High-Throughput Flow-Through Direct Immunoassays for Targeted Bacteria Detection

Abstract

Regulatory authorities require analytical methods for bacteria detection to analyze large sample volumes (typically 100 mL). Currently only the Membrane Filtration and the Most Probable Number assays analyze such large volumes, while other assays for bacteria detection (ELISA, lateral flow assays, etc.) typically analyze volumes 1000 times smaller. This study describes flow-through direct immunoassays (FTDI), a new methodology for the targeted detection of bacteria in liquid samples of theoretically any volume. Flow-through direct immunoassays are performed in fluid-permeable microwells (e.g., wells of a filter well plate) that have a membrane on their bottom where the bacteria are trapped before their detection using a direct immunoassay. Two versions of FTDI assays for the detection of E. coli in 10 mL of sample were developed. A rapid FTDI assay that can be completed in less than 2.5 h can detect E. coli bacteria in levels down to 17 CFU/mL, and an ultrasensitive FTDI assay that employs an additional bacteria culturing step to boost the sensitivity can detect E. coli bacteria in levels lower than 1 CFU/mL in less than 5.5 h. All the steps of the assays, including the immunoassay steps, the culturing step, and the analytical signal measurement step are performed inside the well plate to decrease the chance of contamination and ensure a safe, easy process for the user. The assays were assessed and validated in tap water, river water, and apple juice samples, and the results suggests that the assays are robust, precise, and accurate. When the assays are performed in 96-well filter plates, a filter well plate vacuum manifold and a multichannel peristaltic pump are also used, so multiple samples can be analyzed in parallel to allow highthroughput analysis of samples.

Phage-Displayed Nanobody as a Sensitive Nanoprobe to Enhance Chemiluminescent Immunoassay for Cronobacter sakazakii Detection in Dairy Products

“…Thus, billions of tests are conducted every year to detect possible bacterial contamination. 3 There is now a consensus that bacterial culture technology is the gold standard for foodborne pathogen detection. The methodology for the detection of pathogens is, however, tedious and/or highly professional that cannot meet the requirement of rapid screening for food safety prevention and control.…”

Mycobacteriophage Derived Lipoarabinomannan Binding Protein for Recognizing Non-Tuberculosis Mycobacteria

“…Moreover, the spectrum analysis of SERS requires recondite mathematical processing of the subtle differences between the fingerprints of different bacterial species. 16 Some biological molecules such as antibody and aptamer have been frequently applied as recognition agents for developing various bacteria detection methods, including lateral flow assay, 17,18 ELISA, 19,20 and electrochemiluminescent biosensor. 21,22 They show the merits such as high throughput, satisfactory portability, and improved efficiency.…”

“…Some biological molecules such as antibody and aptamer have been frequently applied as recognition agents for developing various bacteria detection methods, including lateral flow assay, , ELISA, , and electrochemiluminescent biosensor. , They show the merits such as high throughput, satisfactory portability, and improved efficiency. Unfortunately the lack of high-performance antibodies and aptamers for NTM limits their practicality.…”

Isothermal RNA Amplification for the Detection of Viable Pathogenic Bacteria to Estimate the Salmonella Virulence for Causing Enteritis

“…12 Immunoassays use antibodies to directly recognize bacteria, followed with enzymatic amplification, thus allowing a rapid and specific detection of pathogenic bacteria. 13 However, they cannot discriminate viable bacteria from dead ones. The PCR is the most widely used technique for nucleic acid tests and can detect, in principle, any bacteria by designing DNA primers for targeting bacterial genes.…”

“…Current tools for detecting pathogenic bacteria mainly include culture-based methods, immunoassays, , and nucleic acid tests. − Culture-based methods can be reliable and detect viable bacteria but need high labor-intensity and long sample-to-result turnaround time . Immunoassays use antibodies to directly recognize bacteria, followed with enzymatic amplification, thus allowing a rapid and specific detection of pathogenic bacteria . However, they cannot discriminate viable bacteria from dead ones.…”