Evaluation of droplet digital qRT-PCR (dd qRT-PCR) for quantification of SARS CoV-2 RNA in stool and urine specimens of COVID-19 patients

IntroductionThere have been a few reports of viral load detection in stool and urine samples of patients with coronavirus disease 2019 (COVID-19), and the transmission of the virus through faecal oral route. For clinical diagnosis and treatment, the widely used reverse transcription-polymerase chain reaction (qRT-PCR) method has some limitations.MethodsThe aim of our study to assess the presence and concentration of SARS CoV-2 RNA in stool and urine samples from COVID-19 patients with mild, moderate, and severe disease, we compared a traditional qRT-PCR approach with a ddPCR. ddPCR and qRT-PCR-based target gene analysis were performed on 107 COVID-19-confirmed patients paired samples (N1 and N2). The MagMax magnetic beads base method was used to isolate RNA. Real-time qRT-PCR and dd PCR were performed on all patients.Results and DiscussionThe average cycle threshold (Ct) of qRT-PCR was highly correlated with the average copy number of 327.10 copies/l analyzed in ddPCR. In ddPCR, urine samples showed 27.1% positivity while for stool it was 100%.ConclusionThis study’s findings not only show that SARS CoV-2 is present in urine and faeces, but also suggest that low concentrations of the viral target ddPCR make it easier to identify positive samples and help resolve for cases of inconclusive diagnosis

ANTICOAGULANT AND ANTIPLATELET ACTIVITIES OF JACKFRUIT (ARTOCARPUS HETEROPHYLLUS) SEED EXTRACT

  Objective: The current study focuses on the anticoagulant and antiplatelet activities of aqueous seed extract of Jackfruit (AqSEJ).Methods: Anticoagulant effect of AqSEJ was tested using plasma recalcification time, mouse tail bleeding time, Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT). Antiplatelet activity was examined by platelet aggregation studies using agonists such as ADP, Collagen and Epinephrine.Results: The AqSEJ enhanced the clotting time of citrated human plasma from control 200±10 s to 740±14 s. The anticoagulant activity of AqSEJ was further strengthened by in-vivo mouse tail bleeding assay. The i. v. injection of AqSEJ significantly prolonged the bleeding time in a dose dependent manner. The recorded bleeding time was>10 min (P<0.01) at the concentration of 30 μg against the PBS treated control of 1.48±0.10 min with the IC50 values 37.5 μg/ml and 47.5 μg/ml respectively. Interestingly, AqSEJ specifically prolonged the clot formation process of only APTT but not PT, revealing the anticoagulation triggered by the extract could be due to its interference in an intrinsic pathway of the blood coagulation cascade. Furthermore, AqSEJ inhibited the agonists such as ADP, epinephrine and collagen induced platelet aggregation of about 66.7%, 39.2% and 37.0% respectively at the concentration of 200 μg.Conclusion: AqSEJ showed anticoagulant and antiplatelet activities. Hence, it may serve as a better alternative for thrombotic disorders.Â

Detection of Echovirus-18 in Children Suspected With SARS-CoV-2 Infection With Multisystem Inflammatory Syndrome: A Case Report From India

There have been several reports across the globe regarding the presentation of a severe multi-system hyperinflammatory syndrome, resembling Kawasaki disease (KD), in the pediatric population during the SARS-CoV-2 pandemic. The exact pathophysiology is still unclear; however, children typically demonstrate multi-organ dysfunction and less respiratory system involvement compared to adults. The limited literature is available at present for the identification and management of such patients. In this study, we investigated four cases in children ages 11–15 years that fulfilled the case definition for the pediatric multi-system inflammatory syndrome. All were found negative for SARS-CoV-2 from oropharyngeal swabs and stool. As they were having symptoms of diarrhea, tests for bacterial and enteric viral infections were performed after SARS-CoV-2 testing. Molecular analysis revealed that all the children were infected with enterovirus (Echovirus-18). Early and exact diagnosis is vital for timely, effective, and potentially life-saving management of such cases

Vapour phase decomposition of cyclohexanol over mixed metal oxide system

510-512Mixed metal oxides, viz., ferrites have been investigated for their catalytic activity for vapour phase decomposition of cyclohexanol. A good correlation between electronic activation energy and catalytic activity has been observed. The ferrites have been prepared by co- precipitation technique and characterized by XRD, IR and electrical conductivity measurements. The kinetics of the reaction has been studied. The reaction follows typical first order kinetics. The activation energy for cyclohexanol decomposition has been found to be 29.45 kJ/mol

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Not AvailableThe present study was carried out to isolate the Alternative oxidase (aox) gene of Trypanosoma evansi by polymerase chain reaction, clone the amplicons in a suitable bacterial plasmid vector and characterisation of the gene through sequencing. The desired amplicon of aox gene of T. evansi was amplified by PCR using gene specific primers and identified on the basis of size of the gene. The amplicon of expected size was purified from the 1% low melting agarose gel. The DNA fragment of interest was then ligated to the pGEM- T Easy vector and ligated mixture was transformed into Escherichia coli JM109 strains for cloning. After cloning, screening of recombinants was done by Restriction Enzyme digestion of plasmid DNA and by colony PCR. After confirmation of clone, the plasmid DNA was sequenced and coding sequence of aox gene according to the results obtained was of 990 bp. Tree topology of aox gene is based on the Neighbor-Joining method with 100% bootstrap values and identified aox gene sequence showed a close homology with other Trypanosoma spp. gene sequences.Not Availabl

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Not AvailableThe present study was carried out to isolate the Alternative oxidase and Trans-sialidase genes of Trypanosoma evansi using PCR, clone the amplicons in a suitable plasmid vector and then characterization of above genes through sequencing. For this investigation, morphologically suspected T. evansi infected camel was confirmed by examination of Giemsa stained blood smear of camel blood. After confirming infection, DNA isolation from collected pellet of Trypanosoma evansi was done as per the protocols given by ready to use kit from Illustra blood genomic prep. mini kit with slight modifications. The desired amplicons of aox and ts genes were then amplified by PCR using gene specific primers. Amplified PCR products were analyzed on 1.2% agarose gel stained with ethidium bromide and identified on the basis of size of the aox and ts genes. The amplicons of expected size were purified from the 1% low melting agarose gel employing Illustra GFX PCR DNA and Gel Band Purification Kit. The DNA fragment of interest was then ligated to the pGEM- T Easy vector and ligated mixture was transformed into Escherichia coli JM109 strains. The cells containing recombinant plasmid could be identified on the basis of blue/white colony selection on LB agar containing X-Gal, IPTG and ampicillin. Screening of recombinants was done by Restriction Enzyme digestion of plasmid DNAs using EcoRI and found 74 that the release of DNA fragments around 990 bp for aox and 2241 bp for ts gene. Colony PCR was done for quick screening of plasmid inserts directly from E. coli colonies in the presence of insert specific primers. After confirmation of clones of aox and ts genes, the plasmid DNAs were sequenced and coding sequences of aox and ts genes according to the results obtained were of 990 and 2241 bp respectively. The phylogenetic and sequence analysis was done by use of Praline, Clustal X and MEGA5 softwares. Tree topology of aox and ts gene is based on the Neighbor-Joining method and maximum parsimony with 100% bootstrap values. Multiple sequence alignment of obtained protein sequences of aox and ts genes was performed with Praline sequence software. Identified aox and ts gene sequences showed a close homology with other Trypanosoma spp. gene sequences.Not Availabl

Not Available

Not AvailableThe present study was carried out to isolate the Trans-sialidase (ts) gene of Trypanosoma evansi by polymerase chain reaction, clone the amplicons in a suitable bacterial plasmid vector and characterise the gene through sequencing. The desired amplicon of ts gene of T. evansi was amplified by PCR using gene specific primers and identified on the basis of size of the gene. The amplicon of expected size was purified from the 1% low melting agarose gel. The DNA fragment of interest was then ligated to the pGEM- T Easy vector and ligated mixture was transformed into Escherichia coli JM109 strains for cloning. After cloning, screening of recombinants was done by Restriction Enzyme digestion of plasmid DNA and by colony PCR. After confirmation of clone, the plasmid DNA was sequenced and coding sequence of ts gene according to the results obtained was of 2241 bp. Tree topology of ts gene is based on the Neighbour-Joining method with 100% bootstrap values and identified ts gene sequence showed a close homology with other Trypanosoma spp. gene sequences.Not Availabl

Syzygium cumini L. better known as Jamun belonging to the family Myrtaceae is identified to have antidiabetic, anti-inflammatory, anti-pyretic and anti-oxidant activities. Anticancer activity of S. cumini L. fruits has been demonstrated. However, anticancer activity of S. cumini seeds on various types of human cancers has not been explored much. The methanol fraction of ethanol extract from the seeds of S. cumini was found to have significant antibacterial activity. This bioactive fraction was further tested positive for its anticancer activity on various types of human cancer cell lines indicating its potency. Structural characterization of the bioactive fraction was achieved using analysis of high performance liquid chromatography, ultra violet and infra red spectrum.

Syzygium cumini L. better known as Jamun belonging to the family Myrtaceae is identified to have antidiabetic, anti-inflammatory, anti-pyretic and anti-oxidant activities. Anticancer activity of S. cumini L. fruits has been demonstrated. However, anticancer activity of S. cumini seeds on various types of human cancers has not been explored much. The methanol fraction of ethanol extract from the seeds of S. cumini was found to have significant antibacterial activity. This bioactive fraction was further tested positive for its anticancer activity on various types of human cancer cell lines indicating its potency. Structural characterization of the bioactive fraction was achieved using analysis of high performance liquid chromatography, ultra violet and infra red spectrum