Limit of detection of genomic DNA by conventional PCR for estimating the load of Staphylococcus aureus and Escherichia coli associated with bovine mastitis

Chandrashekhar, Kshipra; Isloor, Shrikrishna; Veeresh, B. H.; Hegde, Raveendra; Rathnamma, D.; Murag, Shivaraj; Veeregowda, B.M.; Upendra, H. A.; Hegde, Nagendra R.

doi:10.1007/s12223-015-0384-0

Cited by 12 publications

(6 citation statements)

References 69 publications

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“…Lanes 3, 7, and 11 were biofunctionalized samples subjected to a higher antibody immobilization time (out of standardized conditions) and they were negative to PCR validation. Considering that the sensitivity of a conventional PCR ranges from 0.45 to 194 DNA pg, 44 the obtained genomic DNA was not enough for positive PCR validation. This could have resulted in the blockage of antibody active sites for bacterial epitope recognition confirming that bacteria are unable to bind superficially.…”

Section: Results and Discussionmentioning

confidence: 99%

Antibody Immobilization in Zinc Oxide Thin Films as an Easy-Handle Strategy for Escherichia coli Detection

2020

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The antibody immobilization compatible with low-cost materials and label-free strategies is a challenge for biosensor device fabrication. In this study, ZnO thin film deposition was carried out on corning glass substrates by ultrasonic spray pyrolysis at 200 °C. The thin films were analyzed as platforms for enteropathogenic Escherichia coli ( E. coli EPEC) antibody immobilization. The modification of thin films from the functionalization and antibody immobilization steps was visualized using Fourier transform infrared spectroscopy (FTIR) spectroscopy, and surface changes were observed by atomic force microscopy. The obtained FTIR spectra after functionalization showed a contribution of the amino group (NH 2 ) derived from silane (3-aminopropyltrimethoxysilane). The antibody immobilization showed an amide I conserved signal corresponding to the C=O stretching vibrations and the amide II signal related to the N–H scissor vibration mode. In this way, the signals observed are correlated with the presence of antibody immobilized on the film. The ZnO film morphology changes after every stage of the process and allows observing the antibody distribution on the immobilized surface. In order to validate the antibody recognition capability as well as the E. coli EPEC detection in situ , polymerase chain reaction was used.

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Section: Results and Discussionmentioning

confidence: 99%

Antibody Immobilization in Zinc Oxide Thin Films as an Easy-Handle Strategy for Escherichia coli Detection

2020

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“…Nonetheless, similar studies have found comparable results, whereby metabarcoding using the Illumina platform identi ed more pathogen positive samples than cPCR with Sanger sequencing (31,75,76). Such ndings may represent a diagnostic limit of detection for cPCR as samples can only be Sanger sequenced successfully upon ampli cation and visualisation of a PCR product band, with such bands only observable above a certain threshold concentration of DNA (77)(78)(79). This threshold concentration may be much lower or non-existent for deep sequencing methodologies which can successfully amplify from PCR product, even if no such product is visualisable on a gel (80).…”

Section: Discussionmentioning

confidence: 99%

Development and validation of a long-read metabarcoding platform for the detection of filarial worm pathogens infecting animals and humans

Huggins,

Atapattu,

Young

et al. 2023

Preprint

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Background: Filarial worms are important vector-borne pathogens of a large range of mammalian hosts, including humans and are responsible for some of the most pervasive, and pernicious diseases within the tropics. In humans, lymphatic filariasis caused by Wuchereria bancrofti and Brugia spp., as well as loiasis caused by Loa loa are all categorized as neglected tropical diseases. Moreover, some emerging or difficult-to-eliminate filarioid pathogens are zoonotic using animals like canines as reservoir hosts, for example Dirofilaria sp. ‘hongkongensis’. Diagnosis of filariasis through commonly available methods, like microscopy, can be challenging as microfilaremia may wane below the limit of detection. In contrast, conventional PCR methods are more sensitive and specific but may show limited ability to detect coinfections as well as emerging and/or novel pathogens. Use of deep-sequencing technologies obviate these challenges, providing sensitive detection of entire parasite communities, whilst also being better suited for the characterisation of rare or novel pathogens. Methods: Here we present a novel long-read metabarcoding assay for deep-sequencing the filarial worm cytochrome c oxidase subunit I gene on Oxford Nanopore Technologies’ (ONT) MinIONTM sequencer. We assessed the overall performance of our assay against commonly used diagnostic methods for filarial worm detection, such as conventional PCR (cPCR) with Sanger sequencing and the microscopy-based modified Knott’s test (MKT) Results: We confirmed our metabarcoding assay can characterise filarial parasites from a diverse range of genera, including, Breinlia, Brugia, Cercopithifilaria, Dipetalonema, Dirofilaria, Onchocerca, Setaria, Stephanofilaria and Wuchereria. We demonstrated proof-of-concept for this assay by using blood samples from Sri Lankan dogs, whereby we identified infections with the filarioids Acanthocheilonema reconditum, Brugia sp. Sri Lanka genotype and zoonotic Dirofilaria sp. ‘hongkongensis’. When compared to traditionally used diagnostics, such as the MKT and cPCR with Sanger sequencing, we identified additional filarioid species and numerous additional mono- and coinfections. Conclusions: Our developed metabarcoding assay may show broad applicability for the metabarcoding and diagnosis of the full spectrum of filarioids from a wide range of animal hosts, including mammals and vectors, whilst the utilisation of ONT’ small and portable MinIONTM means that such methods could be deployed for field use.

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“…Hence, targeting a gene which is carried in known number of copies by all the members of a population is critical. We chose three genes for each of these organisms as it was possible that the LOD could be better with one than with the others (Chandrashekar et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

“…Here single copy genes, nuc for Staph. aureus and uidA for E. coli were selected based on its minimum detection limit as mentioned by Chandrashekar et al (2015).…”

Section: Introductionmentioning

confidence: 99%

QUANTIFICATION OF Staphylococcus aureus AND Escherichia coli FROM BOVINE SUBCLINICAL MILK SAMPLES BY CONVENTIONAL PCR

Chandrashekar¹,

Isloor²,

Rathnamma³

et al. 2018

JEBAS

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The present study explores the application of conventional PCR and Mole v/s Avogadro's number using single copy genes of Staphylococcus aureus and Escherichia coli for assessing bacterial load in milk samples from mastitis cases. The nuc and uidA genes produced the lowest detection limit for Staph. aureus and E. coli respectively. The present findings estimated that, as few as 60 and 89 genome copies or organisms of Staph. aureus and E. coli were detected, respectively. Based on the LOD of molecules, standard graphs for these genes were generated and this knowledge was applied to milk samples from the field. Spiking known amount of genomic DNA (which in turn indicates organisms based on Avogadro's number) in LB broth and pasteurized milk was carried out to compare the input with output and simulating field conditions. A total of 90 samples from subclinical cases of mastitis were collected from four organized farms located at various villages. Out of 90 samples, 29 (32.22 %) were showed culture and duplex PCR positive. Of these, 12 (13.33 %) were found to be positive for E. coli alone, nine (10 %) were found to be positive for Staph. aureus alone, and eight (8.88 %) carried a mixed infection for both. In conclusion, this study suggests that quantification of bacteria directly from milk by the conventional PCR can be an alternative to time consuming conventional culture method and expensive real-time PCR, but requires extensive standardization.

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Limit of detection of genomic DNA by conventional PCR for estimating the load of Staphylococcus aureus and Escherichia coli associated with bovine mastitis

Cited by 12 publications

References 69 publications

Antibody Immobilization in Zinc Oxide Thin Films as an Easy-Handle Strategy for Escherichia coli Detection

Antibody Immobilization in Zinc Oxide Thin Films as an Easy-Handle Strategy for Escherichia coli Detection

Development and validation of a long-read metabarcoding platform for the detection of filarial worm pathogens infecting animals and humans

QUANTIFICATION OF Staphylococcus aureus AND Escherichia coli FROM BOVINE SUBCLINICAL MILK SAMPLES BY CONVENTIONAL PCR

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