Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures

Abstract

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.

sRNA‐FISH: versatile fluorescent in situ detection of small RNAs in plants

“…() used a Fast Red substrate instead of NBT/BCIP for detection of miRNAs in mouse brain. Dual‐target fluorescence in situ hybridization assays were also used for detecting pathogens in cell cultures (Shah et al ., ), as well as the specific chromosomal location related to invasive breast tumors (Walker et al ., ). In higher eukaryotes, the fine structure of RNA‐processing bodies, including P‐bodies, D‐bodies and CBs, have been challenging to observe using conventional microscopy because of their small size (300–500 nm in diameter), which is close to the diffraction limit of light (Mito et al ., ).…”

Integrated Electrochemical Biosensors for Detection of Waterborne Pathogens in Low-Resource Settings

“…Recently, a dual-colour fluorescence in situ hybridization (FISH) assay was developed for the analysis of Mycobacterium. The limit of detection for Mycobacterium tuberculosis was determined to be 5.1 × 10 4 CFU/mL and for Mycobacterium avium, 1.5x10 4 CFU/mL [43]. Moreover, Almeida at al.…”

Rapid method for detecting and differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with DNA probes

“…A PNA-based FISH assay has been shown to differentiate MTB from several NTM species, but to have relatively low sensitivity with sputum samples (Kim et al, 2015). The present authors have described two dual fluorescence FISH assaysthe Mycobacterium/Nocardia Genus (MN Genus)-MTBC assay and the MAC-MTBC assaywhich identified MTBC and MAC with specificity and sensitivity of 100% in a large number of cultures and also differentiated them from other NTM and Nocardia species (Shah et al, 2017a). This article reports on a method for applying the MN Genus-MTBC FISH assay to sputum samples to identify MTBC and differentiate them from NTM, as well as the relevant clinical diagnostic parameters.…”

“…Liquid cultures were grown at Kasturba Medical College, Mangaluru, India by inoculating the processed pellets containing bacteria from sputum samples into MGIT tubes in the BACTEC MGIT 320 detection system (Becton Dickinson, Franklin Lakes, NJ, USA), as described previously (Shah et al, 2017a). The processed pellets from sputum samples were also used to initiate cultures on Lowenstein-Jensen (LJ) solid medium slants (BD Diagnostics, Sparks, MD, USA) (Shah et al, 2017a). Mycobacteria grown in liquid and solid cultures were identified to the species level by DNA sequencing.…”

“…Several fluorescence in situ hybridization (FISH) assays that target multiple copies of rRNA present in mycobacterial cells, and therefore do not require NAA, have been described (Yuan et al, 2015;Shah et al, 2017a;Amand St et al, 2005a,b;Stender et al, 1999;Lehtola et al, 2006;Kim et al, 2015;Lefmann et al, 2006). Such FISH assays are based on DNA (Yuan et al, 2015;Shah et al, 2017a;Amand St et al, 2005a,b) or peptide nucleic acid (PNA) (Stender et al, 1999;Lehtola et al, 2006;Kim et al, 2015;Lefmann et al, 2006) probes but are not yet in widespread clinical use. As NAA is not necessary, FISH assays are not affected by inhibitors of NAA and do not require the expensive equipment and infrastructure needed for NAA.…”