Single-tube analysis for ultra-fast and visual detection of Salmonella.

Herein, we developed an ultra-fast and visual single-tube nucleic acid detection approach, which combined the advantages of self-settling characteristics of chitosan-functionalized diatomaceous earth (CDE) and accelerated PCR (AC-PCR). DNA was rapidly extracted by CDE within 3 min for the next nucleic acid amplification based on the nucleic acid attached on the chitosan in pH = 5.0. Under the action of gravity, the DNA-enriched CDE self-sediments to the bottom of the tube could be directly used for AC-PCR to achieve single-tube extraction and amplification. Our method detected Salmonella culture fluids with a detection limit of 1 CFU/mL, which was 100-fold more sensitive than conventional method that have not undergone nucleic acid enrichment. Furthermore, it also displayed high specificity and sensitivity for a variety of spiked samples. The entire process could be completed within 17 min in a single tube, and in particular, the result was visualized by the naked eyes. Overall, it is an all-in-one detection strategy without the requirement of redundant procedure, which greatly improved the detection efficiency, and saved the time and the cost. With these advantages, the approach will supply a promising tool in the field of point-of-care testing for Salmonella and other foodborne pathogens.

A simple methodology for RNA isolation from bacteria by integration of formamide extraction and chitosan-modified silica purification.

RNA isolation from bacteria is technically difficult due to the RNA characteristic of labile and vulnerable degradation. Many reagents were explored for cellular lysis and complete inhibition of RNase. However, the available methods for RNA isolation are either of low efficiency or time-consuming. Here, we developed a rapid and accessible protocol for RNA isolation that combined a simplified cell lysis and RNA release by formamide-based solution and RNA purification by chitosan-modified silica membrane for the first time. With this method, we obtained about ~ 28 µg of total RNA from 108 Escherichia coli cells. The entire procedure can be done within 15 min without redundant pipetting steps. The purity of extracted RNA was comparable to that of commercial kits, but the cost was much lower. Furthermore, the yielded RNA was successfully used in downstream enzymatic reactions, such as reverse transcription and quantitative real-time PCR. This new method would be of benefit for an extensive range of gene expression analyses in bacterial organisms.

Amino-modified silica membrane capable of DNA extraction and enrichment for facilitated isothermal amplification detection of Mycoplasma pneumoniae.

Herein, we developed a facile integrated Mycoplasma pneumoniae diagnosis platform by combining amino-modified silica membrane (AMSM)-based nucleic acids fast extraction and enrichment with colorimetric isothermal amplification detection. AMSM demonstrates a strong ability to capture and enrich nucleic acids in complicated biological matrices, and the purified AMSM/nucleic acids composite could be directly used to perform isothermal amplification including denaturation bubble-mediated strand exchange amplification (SEA) and loop-mediated isothermal amplification (LAMP) reactions. Through comparing clinical specimens, excellent performance of AMSM-based SEA assay with 93.33% sensitivity and 100% specificity relative to real-time PCR was observed, and for AMSM-based LAMP was 96.67% and 100%, respectively. The diagnostic procedure could be completed within 55 min, and the colorimetric-based visual result further alleviates the use of sophisticated equipment. The proposed approach possesses great potential as a simple and time-saving alternative for point-of-care testing (POCT) of M. pneumoniae in resource-limited regions.

An ultra-fast, one-step RNA amplification method for the detection of Salmonella in seafood.

Salmonella is one of the most common pathogens associated with food-borne illness resulting from seafood consumption. Herein, an accelerated strand exchange amplification (ASEA) requiring only a pair of primers and one polymerase was first reported for ultra-fast, one-step RNA amplification detection of Salmonella in seafood. The ASEA method could detect Salmonella typhimurium DNA in dilutions as low as 10 copies per reaction and displayed good specificity for Salmonella under the interference of a variety of food-borne pathogens. In particular, ASEA could detect RNA in one step without additional reverse transcription. The detection limit for Salmonella in artificially contaminated oyster was 1 CFU mL-1 following 12 h of enrichment. Moreover, excellent performance of this assay was observed with 99.02% consistency relative to real-time PCR through actual sample detection. Combined with the rapid nucleic acid extraction method, the entire detection process could be completed within 20 min. Therefore, this assay opens up new prospects for the detection of food-borne pathogens in seafood with its rapidity, which would be very beneficial for food safety supervision and pathogen detection of high-throughput samples.