Rapid Detection of Human Respiratory Syncytial Virus A and B by Duplex Real-Time RT-PCR
August 26, 2021
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Respiratory syncytial virus (RSV) is a typical reason for acute respiratory illness worldwide, particularly in younger youngsters. The World Well being Group (WHO) has initiated an RSV Surveillance Pilot program that goals to carry out worldwide RSV surveillance, requiring the event of dependable and speedy molecular strategies to detect and determine RSV.
A duplex real-time RT-PCR assay developed for simultaneous detection of each A and B subtypes of RSV was included as a part of this program. This duplex assay focused a conserved area of the RSV polymerase gene and was validated for analytical sensitivity, specificity, reproducibility and medical efficiency with a variety of respiratory specimens. The assay was extremely particular for RSV and didn’t react with non-RSV respiratory pathogens, together with the SARS-CoV-2 virus.
Growth of duplex TaqMan-based actual–time PCR assay for the simultaneous detection of Perkinsus olseni and P. chesapeaki in host Manila clam tissue samples
The aetiological agent Perkinsus olseni is globally recognised as a serious menace for shellfish manufacturing contemplating its large geographical distribution throughout Asia, Europe, Australia and South America. One other species, Perkinsus chesapeaki, which has by no means been identified to be related to important mortality occasions, was not too long ago detected alongside French coasts infecting clam populations sporadically in affiliation with P. olseni.
Figuring out potential cryptic infections affecting Ruditapes philippinarum is crucial to develop applicable host useful resource administration methods. Right here, we developed a molecular technique based mostly on duplex real-time quantitative PCR for the simultaneous detection of those two parasites, P. olseni and P. chesapeaki, within the totally different clam tissues: gills, digestive gland, foot, mantle, adductor muscle and the remainder of the comfortable physique.
We firstly checked the presence of potential PCR inhibitors in host tissue samples. The qPCR reactions had been inhibited relying on the character of the host organ. The mantle and the remainder of the comfortable physique have a excessive inhibitory impact from threshold of host gDNA focus of two ng.µL-1, the adductor muscle and the foot have an intermediate inhibition of 5 ng.µL-1, and the gills and digestive gland don’t present any inhibition of the qPCR response even on the highest host gDNA focus of 20 ng.µL-1.
Then, utilizing the gills as a template, the suitability of the molecular method was checked compared with the Ray’s Fluid Thioglycolate Medium methodology advisable by the World Organisation for Animal Well being. The duplex qPCR technique introduced new insights and unveiled cryptic infections because the co-occurrence of P. olseni and P. chesapeaki from in situ tissue samples in distinction to the RFTM analysis. The event of this duplex qPCR technique is a basic work to observe in situ co-infections that can result in optimised useful resource administration and conservation methods to cope with rising illnesses.
Water pre-filtration strategies to enhance environmental DNA detection by actual–time PCR and metabarcoding
Environmental DNA (eDNA) evaluation is a novel strategy for biomonitoring and has been principally utilized in clear water. It’s troublesome to detect eDNA in turbid water as filter clogging happens, and environmental samples include varied substances that inhibit the polymerase chain response (PCR) and have an effect on the accuracy of eDNA evaluation. Due to this fact, we utilized a pre-filtration technique to raised detect the fish species (notably pale chub, Opsariichthys platypus) current in a water physique by measuring eDNA in environmental samples containing PCR inhibitors.
Upon conducting 12S rRNA metabarcoding evaluation (MiFish), we discovered that pre-filtration didn’t have an effect on the quantity or identities of fish species detected in our samples, however pre-filtration by way of pore sizes resulted in considerably diminished variance amongst replicate samples. Moreover, PCR amplification was improved by the pre-filtration of environmental samples containing PCR inhibitors resembling humic substances. Though this examine could look like a conservative and ancillary experiment, pre-filtration is an easy method that may not solely enhance the bodily properties of water, resembling turbidity, but in addition the standard of eDNA biomonitoring.
A comparative examine of actual–time RT-PCR-based SARS-CoV-2 detection strategies and its utility to human-derived and floor swabbed materials
Actual-time reverse transcription polymerase chain response (RT-PCR) stays a gold customary in detection of assorted viral illnesses. Within the COVID-19 pandemic, a number of RT-PCR based mostly checks had been developed to display for viral an infection. As an emergency response to rising testing demand, we established a SARS-CoV-2 PCR diagnostics platform for which we in contrast totally different industrial and in-house RT-PCR protocols. 4 industrial (CDC 2019-nCoV, Utilized BiosystemsTM 2019-nCoV Assay Equipment v1 TF-SinglePlex, 2019-nCoV Assay Equipment v2 TF-MultiPlex, and EURORealTime SARS-CoV-2), one custom-made (Institute Pasteur), and one in-house RT-PCR protocols had been evaluated with 92 SARS-CoV-2 optimistic and 92 SARS-CoV-2 adverse samples.
Moreover, economical and sensible traits of those protocols had been in contrast. Moreover, a extremely delicate digital droplet PCR (ddPCR) technique was developed and utility of RT- and ddPCR strategies on SARS-CoV-2 environmental samples was examined. Very low limits of detection (1 or 2 viral copies/μL), excessive sensitivities (93.6-97.8%) and specificities (98.7-100%) for the examined RT-PCR protocols had been discovered.
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Moreover, the feasibility of downscaling two of the industrial protocols, which might optimize testing capability was demonstrated. Examined industrial and customised RT-PCR detection kits present superb and comparable sensitivity, and specificity, and the kits may very well be additional optimized to be used on SARS-CoV-2 viral samples derived from human and floor swabbed samples.