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Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease.

ABSTRACT: Background:Several pathogens have been debated to play a role in inflammatory bowel disease (IBD) including Crohn's disease (CD). None of these pathogens have been investigated together in same clinical samples. We developed a multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) protocols for simultaneous detection of CD-associated pathogens including Mycobacterium avium subspecies paratuberculosis (MAP), Klebsiella pneumoniae, and adherent-invasive Escherichia coli strain LF82. Methods:The multiplex PCR is based on 1-h DNAzol® extraction protocol modified for rapid extraction of bacterial DNA from culture, blood, and intestinal biopsies. Oligonucleotide primers sequences unique to these pathogens were evaluated individually and in combinations using bioinformatics and experimental approaches. m-FISH was based on fluorescent-tagged oligonucleotides and confocal scanning laser microscopy (CSLM). Results:Following several attempts, the concentration of the oligonucleotide primers and DNA templates and the PCR annealing temperatures were optimized. Multiplex PCR analyses revealed excellent amplification signal in trials where a single primer set and combinations of two and three primers sets were tested against a mixture of DNA from three different bacteria or a mixture of three bacterial cultures mixed in one tube before DNA extraction. Slides with individual and mixtures of bacterial cultures and intestinal tissue sections from IBD patients were tested by m-FISH and the CSLM images verified multiplex PCR results detected on 3% agarose gel. Conclusion:We developed a 4-h multiplex PCR protocol, which was validated by m-FISH images, capable of detecting up to four genes from major pathogens associated with CD. The new protocol should serve as an excellent tool to support efforts to study multi-pathogens involved in CD and other autoimmune disease.

SUBMITTER: Sharp RC

PROVIDER: S-EPMC6280354 | biostudies-other | 2018

REPOSITORIES: biostudies-other

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Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease.

Sharp Robert C RC Naser Ebraheem S ES Alcedo Karel P KP Qasem Ahmad A Abdelli Latifa S LS Naser Saleh A SA

Gut pathogens 20181205

<h4>Background</h4>Several pathogens have been debated to play a role in inflammatory bowel disease (IBD) including Crohn's disease (CD). None of these pathogens have been investigated together in same clinical samples. We developed a multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) protocols for simultaneous detection of CD-associated pathogens including Mycobacterium avium subspecies paratuberculosis (MAP), Klebsiella pneumoniae, and adherent ...[more]

PMID: 30534203

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Project description:Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for M. tuberculosis was determined to be 5.1x104 cfu per ml and for M. avium 1.5x104 cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries.

Project description:Aneuploidy is one of the main causes of fetal and embryonic mortality in mammals. Nonetheless, its incidence in domestic ruminants has been investigated little. Indeed, no incidence data have ever been reported for water buffalo. To establish the incidence of aneuploidy in this species, we analysed in vitro matured metaphase II (MII) oocytes with corresponding first polar bodies (I PB) of the river (2n = 50) and swamp (2n = 48) buffaloes. For the first time, six river type probes (corresponding to chromosomes 1-5 and heterosome X), were tested on swamp buffalo metaphases using Multicolor-Fluorescent In Situ Hybridization (M-FISH) before their use on oocytes MII metaphases. Of the 120 total Cumulus Oocyte Complexes (COCs, 60 for each buffalo type) subjected to in vitro maturation, 104 reached the MII stage and were analysed by M-FISH. Haploid chromosome arrangement and visible I PB were observed in 89 of the oocytes (45 in river and 44 in swamp type). In the river type, the analysis revealed one oocyte was disomic for the chromosome X (2.22%). In the swamp type, one oocyte was found to be nullisomic for chromosome X (2.27%); another was found to be nullisomic for chromosome 5 (2.27%). We also observed one oocyte affected by a premature separation of sister chromatids (PSSC) on the chromosome X (2.27%). In both buffalo types, no abnormalities were detected in other investigated chromosomes. Based on merged data, the overall aneuploidy rate for the species was 3.37%. Oocytes with unreduced chromosomes averaged 1.92% across the two types, with 1.96% in river and 1.88% in swamp. The interspecies comparison between these data and cattle and pig published data revealed substantial difference in both total aneuploidy and diploidy rates. Reducing the negative impact of the meiotic segregation errors on the fertility is key to more sustainable breeding, an efficient embryo transfer industry and ex-situ bio-conservation. In this respect, additional M-FISH studies are needed on oocytes of domestic species using larger sets of probes and/or applying next generation sequencing technologies.

Project description:BackgroundWhole-mount in situ hybridization (WISH) is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Two-color fluorescent in situ hybridization (FISH) can be used to compare different transcript distributions at cellular resolution. Conventional two-color FISH protocols require two separate rounds of horseradish peroxidase (POD)-based transcript detection, which involves tyramide signal amplification (TSA) and inactivation of the first applied antibody-enzyme conjugate before the second detection round.ResultsWe show here that the alkaline phosphatase (AP) substrates Fast Red and Fast Blue can be used for chromogenic as well as fluorescent visualization of transcripts. To achieve high signal intensities we optimized embryo permeabilization properties by hydrogen peroxide treatment and hybridization conditions by application of the viscosity-increasing polymer dextran sulfate. The obtained signal enhancement allowed us to develop a sensitive two-color FISH protocol by combining AP and POD reporter systems. We show that the combination of AP-Fast Blue and POD-TSA-carboxyfluorescein (FAM) detection provides a powerful tool for simultaneous fluorescent visualization of two different transcripts in the zebrafish brain. The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day. Inactivation of the first applied reporter enzyme became unnecessary, so that false-positive detection of co-localization by insufficient inactivation, a problem of conventional two-color FISH, could be eliminated.ConclusionSince POD activity is rather quickly quenched by substrate excess, less abundant transcripts can often not be efficiently visualized even when applying TSA. The use of AP-Fast Blue fluorescent detection may provide a helpful alternative for fluorescent transcript visualization, as the AP reaction can proceed for extended times with a high signal-to-noise ratio. Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level. The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.

Dataset Information

Development of multiplex PCR and multi-color fluorescent in situ hybridization (m-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease.

Publications

Development of multiplex PCR and multi-color fluorescent in situ hybridization (<i>m</i>-FISH) coupled protocol for detection and imaging of multi-pathogens involved in inflammatory bowel disease.

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