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Резюме

To facilitate the rapid and precise detection of Acinetobacter baumannii, we present a protocol that employs Recombinase Polymerase Amplification (RPA) in conjunction with LbaCas12a endonuclease for identifying A. baumannii infections.

Аннотация

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.

Введение

In clinical microbiology, detecting Acinetobacter baumannii infections presents a significant challenge. This gram-negative bacterium can cause infections with severe clinical symptoms, even with high mortality rates, particularly among immunocompromised patients1. Traditional culture-based detection methods for pathogen infections are time-consuming and may lack sensitivity, which may delay antibiotic treatment and compromise patient outcomes. Rapid and accurate identification of A. baumannii is crucial for effective treatment and outbreak control. Molecular techniques employing nucleic acid amplification have been explored to meet this need. However, these approaches often need sophisticated thermal cycling equipment and may be limited by well-trained technicians and well-established laboratories. To overcome these challenges, research is increasingly focused on developing isothermal amplification methods2,3,4.

Recombinase Polymerase Amplification (RPA) is a method established by Piepenburg et al 5 and used to amplify DNA, similar to polymerase chain reaction (PCR) but without the need for temperature cycling. This method includes 50 mM Tris (pH 7.5), 100 mM potassium acetate, 14 mM magnesium acetate, 2 mM DTT, 5% PEG20000 (a high molecular weight polyethylene glycol), 200 µM dNTPs,3 mM ATP, 50 mM phosphocreatine, 100 µg/mL creatine kinase, 120 µg/mL UvsX, 30 µg/mL UvsY, 900 µg/mL Gp32, 30 µg/mL Bsu LF, 450 nM primers, and DNA templates. The amplification process begins with the recombinase protein UvsX binding to primers in the presence of 3 mM ATP and 5% PEG20000 forming a recombinase-primer complex. This complex then facilitates the combination of primers with the homologous sequences on the double-stranded DNA.

With the help of UvsY, the recombinase UvsX facilitates the exchange between the primer and the template strand, resulting in the displacement of one strand of the target DNA. Gp32 helps maintain the single-stranded DNA structure. Finally, the recombinase dissociates, and a DNA polymerase (Bsu LF) capable of displacing DNA strands binds to the 3' end of the primer, elongating it in the presence of deoxyribonucleoside triphosphates (dNTPs). This process is repeated cyclically, achieving exponential amplification. All amplification processes can be completed within 20-40 min and at relatively constant temperatures between 37 °C and 42 °C. This temperature range is approximately identical to physiological temperatures, which permits RPA to be conducted in a minimalist environment, making RPA a versatile and efficient tool for DNA detection and analysis.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR-Cas) system functions as an adaptive immune mechanism in bacteria and archaea, encompassing Class I and Class II systems. Class II includes proteins such as Cas12 (a, b, f), Cas13 (a, b), and Cas14, which identify and cleave target DNA or RNA guided by CRISPR RNA (crRNA)6,7,8 . LbaCas12a (or Cpf1) is a crRNA-guided DNA endonuclease. The crRNA serves as a guiding RNA within the CRISPR-Cas system, where it complexes with Cas proteins. It leverages its spacer region to pair with the target DNA, effectively steering the protein complex to the target sequences. The sequence 5'-UAAUUUCUACUAAGUGUAGAU-3' serves as the conserved crRNA repeat, a constant element in all LbaCas12a crRNAs. Following this repeat is a target-specific segment that differs based on the intended DNA target. This targeting sequence ranges from 18 to 24 nucleotides in length.

The PAM (Protospacer-Adjacent Motif) sequence (TTTV, where V can be A, C, or G) is located at the 5' end of the non-complementary strand of the DNA target. The Cas12a cuts target double-stranded DNA at the PAM sequence. Subsequently, the activated Cas proteins execute non-specific trans-cleavage of single-stranded DNA (ssDNA)9,10,11,12. Thus, ssDNA labeled with a fluorophore and quencher undergoes cleavage, resulting in the emission of a fluorescent signal following collateral cleavage8,13. The fluorescence intensity can be quantified using a fluorescence reader for precise nucleic acid detection14.Notably, Cas12a is extensively utilized for DNA analysis, with its binding and cleavage of target sequences contingent upon the recognition of the Protospacer-Adjacent Motif (PAM), while Cas13a operates independently of such a requirement.

CRISPR-Cas systems15,16,17 facilitate cleavage within a single reaction18,19,20,21. Combining the above two can create a robust tool known as A. baumannii-DETECTR (LbaCas12a-Enabled Detection of Targeted A. baumannii) for nucleic acid detection22,23,24.This protocol demonstrates the use of RPA in conjunction with the DNase activity of Cas12a to specifically target and cleave a crRNA-guided sequence within the 16s rDNA gene of A. baumannii, thereby enabling sensitive and precise detection of the pathogen.

The A. baumannii-DETECTR method presents several advantages over conventional detection methods25,26. Firstly, the isothermal characteristic of RPA streamlines operational procedures and reduces costs through its thermal cycler-independent amplification mechanism5. Secondly, the Cas12a endonuclease increases the assay's specificity by means of crRNA-mediated targeting and Watson-Crick base pairing7. Lastly, using the one-tube method of A. baumannii-DETECTR not only saves time but also significantly reduces the risk of amplicon contamination19.

The protocol outlines steps for the extraction of DNA extraction, the selection of target DNA sequences, the design of primers and crRNAs, the construction of positive recombinant plasmids, the establishment of setting up the Cas12a-RPA assay, the optimization of optimizing RPA amplification, and the visualization of visualizing results with using fluorescence detection instruments tools such as a Real-time PCR machine, complete with sensitivity and specificity evaluations.

The A. baumannii-DETECTR method holds promise in enhancing patient outcomes by facilitating timely and effective treatment, robust infection control, and the containment of antibiotic resistance spread. This protocol can serve as a comprehensive guideline for the implementation of Cas12a-RPA technology, enhancing its utility in various healthcare settings. However, it is crucial to note that every step should be optimized based on specific lab conditions and the variety of samples to ensure consistent and reliable results.

протокол

1. Construction of the A. baumannii -DETECTR

NOTE: The construction of A. baumannii-DETECTR is a four-step process involving the design of primer and crRNA, the construction of positive recombinant plasmid construction, the preparation of reaction solutions, isothermal DNA amplification by RPA, and the visualization of the RPA-CRISPR/Cas12a assay. The schematic of the DETECTR assay is illustrated in Figure 1.

  1. Design of primer and crRNA (see Supplemental File 1)
    1. Design 12 pairs of primers using design toolson the basis of Primer design principles. Obtain four forward primers (F0-F3) and three reverse primers (R1-R3) to compose twelve pairs of primers (F0R1, F0R2, F0R3, F1R1, F1R2, F1R3, F2R1, F2R2, F2R3, F3R1, F3R2, F3R3).
      1. Ensure that the primer's 5' end contains C or T bases within the first 3-5 nucleotides instead of a continuous G stretch. Additionally, make sure the last three nucleotides at the 3' end are ideally G and C bases to enhance amplification.
      2. Design primers without self-complementary sequences to avoid hairpin formation and with no more than four complementary or homologous bases between them. Steer clear of 3' end complementarity to avoid primer dimerization.
    2. Evaluate each primer pair for specificity using the Primer-BLAST tool of National Center for Biotechnology Information (NCBI) to assess the efficacy and specificity of the primers.
      NOTE: Primer-BLAST can utilize the NCBI database to find sequences that match the designed primers, helping users assess the efficacy and specificity of the primers.
    3. Following the primer screening, design the CRISPR RNA (crRNA) or guide RNA (gRNA) using the LbaCas12a crRNA scaffold sequence (5'-UAAUUUCUACUAAGUGUAGAU-3') as the fixed sequence. Incorporate a target-specific segment (5'-GUUAAUACCUAGAGAUAGUG-3'), ensuring that the PAM motif is located at the 5' end of the non-complementary strand.
      NOTE: The sequences of all oligonucleotides and crRNA are presented in Table 1.
  2. Positive recombinant plasmid construction
    1. Amplify the 16s rDNA segment using the primer set A. baumannii 16s rDNA-F (forward primer) and A. baumannii 16s rDNA-R (reverse primer).
      1. Add 1 µL of 10 µM F and 10 µM R, 10 µL of 5x FastpFu Buffer, 1 µL of FastpFu Polymerase, 4 µL of 2.5 mM dNTPs, 1 µL of 10 ng/µL A. baumannii genomic DNA, and 32 µL of Nuclease-free Water to the 50 µL PCR system.
      2. Set the PCR cycling conditions as follows: initial denaturation at 95 °C for 5 min, followed by 33 cycles of denaturation at 95 °C for 30 s, annealing at 55°C for 30 s, and extension at 72 °C for 1 min and 20 s. Finally, perform 10 min at 72°C for the final extension.
    2. To purify the positive PCR product, use a DNA Purification Kit according to the manufacturer's instructions. Thereafter, clone the purified DNA into the pUC57 vector, which has been digested with BamHI and SalI enzymes.
      NOTE: This process will result in the creation of a positive recombinant plasmid containing a conserved region.
    3. Dilute the positive recombinant plasmid serially in tenfold increments with sterilized ddH2O to achieve a concentration of one copy/µL to 107 copies/µL. Store the diluted plasmid at -20 °C for subsequent experiments.
  3. Solution preparation
    1. Prepare Solution A as follows: for each reaction, add 29.5 µL of Primer Free Rehydration buffer, 2.4 µL of 10 µM A. baumannii-F and 10 µM A. baumannii-R, and 12.2µL of ddH2O to achieve a final volume of 46.5 µL. Vortex and spin briefly. Next, add the reaction mixture to the enzyme reaction tubes from the RPA Kit. Pipette to mix.
      NOTE: The primers of A. baumannii-F and R are used in the assay that targets the A. baumannii gene. Please refer to Table 1 for the sequence.
    2. Prepare Solution B as follows: for each reaction, add 0.24 µL of 10 µM ssDNA reporter, 2 µL of 10x Cas12a Reaction Buffer, 1 µL of 1 µM crRNA, 1 µL of 1 µM Cas12a, and 10.76µL of ddH2O to achieve a final volume of 15 µL.
      NOTE: The probes (ssDNA reporter) were purchased and consisted of a single-stranded DNA reporter that was tagged with a 5-Carboxyfluorescein (FAM) fluorophore at the 5' end and a Black Hole Quencher 1 (BHQ1) quencher at the 3' end. In the case of numerous samples needing detection, prepare a substantial amount of solution B, and then divide the samples into aliquots later.
  4. Isothermal DNA amplification (RPA)
    1. Add 1 µL of the positive recombinant plasmid of 107 copies/µL to solution A with different primer pairs; mix thoroughly. Then, add 2.5 µL of 280 mM Magnesium Acetate (MgOAc) to the solution and mix well.
      NOTE: RPA reactions start as soon as MgOAc is added.
    2. Incubate at 39 °C for 20 min. After 20 min, purify the products amplified with different primers using RPA with a purification kit, and then run the clean amplicons on a 1% agarose gel at 120 V for 10 min to select the brightest and broadest band as the best primer pair.
      NOTE: For low template copy number, after 4 min, remove the EP tube, vortex, and spin briefly; then replace it in the heating device for another 16 min. After amplification, tubes should be opened with extreme caution to avoid contaminating work surfaces with amplicons. This precaution will reduce the risk of inducing false positive results in subsequent experimental procedures.
  5. Optimization of RPA amplification system
    NOTE: The optimal primer pair F3R3 selected in the previous step was set to different final concentrations for RPA amplification reaction to screen out the optimal primer concentration and time. NC is a blank control for the corresponding primer concentration.
    1. Maintain the concentrations of the other components constant and the total volume of46.5 µL in Solution A. Adjust the volume of RNase-free ddH2O accordingly to achieve the final primer concentrations of 10 µM F3 and 10 µM R3 at 0.40 µM (2.0 µL), 0.44 µM (2.2 µL), 0.48 µM (2.4 µL), and 0.52 µM (2.6 µL), respectively.
    2. Incubate at 39 °C for different times:10 min, 20 min, 30 min.
    3. Clean the RPA products using a DNA Purification Kit and perform electrophoresis on a 1% agarose gel at 120 V for 10 min to identify the optimal primer concentration and incubation times by analyzing the characteristics of the bands.
  6. Visualization of the RPA-CRISPR/Cas12a assay
    1. Add 5 µL of the RPA product into solution B and mix thoroughly until homogeneous.
    2. Place the samples in the fluorescence quantitative PCR instrument, set the FAM channel, and read the values every minute for 60 min at 37 °C, 60x for a total of 60 min.
      NOTE: Green fluorescence signal can last several days. To make sure the system works effectively, it is better to perform a DNA and RNase free test.

2. Specificity evaluation of the RPA-CRISPR/Cas12a-based detection platform

NOTE: To test the specificity of A. baumannii-DETECTR, the nucleic acids of A. baumannii, Streptococcus pneumoniae, Staphylococcus aureus, Rickettsia mooseri, Enterobacter, Escherichia coli, Pseudomonas aeruginosa, Klebsiella, Chlamydia psittaci, Legionella, Cockerella beckeri, Serratia, and human samples were subjected to the DETECTR test.

  1. Use DNA templates obtained from various species by using the referenced DNA Kit or isolating them by boiling the bacteria in the water. Ensure that all templates have equal concentrations. Use water as a negative control.
  2. Add 10 ng or 1 µL of DNA templates to Solution A containing F3R3 primers. Add 2.5 µL of MgOAc, mix thoroughly, and incubate the mixture at 39 °C for 20 min.
  3. After 20 min, add 5 µL of the product from the previous step to solution B and mix.
  4. Place in the fluorescence quantitative PCR instrument, set the channel to FAM, and read the value once every minute at 37 °C, for a total of 60 x for 60 min.

3. Sensitivity evaluation of the RPA-CRISPR/Cas12a-based detection platform

  1. Use a positive recombinant plasmid, which has been prepared to a concentration of 1-107 copies/µL, as the template for the reaction. Use water for the negative control.
  2. Perform the method by following steps 2.2-2.4.
    NOTE: The primer set RPA-F3 and RPA-R3 is designed to amplify a shorter fragment of 174 base pairs from the positive recombinant plasmid.

4. Preparations while the sample is under UV light and LFS detection

  1. Take out the required number of test strips and label them appropriately.
  2. Transfer 10 µL of the amplified products from steps 2 and 3 into separate PCR tubes, respectively. Then, add 40 µL of ddH2O to each tube.
    NOTE: If using a self-prepared reaction system, it is recommended to explore the optimal dilution ratio.
  3. Perform the Test.
    1. Subject the PCR tube containing the amplified products to a transilluminator to assess the presence of fluorescence.
    2. Insert the test strips into the PCR tubes with the sample pad end facing downwards.
    3. Let it stand at room temperature for 5-10 min to read the result. After this time, ensure the liquid level does not exceed the maximum line before proceeding further.
    4. Interpret the results as negative if the T line does not show color. Interpret the results as positive if the T line is visible to the naked eye, indicating that the nucleic acid probe has been cleaved by the Cas enzyme, and the Cas enzyme has been activated. Consider the result to be invalid if neither the C line nor the T line shows color.

Результаты

In this study, we introduce a novel, portable diagnostic platform named A. baumannii-DETECTR, which integrates the isothermal amplification efficiency of RPA and CRISPR-Cas12a system for rapid and reliable field identification of A. baumannii. The schematic of the DETECTR assay is illustrated in Figure 1.

Twelve pairs of primers were designed using design toolson the basis of prime...

Обсуждение

Traditional diagnostic methods for A. baumannii infections have various restrictions that make them less accessible and feasible for point-of-care testing27. For example, PCR has good sensitivity and specificity but necessitates specialized thermal cycling equipment and complex workflows that must be operated by professionals. The novel method presented here, which couples RPA and CRISPR-LbaCas12a genome editing with recombineering, known as the A. baumannii-DETECTR assay, allows...

Раскрытие информации

The authors declare that they have no competing interests.

Благодарности

This work was supported by Science and Technology Development Plan Project of Jilin Province, China (20240305027YY) and Jilin Provincial Department of Finance (JLSWSRCZX2023-55, JLSWSRCZX2021-041).

Материалы

NameCompanyCatalog NumberComments
 -20 °C FreezerHaierHYCD-290China
Agarose BasicBioFroxx1110GR100China
All oligonucleotides and crRNA were synthesized by companywww.comatebio.com
Cas12a cutting substrate - ssDNA - fluorescent typeEZassay Biotech. Co. Ltd.DNA-FAM-BHQChina
Cas12a cutting substrate - ssDNA - test paper typeEZassay Biotech. Co. Ltd.DNA-FAM-BIOChina
Electrophoresis apparatusBIO-RADPOWER PAC1000USA
Fluorescence quantitative PCR instrumentBIO-RADCFX ConnectUSA
Gel Imaging SystemBIO-RADGel Doc 2000USA
https://ezassay.com/rna
https://www.ezassay.com/primer  
https://www.ncbi.nlm.nih.gov/tools/primer-blast
Lateral flow paper strip (Biotin/FAM)EZassay Biotech. Co. Ltd.HD-FMBOChina
LbaCas12a (Cpf1) enhanced proteinEZassay Biotech. Co. Ltd.CAS-12E-001China
LED TransilluminatorLABGICBL-20China
Magnesium acetate, MgOAcTwistDxTABAS03KITUK
MicrocentrifugeallshengMini-6kChina
PCR strip tubesPCR strip tubesPST-0208-FT-CChina
TGrade Dry Bath IncubatorTiangen biochemical technology OSE-DB-01China
Tianamp Bacteria DNA KitTiangen biochemical technology DP302-02China
TIANamp Bacteria DNA Kit TIANGEN BIOTECH (BEIJING) CO.; LTD.DP302 China 
TransStart FastPfu DNA Polymerase TransGen Biotech. Co. Ltd.AP221 China 
TwistAmp Basic KitTwistDXTABAS03KITUK
Universal DNA Purification KitTiangen biochemical technology DP214-03China

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