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

BackgroundSeveral 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.MethodsThe 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).ResultsFollowing 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.ConclusionWe 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.

Acute Ulcerative Enterocolitis With Severe Protein Loss Due to Mucosal Invasion With Enterococcus spp. in a Dog With Exocrine Pancreatic Insufficiency: A Case Report

“…As FISH is not a routine diagnostic tool, the frequency of this is unknown. FISH should be considered more frequently as part of routine diagnostics like in human IBD (43)(44)(45), particularly in severe or difficult to control cases. Results may identify different underlying phenotypes of CIE, that more closely resemble human CD.…”

Anti-MAP Triple Therapy Supports Immunomodulatory Therapeutic Response in Crohn’s Disease through Downregulation of NF-κB Activation in the Absence of MAP Detection

“…In contrast, other studies have reported lower incidence of MAP infection among CD patients in comparison to healthy controls, which might be due to the fact that different methodology was used for MAP detection in human samples [15]. Detection of MAP in cultures of blood, breast milk, and intestinal tissue was reported using RT-PCR and nested PCR techniques [16]. At least one third of CD patients may not have MAP infection or they may have it at levels below the limit of detection provided by available techniques.…”

“…After 24 h of infection with MAP, monocyte-derived macrophages were washed twice with PBS to remove extracellular bacteria, and then antibiotics were added, and the cells were incubated for an additional 24 h. Monocyte-derived macrophages were collected at three time points: 24, 48, and 72 h. The cells were lysed, and then the samples were centrifuged at 14,000 rpm for 20 min. Supernatants were collected and the LIVE/DEAD™ BacLight™ Bacterial Viability Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used according to the manufacturer's protocol as described earlier [16]. Briefly, 100 µL of each bacterial suspension was pipetted into separate wells of a 96-well flat-bottom microplate in triplicate and mixed with 100 µL of stain solution.…”

Divergent Effect of Cigarette Smoke on Innate Immunity in Inflammatory Bowel Disease: A Nicotine-Infection Interaction

“…One of the most debated explanations for the contradictory effect of nicotine is the association of CD with microbial infection, which lacks in UC. Among the most discussed pathogens associated with CD is MAP [7,8,[49][50][51][52][53][54][55][56][57][58].…”