in 64 of 140 cases sequenced (46%): in 62 cases, the detection of a disease-defining fusion confirmed the morphologic impression; in 2 cases, a germline TFG-GPR128 polymorphic fusion variant was detected. Several genes in the panel partnered with multiple fusion partners specific for different diagnoses, for example, EWSR1, NR4A3, FUS, NCOA2, and TFE3. Interesting examples are presented to highlight how fusion detection or lack thereof was instrumental in establishing accurate diagnoses. Novel fusion partners were detected for 2 cases of solid aneurysmal bone cysts (PTBP1-USP6, SLC38A2-USP6).
Conclusions.—: Multiplex detection of fusions in total nucleic acid purified from FFPE specimens facilitates diagnosis of bone and soft tissue tumors. This technology is particularly useful for morphologically challenging entities and in the absence of prior knowledge of fusion partners, and has the potential to discover novel fusion partners.

Performance Evaluation of a Commercial Real-Time PCR Assay and of an In-House Real-Time PCR for Trypanosoma cruzi DNA Detection in a Tropical Medicine Reference Center, Northern Italy

 

Chagas disease, a neglected protozoal disease endemic in Latin America, is also currently considered an emerging threat in nonendemic areas because of population movements. The detection of Trypanosoma cruzi DNA is increasingly being considered as important evidence to support Chagas disease diagnoses.
However, further performance evaluation of molecular assays is useful for a standardization of strategy considering the whole process in routine diagnosis, especially for the different settings such as endemic and nonendemic countries. Seventy-five samples were collected from subjects screened for Chagas disease in Italy.
The DNA was isolated from blood using automated extraction. We evaluated the performance of the commercial RealCycler® CHAG kit (pmPCR) based on satellite DNA (SatDNA) and of an in-house real-time PCR (ihPCR) targeting Sat and kinetoplast (k) DNAs, using the concordance of two serology assays as a reference standard. The sensitivity of kDNA and SatDNA tests by ihPCR and SatDNA by pmPCR were 14.29% (95% confidence interval (CI) 6.38 to 26.22), 7.14% (95% CI 1.98 to 17.29), and 7.14% (95% CI 1.98 to 17.29), respectively.
Specificity was 100% for all PCR assays and targets. Overall, our results suggest that the preferred approach for clinical laboratories is to combine the kDNA and SatDNA as targets in order to minimize false-negative results increasing sensitivity.

Selection of DNA Aptamers for Subcellular Localization of RBSDV P10 Protein in the Midgut of Small Brown Planthoppers by Emulsion PCR-Based SELEX

Rice black-streaked dwarf virus (RBSDV), classified under the Reoviridae, Fijivirus genus, caused an epidemic in the eastern provinces of China and other East Asian countries and resulted in severe yield loss in rice and wheat production. RBSDV is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus Fallén) in a persistent manner.
In order to provide a stable and cost-effective detection probe, in this study we selected three DNA aptamers (R3, R5 and R11) by an optimized, standardized and time saving emulsion PCR-based SELEX, for the detection of RBSDV outer-shell P10 protein for in situ localization studies in the midgut of SBPH. The specificity of these three DNA aptamers was tested through detection of the P10 protein using an enzyme-linked oligonucleotide assay (ELONA) and aptamer-based dot-blot ELISA.
All three DNA aptamers can be used to detect RBSDV P10 protein by immunofluorescent labeling in the midgut of RBSDV-infected SBPH. These data show that the selected aptamers can be used for the detection of RBSDV P10 protein in vitro and in vivo. This is the first report of aptamers being selected for detection of a rice virus capsid protein.

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HCV9057 11 X 1 mL
EUR 1548.64
Description: Please contact Gentaur in order to receive the datasheet of the product.

HCV Seroconversion Panel Donor# 66647 (5 X 1 mL)
HCV9058 5 X 1 mL
EUR 1548.64
Description: Please contact Gentaur in order to receive the datasheet of the product.

HCV Seroconversion Panel Donor# 22708D (5 X 1 mL)
HCV9094 5 X 1 mL
EUR 1548.64
Description: Please contact Gentaur in order to receive the datasheet of the product.

HCV Seroconversion Panel Donor# 1038851 (5 X 1 mL)
HCV9095 5 X 1 mL
EUR 1548.64
Description: Please contact Gentaur in order to receive the datasheet of the product.

HCV Pre/Post Seroconversion Panel Donor# 66822 (5 X 1 mL)
HCV10235 5 X 1 mL
EUR 512.8
Description: Please contact Gentaur in order to receive the datasheet of the product.

Positive control tissue section for each antibody; Based on availability INQUIRE
Control-Slides Set of 5
EUR 176

Zika Virus Seroconversion Panel (7 X 0.5mL)
KZMC047 7 X 0.5mL
EUR 2367.12
Description: Please contact Gentaur in order to receive the datasheet of the product.

HIV Seroconversion Panel Donor# 65404 (7 X mL)
HIV9013 7 X mL
EUR 1548.64
Description: Please contact Gentaur in order to receive the datasheet of the product.

HCV Genotype Panel (14 X 0.5 mL)
KZMC049 14 X 0.5 mL
EUR 2191.36
Description: Please contact Gentaur in order to receive the datasheet of the product.

EBV Seroconversion Panel Donor# 283843421 (14 X 1 mL)
EBV9097 14 X 1 mL
EUR 1548.64
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 64090 (5 X 1 mL)
HBV10231 5 X 1 mL
EUR 582.48
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 64017 (6 X 1 mL)
HBV10232 6 X 1 mL
EUR 582.48
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 65449 (9 X 1 mL)
HBV11000 9 X 1 mL
EUR 859.12
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 65524 (8 X 1 mL)
HBV11001 8 X 1 mL
EUR 859.12
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 65556 (6 X 1 mL)
HBV11002 6 X 1 mL
EUR 859.12
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 65584 (8 X 1 mL)
HBV11003 8 X 1 mL
EUR 859.12
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 65732 (8 X 1 mL)
HBV11004 8 X 1 mL
EUR 859.12
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 65777 (14 X 1 mL)
HBV11005 14 X 1 mL
EUR 859.12
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 66201 (17 X 1 mL)
HBV11006 17 X 1 mL
EUR 859.12
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 66537 (14 X 1 mL)
HBV11007 14 X 1 mL
EUR 859.12
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 67303 (18 X 1 mL)
HBV11008 18 X 1 mL
EUR 859.12
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 67449 (23 X 1 mL)
HBV11009 23 X 1 mL
EUR 859.12
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 67457 (18 X 1 mL)
HBV11010 18 X 1 mL
EUR 859.12
Description: Please contact Gentaur in order to receive the datasheet of the product.

HBV Seroconversion Panel Donor# 67694 (14 X 1 mL)
HBV11011 14 X 1 mL
EUR 859.12
Description: Please contact Gentaur in order to receive the datasheet of the product.
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