a6e6547e0f80d642cf302ff2b1650549 pmc_34577201.pdf 52f7a8d89c04fa76493b203f07dda9bb2528e160 pmc_34577201.pdf 12e61bf3113f41b5334229e3d14e0834046072249ebdfccfff24e24deb45d33f pmc_34577201.pdf Title: Immunomagnetic Separation Improves the Detection of Mycobacteria by Paper-Based Lateral and Vertical Flow Immunochromatographic Assays Subject: This work addresses a method that combines immunomagnetic separation (IMS) and paper-based nucleic acid immunochromatographic assay for the sensitive detection of Mycolicibacterium fortuitum (basonym Mycobacterium fortuitum) In particular, the preconcentration of the bacteria was achieved by using magnetic particles modified with an antibody specific towards mycobacteria. Following the IMS, the bacteria were lysed, and the genome was amplified by double-tagging PCR, using a set of primers specific for the 16S rRNA gene for Mycobacterium. During the amplification, the amplicons were labeled with biotin and digoxigenin tags. Moreover, a comparative study of paper-based immunochromatographic platforms, relying on vertical and lateral flow and on the use of streptavidin gold nanoparticles as a signal generating system, was also performed. The visual readout was achieved when the gold-modified amplicons were captured by the anti-DIG antibody in the test line. The analytical performance of both methods, nucleic acid vertical flow (NAVF) and nucleic acid lateral flow (NALF), is also discussed. Although NALF showed lower limit of detections (LODs), both NALF and NAVF combined with IMS were able to detect the required LOD in hemodialysis water, becoming two promising and useful techniques for the rapid screening of water supplies in hemodialysis centers, to prevent the exposure of immunosuppressed patients to contaminated sources. Keywords: rapid diagnostic tests; lateral flow assay; vertical flow assay; gold nanoparticles; mycobacteria; double-tagging PCR Author: Alejandra Ben Aissa, Barbara Araújo, Esther Julián, Maria Valnice Boldrin Zanoni and María Isabel Pividori Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Sep 9 09:31:13 2021 CEST ModDate: Thu Sep 9 09:43:35 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2683351 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- RZWHZC+VnURWPalladioL Type 1 Custom yes yes no 10 0 FEPSCO+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 WLKLBJ+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 YMFNXL+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 JGBJTI+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 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Custom yes yes no 236 0 WLKLBJ+URWPalladioL-Bold Type 1 Custom yes yes no 240 0 YMFNXL+URWPalladioL-Roma Type 1 Custom yes yes no 244 0 QKYDFU+MSAM10 Type 1 Custom yes yes no 248 0 YGOXWR+CMSY10 Type 1 Custom yes yes no 252 0 DKAHAM+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 259 0 DKAHAN+PalatinoLinotype CID TrueType Identity-H yes yes yes 262 0 DKAHAO+PalatinoLinotype TrueType WinAnsi yes yes no 268 0 DKAHCA+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 271 0 DKAPBC+TimesNewRoman CID TrueType Identity-H yes yes yes 274 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-27 02:56:44 CEST RepresentationInformation: pmc_34577201.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-31 12:05:47 CEST Size: 2683351 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 440 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Instrumentation and Materials Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Chemicals and Biochemical Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Oligonucleotides Sequences Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Preparation of the Devices in Vertical Flow Configuration Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Preparation of Devices in Lateral Flow Configuration Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Covalent Immobilization of Antibodies on Magnetic Particles Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Bacterial Strain and Culture Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Immunomagnetic Separation and DNA Extraction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Double-Tagging PCR for the Amplification of Mycobacteria Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Nucleic Acid Vertical Flow Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Nucleic acid Lateral Flow Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Data Interpretation and Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Results and Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Children: Item: Title: Double-Tagging PCR for the Amplification of Mycobacteria Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Nucleic Acid Vertical Flow Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Nucleic Acid Lateral Flow Immunoassay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Info: Title: Immunomagnetic Separation Improves the Detection of Mycobacteria by Paper-Based Lateral and Vertical Flow Immunochromatographic Assays Author: Alejandra Ben Aissa, Barbara Araújo, Esther Julián, Maria Valnice Boldrin Zanoni and María Isabel Pividori Subject: This work addresses a method that combines immunomagnetic separation (IMS) and paper-based nucleic acid immunochromatographic assay for the sensitive detection of Mycolicibacterium fortuitum (basonym Mycobacterium fortuitum) In particular, the preconcentration of the bacteria was achieved by using magnetic particles modified with an antibody specific towards mycobacteria. Following the IMS, the bacteria were lysed, and the genome was amplified by double-tagging PCR, using a set of primers specific for the 16S rRNA gene for Mycobacterium. During the amplification, the amplicons were labeled with biotin and digoxigenin tags. Moreover, a comparative study of paper-based immunochromatographic platforms, relying on vertical and lateral flow and on the use of streptavidin gold nanoparticles as a signal generating system, was also performed. The visual readout was achieved when the gold-modified amplicons were captured by the anti-DIG antibody in the test line. The analytical performance of both methods, nucleic acid vertical flow (NAVF) and nucleic acid lateral flow (NALF), is also discussed. Although NALF showed lower limit of detections (LODs), both NALF and NAVF combined with IMS were able to detect the required LOD in hemodialysis water, becoming two promising and useful techniques for the rapid screening of water supplies in hemodialysis centers, to prevent the exposure of immunosuppressed patients to contaminated sources. 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