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

快速、准确、低成本的粪便艰难梭菌基因检测

• 批准号: 8715249
• 负责人: Wan Y. Shih
• 金额: $ 30万
• 依托单位: LENIMA FIELD DIAGNOSTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-15 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8715249
• 关键词: Antibiotics; Antigens; Bacteria; Binding; Biological Assay; Blinded; Caring; Cells; Cessation of life; Clinical; Clostridium difficile; Contracts; Cytolysis; Cytotoxin; DNA; DNA amplification; Detection; Diagnosis; Diarrhea; Enzyme Immunoassay; Feces; Film; Fluorescent Probes; Frequencies; Gene Amplification; Genes; Genetic; Genetic screening method; Goals; Gold; Healthcare; High temperature of physical object; Hospitals; Hour; In Situ; Infection; Intestines; Label; Lead; Link; Magnesium; Mediating; Medical; Monitor; Nucleic Acid Amplification Tests; Nucleic Acids; Nursing Homes; Outpatients; Patients; Persons; Phase; Physicians; Polymerase Chain Reaction; Prevalence Study; Recurrence; Relative (related person); Risk; Sampling; Sensitivity and Specificity; Severities; Specificity; Stress; Surface; System; Testing; Time; Toxin; cold temperature; combat; cost; galactitol 2-dehydrogenase; lead titanate; mortality; patient safety; point of care; prototype; public health relevance; receptor; sensor; solid solution

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.

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，在美国每年导致 14,000 人死亡。 CDI 是一种抗生素相关感染，也是一种医疗保健相关感染。目前的 CDI 诊断依赖于 CD 毒素酶联免疫分析 (EIA) 和抗原 (GDH) EIA。尽管粪便毒素EIA具有特异性且相对快速（数小时至1天），但毒素EIA的敏感性仅为60%。核酸扩增试验（NAAT）采用定量实时聚合酶链式反应（qPCR）或环介导等温扩增（LAMP）来检测毒素基因tcdB或tcdA，灵敏且特异。然而，由于高毒力菌株的出现，仅检测tcdA或tcdB并不能判断CDI的严重程度，也不足以帮助医生做出明智的CDI治疗决定。由高毒力菌株引起的 CDI 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 由cdtB和cdtA编码的毒素。所有 CDI 都涉及 tcdB，但不涉及 tcdA。因此，如果有一种基因 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 的严重程度和复发风险，以帮助医生做出治疗决策。 (PbMg1/3Nb2/3O3)0.65(PbTiO3)0.35 (PMN-PT) 压电板传感器 (PEPS) 是 Shih 和 Shih 开发的独特传感器，它使用相对共振频移 Df/f 来检测目标与PEPS 表面的受体。 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 增强了 1000 倍。因此，PEPS 在 DNA 检测中表现出类似于 PCR 的灵敏度，但不需要 DNA 提取和 DNA 扩增。该研究的目标是使用 3-PEPS 阵列开发一种快速、准确且低成本的 CDI 测试——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敏感性和特异性，有助于诊断 CDI 并评估 CDI 严重程度和复发风险。在 20 名盲法患者粪便中检测 tcdB 的令人鼓舞的初步结果表明，PEPS 使用简单的电气手段在 40 分钟内正确识别了 11/11 qPCR 阳性样本和 7/9 qPCR 阴性样本，表明 PEPS 测试的敏感性。此外，PEPS 易于复用，并且简单的电检测使测试成本低廉，因此可以广泛使用。

项目成果

Specific in situ hepatitis B viral double mutation (HBVDM) detection in urine with 60 copies ml(-1) analytical sensitivity in a background of 250-fold wild type without DNA isolation and amplification.

尿液中特异性原位乙型肝炎病毒双突变 (HBVDM) 检测，在 250 倍野生型背景下具有 60 拷贝 ml(-1) 分析灵敏度，无需 DNA 分离和扩增。

• DOI: 10.1039/c4an01885k
• 发表时间: 2015-03-07
• 期刊: The Analyst
• 作者: Kirimli CE;Shih WH;Shih WY
• 通讯作者: Shih WY

A model study of 3-dimensional localization of breast tumors using piezoelectric fingers of different probe sizes.

使用不同探头尺寸的压电手指进行乳腺肿瘤三维定位的模型研究。

• 发表时间: 2019-01
• 作者: Xu, Xin;Shih, Wei;Shih, Wan Y
• 通讯作者: Shih, Wan Y

DNA hybridization detection with 100 zM sensitivity using piezoelectric plate sensors with an improved noise-reduction algorithm.

使用具有改进的降噪算法的压电板传感器进行 DNA 杂交检测，灵敏度为 100 zM。

• DOI: 10.1039/c4an00215f
• 发表时间: 2014-06-07
• 期刊: The Analyst
• 作者: Kirimli CE;Shih WH;Shih WY
• 通讯作者: Shih WY

Piezoelectric Plate Sensor (PEPS) for Analysis of Specific KRAS Point Mutations at Low Copy Number in Urine Without DNA Isolation or Amplification.

压电板传感器 (PEPS)，用于分析尿液中低拷贝数的特定 KRAS 点突变，无需 DNA 分离或扩增。

• 发表时间: 2017
• 作者: Kirimli, Ceyhun E;Shih, Wei;Shih, Wan Y
• 通讯作者: Shih, Wan Y

Rapid, label-free genetic detection of enteropathogens in stool without genetic isolation or amplification.

对粪便中的肠道病原体进行快速、无标记的基因检测，无需基因分离或扩增。

• DOI: 10.1016/j.bios.2019.01.025
• 发表时间: 2019-04-01
• 期刊: Biosensors & bioelectronics
• 影响因子: 12.6
• 作者: Song Han;M. Soylu;Ceyhun E. Kirimli;Wei Wu;Bhaswati Sen;S. Joshi;Christopher L. Emery;Giang H. T. Au;Xiaomin Niu;Richard Hamilton;Kyle Krevolin;W. Shih;W. Shih
• 通讯作者: W. Shih