J Vet Sci.  2009 Mar;10(1):35-42. 10.4142/jvs.2009.10.1.35.

A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples

• Kumanan V
• Nugen SR
• Baeumner A
• Chang YF

• KMID: 1089344
• DOI: http://doi.org/10.4142/jvs.2009.10.1.35

Abstract

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR.

Keyword

feces; lateral flow biosensor assay; liposomes; Mycobacterium avium subsp. paratuberculosis; RT-PCR

MeSH Terms

Animals
Bacteriological Techniques
Biosensing Techniques/*veterinary
Cattle
Feces/*microbiology
Mycobacterium avium subsp. paratuberculosis/*isolation & purification
RNA, Bacterial/classification/isolation & purification
Reverse Transcriptase Polymerase Chain Reaction/veterinary
Sensitivity and Specificity

Figure

• 

  Fig. 1 Dose-response curve of the optimized lateral flow biosensor assay using quantified synthetic DNA target sequence. The intensity of the signals increased as the concentration of the target sample increased. Assays were run in triplicate. The value for the negative control was 1.04 ± 3.

• 

  Fig. 2 Biosensor assay done with the RT-PCR product of RNA isolated from fecal samples spiked with 101 to 106 MAP organisms. Three strips were used for each dilution (101 to 106) of sample. One strip each for positive control (PC) and negative control (NC) were used. Positive signals are seen at the capture zone even with the RT-PCR product of RNA extracted from fecal samples containing 10 organisms of MAP. RFR: reflectometer reading, CFU: colony forming units.

• 

  Fig. 3 Effect of synthetic DNA target concentration (0-1,000 nM) on the fluorescence signal assessed by microtiter plate assay. Each point is the average of triplicate determinations at each of the target concentrations tested and the error bars represent one standard deviation. A detection limit of 0.1 nM was obtained based on the value of the lowest concentration tested to be above the value of the negative control plus three times the standard deviation of the negative control.

• 

  Fig. 4 Effect of RT-PCR products of RNA extracted from spiked fecal samples (containing 101 to 106 organisms) on the fluorescence signal assessed by microtiter plate assay. Each point is the average of 3 determinations with error bars representing one standard deviation. The detection limit was found to be as low as 10 CFU based on the value of the lowest CFU tested to be above the value of the negative control plus three times the standard deviation of the negative control.

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