Novel potential diagnostic test for <i>Mycobacterium tuberculosis</i>
 complex using combined immunomagnetic separation (IMS) and PCR-CTPP

Intorasoot, Sorasak; Tharinjaroen, Chayada Sitthidet; Phunpae, Ponrut; Butr-Indr, Bordin; Anukool, Usanee; Intachai, Kannaporn; Orrapin, Santhasiri; Apiratmateekul, Napaporn; Arunothong, Surachet; Suthachai, V.; Saengsawang, Kanokwan; Khamnoi, Phadungkiat; Pata, Supansa; Tragoolpua, Khajornsak

doi:10.1111/jam.13157

Cited by 4 publications

(3 citation statements)

References 39 publications

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“…In this study, we developed a novel method using a combination of immunomagnetic beads separation and enrichment followed by RAA. Studies have reported that immunomagnetic separation technology is also used for the detection and enrichment of Listeria monocytogenes, Vibrio parahaemolyticus , Mycobacterium tuberculosis and Escherichia coli (Kraft et al 2017 ; Intorasoot et al 2016 ; Jiang et al 2020 ; Stewart et al 2017 ; Wadud et al 2010 ; Zeng et al 2014 ). These researchers used specific antibodies combined with magnetic beads to capture the target pathogen, which was then combined with PCR, LAMP and other techniques to perform nucleic acid amplification.…”

Section: Discussionmentioning

confidence: 99%

Detection of low-load Epstein-Barr virus in blood samples by enriched recombinase aided amplification assay

Chen

Tie

et al. 2022

AMB Expr

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Epstein-Barr virus (EBV), a common human γ-herpesvirus, infects more than 90% of adults worldwide. The purpose of this study was to establish a novel EBV detection method by combining the recombinase aided amplification (RAA) assay with an initial enrichment step that utilizes magnetic beads coated with a recombinant human mannan-binding lectin (rhMBL, M1 protein). An M1 protein–protein A magnetic bead complex (M1 beads) was prepared and used to achieve separation and enrichment of EBV from blood. After nucleic acid extraction, DNA was amplified by RAA. Using 388 whole blood samples and 1 serum sample, we explored the specificity, sensitivity and applicability of the newly developed detection method and compared it with commercial quantitative real-time polymerase chain reaction (qPCR) following M1 bead enrichment, traditional qPCR and traditional RAA. After enrichment, the positivity rate of EBV was increased from 15.94% to 17.74% by RAA (P < 0.05) and from 7.20% to 15.17% by qPCR (P < 0.05). The viral loads after enrichment were increased by 1.13 to 23.19-fold (P < 0.05). Our data demonstrates that an RAA assay incorporating M1 bead enrichment is a promising tool for detecting low EBV viral loads in blood samples that will facilitate an early response to EBV infection. Graphical abstract

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Section: Discussionmentioning

confidence: 99%

Detection of low-load Epstein-Barr virus in blood samples by enriched recombinase aided amplification assay

Chen

Tie

et al. 2022

AMB Expr

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“…Ag85 complex proteins specifically interact with fibronectin [ 34 ] and exhibit robust sequence conservation across mycobacterium species, which may affect sensitivity and specificity of their detection by immunoassay methods. One recent study isolated mycobacteria from sputum samples using an Ag85B immunomagnetic enrichment approach prior to an Mtb -specific PCR analysis in an attempt to improve Mtb detection efficiency [ 35 ]. The sensitivity of this relatively labor-intensive approach for Mtb cases did not surpass Xpert-MTB/RIF assays results (89.9 vs. 95.2%), and this study did not examine the diagnostic sensitivity of this method for common NTM species causing pulmonary disease.…”

Section: Discussionmentioning

confidence: 99%

Antigen 85B peptidomic analysis allows species-specific mycobacterial identification

et al. 2018

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BackgroundNontuberculous mycobacteria (NTM)-mediated infections are a growing cause of worldwide morbidity, but lack of rapid diagnostics for specific NTM species can delay the initiation of appropriate treatment regimens. We thus examined whether mass spectrometry analysis of an abundantly secreted mycobacterial antigen could identify specific NTM species.MethodsWe analyzed predicted tryptic peptides of the major mycobacterial antigen Ag85B for their capacity to distinguish Mycobacterium tuberculosis and three NTM species responsible for the majority of pulmonary infections caused by slow-growing mycobacterial species. Next, we analyzed trypsin-digested culture supernatants of these four mycobacterial species by liquid chromatography–tandem mass spectrometry (LC–MS/MS) to detect candidate species-specific Ag85B peptides, the identity of which were validated by LC–MS/MS performed in parallel reaction monitoring mode.ResultsTheoretical tryptic digests of the Ag85B proteins of four common mycobacterial species produced peptides with distinct sequences, including two peptides that could each identify the species origin of each Ag85B protein. LC–MS/MS analysis of trypsinized culture supernatants of these four species detected one of these species-specific signature peptides in each sample. Subsequent LC–MS/MS analyses confirmed these results by targeting these species-specific Ag85B peptides.ConclusionsLC–MS/MS analysis of Ag85B peptides from trypsin-digested mycobacterial culture supernatants can rapidly detect and identify common mycobacteria responsible for most pulmonary infections caused by slow-growing mycobacteria, and has the potential to rapidly diagnose pulmonary infections caused by these mycobacteria through direct analysis of clinical specimens.Electronic supplementary materialThe online version of this article (10.1186/s12014-017-9177-6) contains supplementary material, which is available to authorized users.

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“…Magnetic-separation methods, already established as routine methods to detect Listeria monocytogenes (24), Salmonella spp. (25), and Escherichia coli O157:H7 (26) (27) in food and veterinary clinical samples, are promising alternatives to selectively separate and concentrate MTB (28)(29)(30)(31)(32) and MAP (33,34). Magnetic bead characteristics (composition, size, concentration, and surface modification) and the nature of coating ligands (antibodies, peptides, chemicals) are important factors enabling the concentration of the target bacteria and increasing the performance of further complementary detection methods such as culture, PCR, microscopy, antigen detection immunoassay, or phage assay (33,35,36).…”

Section: Introductionmentioning

confidence: 99%

Use of untargeted magnetic beads to capture Mycobacterium smegmatis and Mycobacterium avium paratuberculosis prior detection by mycobacteriophage D29 and Real-Time-PCR

Rojas-Ponce

Sauvageau

Zemp

et al. 2021

Preprint

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Dynabeads® M-280 Tosylactivated (untargeted magnetic beads) were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Untargeted magnetic beads added to the spiked samples were slightly mixed for 1 hour and separated in a magnetic rack for further detection. Beads recovered more M. smegmatis cells from PBS suspension that the centrifugation method; these results were confirmed by the recovery of 96.31% of 1.68 x 104 CFU/mL viable M. smegmatis by beads and 0% by centrifugation. Likewise, the F57-qPCR detection of MAP cells, after being recovered by beads and centrifugation, were different; cycle threshold (Ct) was lower (p<0.05) for the detection of MAP cells recovered by beads than centrifugation. Magnetic separation of MAP cells from milk, urine, and feces specimens were detected by amplifying F57 and IS900 sequences. Ct values demonstrated that beads captured no less than 109 CFU/mL from feces and no less than 104 CFU/mL of MAP cells from milk and urine suspensions. Milk proteins were denatured by Proteinase k before capturing MAP cells by magnetic beads. M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29; plaque former units were observed clearly from urine containing 2 x 105 and 2 x 103 CFU/mL M. smegmatis in 24 hours. The results of this study encourage further effort to rule out the use of untargeted beads as a simple tool for diagnosis of Johne′s disease and other mycobacterial diseases such as tuberculosis.

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Novel potential diagnostic test for Mycobacterium tuberculosis complex using combined immunomagnetic separation (IMS) and PCR-CTPP

Abstract: This study is the first report emphasizing the combination of IMS and PCR-CTPP for the detection of MTC and simultaneous identification of Myco. bovis from sputum. It could be used for TB diagnosis in resource-limited countries with high TB burden.

Cited by 4 publications

References 39 publications

Detection of low-load Epstein-Barr virus in blood samples by enriched recombinase aided amplification assay

Detection of low-load Epstein-Barr virus in blood samples by enriched recombinase aided amplification assay

Antigen 85B peptidomic analysis allows species-specific mycobacterial identification

Use of untargeted magnetic beads to capture Mycobacterium smegmatis and Mycobacterium avium paratuberculosis prior detection by mycobacteriophage D29 and Real-Time-PCR

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