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

Kumanan V, Nugen SR, Baeumner AJ, Chang YF - J. Vet. Sci. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Animal Health Diagnostic Center, Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.

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 (10(1) to 10(6)) 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. fortutitum, M. scrofalaceum, M. intracellular, 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.

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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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Figure 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.

Mentions: The lateral-flow biosensor assay was compared with the microtiter plate assay employing the same probe and target sequences for the detection of RNA extracted from fecal samples. The microtiter plate assay was performed with the RT-PCR product of the IS900 gene after denaturation at 95℃ for 5 min and hybridized at 60℃. Positive signals were obtained when 1.5 µl of target was used in the assay. The detection limit was found to be as low as 10 CFU when RT-PCR product of RNA extracted from fecal samples spiked with 101 to 106 organisms (Fig. 4). This was the same limit of detection obtained for the simple LF assay.


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

Kumanan V, Nugen SR, Baeumner AJ, Chang YF - J. Vet. Sci. (2009)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2801105&req=5

Figure 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.
Mentions: The lateral-flow biosensor assay was compared with the microtiter plate assay employing the same probe and target sequences for the detection of RNA extracted from fecal samples. The microtiter plate assay was performed with the RT-PCR product of the IS900 gene after denaturation at 95℃ for 5 min and hybridized at 60℃. Positive signals were obtained when 1.5 µl of target was used in the assay. The detection limit was found to be as low as 10 CFU when RT-PCR product of RNA extracted from fecal samples spiked with 101 to 106 organisms (Fig. 4). This was the same limit of detection obtained for the simple LF assay.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Animal Health Diagnostic Center, Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.

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 (10(1) to 10(6)) 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. fortutitum, M. scrofalaceum, M. intracellular, 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.

Show MeSH
Related in: MedlinePlus