Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

My research focuses on respiratory diseases and infection. I will address current technologies advancing in this field and the whole project field's research gaps. Key technologies we are talking about here include next-generation sequencing, CRISPR, single-cell RNA sequencing, and organ analysis models. They have a hard figure regard to diagnostics on standard heart diseases work and come up with better treatment. My protocol addresses the need for rapid and specific detection of Acinetobacter baumannii, overcoming limitation in current diagnostic matters like time-consuming and culture-based techniques.

[Narrator] To begin, design 12 pairs of primers using primer design tools based on standard design principles. Obtain four forward primers and three reverse primers to compose 12 primer pairs. Evaluate each primer pair for specificity and efficacity using the Primer-BLAST tool from the National Center for Biotechnology Information. Then design the CRISPR RNA or guide RNA or gRNA using the LbaCas12a CRISPR RNA scaffold sequence as the fixed sequence. Incorporate a target-specific segment, ensuring that the protospacer adjacent motif or PAM is located at the five-prime end of the non-complementary strand. For the construction of the positive recombinant plasmid, first amplify the Acinetobacter baumannii 16S rDNA segment using the forward and reverse primer sets. Prepare the 50-microliter PCR reaction mix as given on screen. Set the PCR cycling conditions on the PCR machine. After purifying the positive PCR product, clone it into a PUC57 vector that has been digested with BamHI and SalI enzymes. Dilute the positive recombinant plasmid serially in tenfold increments with sterilized double-distilled water. Store the diluted plasmid at minus 20 degrees Celsius for further experimentation. Next, mix together primer-free rehydration buffer, forward and reverse primers of A. baumannii, and double-distilled water to a final volume of 465 microliters. Vortex and briefly spin the mixture before transferring it to enzyme reaction tubes from the RPA kit. Mix thoroughly and add 25 microliters of 280 millimolar magnesium acetate. Next, pipette one microliter of the positive recombinant plasmid, containing 10⁷ copies per microliter to solution A with different primer pairs. Incubate the solution at 39 degrees Celsius for 20 minutes. Then purify the amplified products using an RPA purification kit. Run the purified product on a 1% agarose gel at 120 volts for 10 minutes to identify the primer pair with the brightest and broadest band. For optimizing the RPA amplification system, maintain the concentrations of all other reaction components constant while adjusting the final volume of solution A to 465 microliters. Then add appropriate volumes of RNA-free double-distilled water to obtain final primer concentrations as given and incubate. After purifying and electrophoresing the RPA product, identify the optimal primer concentration and incubation time by analyzing band intensity and distribution. To prepare solution B, mix together single-stranded DNA reporter, CAST12a reaction buffer, CRISPR RNA CAST12a, and double-distilled water to a final volume of 15 microliters. Add five microliters of the recombinase polymerase amplification or RPA product into solution B. Mix thoroughly until homogeneous. Place the samples into a fluorescence quantitative PCR instrument. For specificity testing, obtain DNA templates from various species using a reference DNA extraction kit or by boiling bacteria in water. Use water as a negative control. Transfer 10 nanograms, or one microliter, of the DNA templates into solution A containing forward and reverse primers. Then pipette 25 microliters of 280 millimolar magnesium acetate. Mix thoroughly and incubate at 39 degrees Celsius for 20 minutes. After incubation, add five microliters of the reaction product to solution B and mix. Place the samples in a fluorescence quantitative PCR instrument. Set the channel to FAM and read the fluorescent signal every minute at 39 degrees Celsius for a total of 60 minutes. For sensitivity evaluation of the RPA-CRISPR CAST12a-based detection platform, use a positive recombinant plasmid diluted to concentrations ranging from one to 10⁷ copies per microliter as the template for the reaction. Use water as a negative control. Transfer plasmid to solution A, add magnesium acetate, and incubate as demonstrated earlier. After incubation and transfer to solution B, read the fluorescent signal. The primer pair F3-R3 was identified as the most effective for recombinase polymerase amplification, displaying the brightest and densest bands on agarose gel electrophoresis. The optimal primer concentration for amplification was determined to be 0.44 micromolar, and the optimal reaction time was 20 minutes. The fluorescence intensity of CRISPR RNA1 increased over time, while CRISPR RNA2 did not, leading to the selection of CRISPR RNA1 for A. baumannii detection. The specificity analysis confirmed that the A. baumannii detector system exclusively detected A. baumannii DNA, with no fluorescence observed for other bacterial DNA samples. The system could detect as low as one copy per microliter of DNA, making it highly sensitive. The lateral flow strip assay confirmed the high specificity of the detection method, showing no cross-reactivity with other bacterial species. The A. baumannii assay successfully detected DNA across dilutions from 10⁷ copies per microliter down to a single copy per microliter, confirming its robustness under UV light.