Rapid, Accurate, and Low-Cost Stool Clostridium difficile Genetic Test

描述(申请人提供)：艰难梭菌(CD)是一种引起腹泻和其他肠道问题的细菌，在美国每年导致14,000人死亡。CDI是一种与抗生素相关的感染，也是一种与医疗保健相关的感染。目前CDI的诊断依赖于Cd毒素酶免疫分析(EIA)和抗原(GdH)EIA。虽然粪便毒素EIA具有较高的特异性和相对较快的检测速度(数小时到1天)，但其敏感性仅为60%。核酸扩增试验(NAAT)采用实时定量聚合酶链式反应(QPCR)或环介导的等温扩增(LAMP)技术检测毒素基因TcdB或TcdA，具有较高的敏感性和特异性。然而，由于超强毒株的出现，单独检测TcdA或TcdB不能判断CDI的严重程度，也不足以帮助医生做出明智的CDI治疗决策。由超强毒力菌株引起的CDI的30天死亡率和复发率较高，并与第三种毒素有关，即二元毒素 由cdtB和cdtA编码的毒素。所有CDI都涉及Tcdb，但不涉及TcdA。因此，如果有一种遗传CD测试可以在一次测试中同时检测TcdB和CDtB，那么不仅可以诊断CDI，还可以确定CDI的严重性和复发风险，以帮助医生做出治疗决定。(PbMg1/3Nb2/3O3)0.65(PbTiO3)0.35(PMN-PT)压电板传感器(PEPS)是Shih和Shih开发的一种独特的传感器，它利用相对共振频移df/f来检测靶标与PEPS表面受体的结合。PMN-PT PEPS的独特之处在于，由于PMN-PT层内独特的偏振切换机制，检测df/f提高了1000倍。因此，PEPS在DNA检测中表现出与PCR相似的敏感性，而不需要DNA提取和DNA扩增。这项研究的目标是开发一种快速、准确和低成本的CDI检测方法，使用3-PEPS阵列--一个PEPS检测TcdB，第二个PEPS检测CDtB，第三个PEPS作为阴性对照，具有高度的敏感性和特异性，以帮助诊断CDI，评估CDI的严重程度和复发风险。对20例盲人粪便进行TcdB检测的初步结果显示，PEPS能在40min内正确识别11/11qPCR阳性样本和7/9qPCR阴性样本，说明PEPS试验的敏感性。此外，PEPS易于多路复用，简单的电气检测使测试成本低，因此可以广泛使用。

DESCRIPTION (provided by applicant): Clostridium difficile (CD) is a bacterium causing diarrhea and other intestinal problems linked to 14,000 annual deaths in the US. CDI is an antibiotic-associated infection as well as a health-care-associated infection. Current CDI diagnosis relies on CD toxins enzyme immunoassay (EIA) together with antigen (GDH) EIA. Although stool toxin EIA is specific and relatively fast (hours to 1 day), the sensitivity of toxinEIA is only 60%. Nucleic acid amplification test (NAAT) using quantitative real-time polymerase chain reaction (qPCR) or loop-mediated isothermal amplification (LAMP) to detect the toxin gene tcdB or tcdA is sensitive and specific. However, due to the emergence of hypervirulent strains, detecting tcdA or tcdB alone cannot tell the severity of CDI, and is not sufficient to hel physician make informed CDI treatment decisions. The 30-day mortality rate and recurrence rate for CDI caused by hypervirulent strains are higher and are linked to a third toxin, the binary toxin which is encoded by cdtB and cdtA. All CDI involve tcdB but not tcdA. Therefore, if there is a genetic CD test that can detect tcdB and cdtB simultaneously in one test it would be possible to not only diagnose CDI but also determine the severity and recurrence risk of the CDI to help physicians make treatment decisions. (PbMg1/3Nb2/3O3)0.65(PbTiO3)0.35 (PMN-PT) piezoelectric plate sensor (PEPS) is a unique sensor developed by Shih and Shih, which uses relative resonance frequency shift, Df/f to detect binding of a target to the receptor on the PEPS surface. What is unique about PMN-PT PEPS is that detection Df/f is enhanced by 1000 times due to the unique polarization switching mechanism within the PMN-PT layer. As a result, PEPS has demonstrated sensitivity similar to PCR in DNA detection but without the need of DNA extraction and DNA amplification. The goal of the study is to develop a rapid, accurate, and low-cost CDI test using a 3- PEPS array--one PEPS to detect tcdB, the second PEPS to detect cdtB, and the third PEPS to serve as a negative control with high sensitivity and specificity to help diagnose CDI and assessing CDI severity and recurrence risk. Encouraging preliminary results of detecting tcdB in 20 blinded patient stools showed that PEPS correctly identified 11/11 qPCR positive samples and 7/9 qPCR negative samples within 40 min using simple electrical means, indicating the sensitivity of the PEPS test. In addition, PEPS is easily multiplexed and the simple electrical detection makes the test low-cost and thus can be widely available.