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

描述（由申请人提供）：艰难梭菌（CD）是一种引起腹泻和其他肠道问题的细菌，在美国每年有14,000人死亡。CDI是一种抗生素相关感染，也是一种卫生保健相关感染。目前的CDI诊断依赖于CD毒素酶免疫测定（EIA）和抗原（GDH）免疫测定。虽然粪便毒素EIA具有特异性且相对较快（数小时至1天），但毒素ia的敏感性仅为60%。核酸扩增试验（NAAT）采用定量实时聚合酶链反应（qPCR）或环介导等温扩增（LAMP）检测毒素基因tcdB或tcdA具有敏感性和特异性。然而，由于高毒性菌株的出现，仅检测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。由高毒力菌株引起的CDI的30天死亡率和复发率较高，并且与第三种毒素二元毒素有关

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.