Kumanan Vijayarani, Nugen Sam R, Baeumner Antje J, Chang Yung-Fu
Animal Health Diagnostic Center, Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
J Vet Sci. 2009 Mar;10(1):35-42. doi: 10.4142/jvs.2009.10.1.35.
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.
一种基于膜条的简单侧向流动（LF）生物传感器检测方法和一种高通量微孔板检测方法已与逆转录聚合酶链反应（RT-PCR）相结合，用于检测粪便样本中少量（十个）活的鸟分枝杆菌副结核亚种（MAP）细胞。这些检测方法基于对MAP的IS900元件RNA的鉴定。在该检测中，从接种了已知数量（10¹至10⁶）MAP细胞的粪便样本中提取RNA，使用RT-PCR进行扩增，并通过LF生物传感器和微孔板检测进行鉴定。虽然LF生物传感器检测仅需30分钟的检测时间，但检测10个活细胞的整个过程耗时10小时。这些检测方法基于寡核苷酸夹心杂交检测形式，使用与靶序列杂交的固定化DNA探针的膜流通系统或微孔板孔。当靶序列与已偶联至包裹染料磺罗丹明B的脂质体的第二个DNA探针杂交时，可实现信号放大。脂质体中的染料提供的信号可通过肉眼读取、用手持式反射仪定量或用荧光读数器定量。检测方法的特异性分析表明，与其他分枝杆菌无交叉反应，如鸟分枝杆菌复合群、溃疡分枝杆菌、海分枝杆菌、堪萨斯分枝杆菌、脓肿分枝杆菌、亚洲分枝杆菌、草分枝杆菌、偶然分枝杆菌、瘰疬分枝杆菌、胞内分枝杆菌、耻垢分枝杆菌和牛分枝杆菌。检测活MAP生物体的整体检测方法相对便宜且快速，特别是与需要6至8周培养时间的标准MAP培养检测方法相比，并且比实时PCR便宜得多。
