Point-of-care detection of 16S rRNA of Staphylococcus aureus based on multiple biotin-labeled DNA probes
Introduction
Many pathogenic bacteria found in the environment, food, water, and soil can often spread to humans and cause food-borne diseases. A total of 31 major pathogens have been identified to cause food-borne diseases in millions of people each year through contaminated-food consumption [1]. Among them, Staphylococcus aureus is a widely distributed pathogenic bacterium in nature, which is considered as one of the most serious threats to public health [2,3], affecting people all over the world, especially those living in poor areas [4]. Therefore, to control infectious diseases caused by S. aureus, there is an urgent need to develop a simple and cost-effective point-of-care test (POCT) for S. aureus detection.
Owing to their high sensitivity, ideal specificity, and good reliability, bacterial culture and colony counting method are used as the gold standard for the identification and quantitative analysis of S. aureus; however, they are not suitable for POCT because of the requirement of 3–5 days to yield results. In contrast, immunoassays such as conventional ELISA, as a popular and widely used technique for detecting pathogenic bacteria, require shorter detection time of several hours, but present sensitivity of 106–107 CFU/mL [5]. Specific nucleic acid sequence detection plays an important role in pathogen detection, because the nucleic acid sequence is individualized and associated with pathogenic specificity. The most widely used PCR is a valuable tool for the detection and analysis of pathogenic nucleic acids, PCR could be more sensitive than immunoassays because of its ability to amplify trace amounts of DNA to detectable levels. However, the high cost and confined resources limit the use of PCR technology in developing and underdeveloped countries. Several low cost nucleic acid detection methods based on isothermal amplification (e.g., loop-mediated amplification, nucleic acid sequence based amplification), as an alternative approach, have the ability to provide a platform for POCT in a low resource environment because of their high sensitivity and specificity [6]. These methods are performed under the condition of constant temperature, which simplifies the reaction process and is conducive to POCT.
While target nucleic acid amplification assays are sensitive, they require enzymatic amplification and are therefore easily affected by inhibitors and prone to false-positives. In contrast, signal amplification assays have certain specific advantages; for example, they typically do not require target amplification step, thus simplifying the detection workflow, and are more resistant to inhibitors and less prone to false-positives in the absence of enzymes [7]. In the past few decades, various signal amplification methods have been developed for direct detection of specific nucleic acids, such as fluorescence assay, electrochemical analysis, and biosensor. Furthermore, considerable efforts have been made to develop sensitive biosensors for nucleic acid detection based on signal amplification [8]. Direct multiple labeling of target nucleic acid is an effective signal amplification method. In a previous study, 64 DNA strands were directly hybridized with 16S rRNA to form 32 deoxy ribozyme catalytic nuclei to generate fluorescence signals, and achieved detection of about 3 × 104 bacterial cells [9]. Furthermore, to image mRNA expression patterns in fixed biological specimens, fluorescence amplification polymers formed by self-assembly of hybrid chain reaction were added to the target mRNA of interest through complementary DNA probes [[10], [11], [12]].
In this study, based on 16S rRNA appended with multiple biotin-labeled DNA probes and lateral flow nucleic acid biosensor (LFNAB), a visual POCT method for S. aureus detection was developed. It must be noted that 16S rRNA has been widely used in bacterial classification and identification, mainly because of its bacterial specificity, with each bacterium containing 5,000–75,000 ribosomes. In the present study, the 16S rRNA extracted from S. aureus was hybridized with two specific capture probes (CPs) and multiple bridge probes (BPs), which were connected with prefabricated biotinylated report probes (RPs), and the signal was output by LFNAB. Each target 16S rRNA yielded multiple biotinylated DNA probes in this process, thus functionalizing the signal structure, and numerous streptavidin (SA)-labeled gold nanoparticles (AuNPs) were introduced in the LFNAB for visual detection. The images of the lateral flow detection results, obtained by using a smartphone, were transmitted to a computer via Wi-Fi or Bluetooth connection for quantitative processing by ImageJ software (Fig. 1).
Section snippets
Reagents
Bacterial RNA Extraction Kit was supplied by BioFlux (Japan), lysozyme was purchased from Biosharp (Guangzhou, China), and biotin-11-dUTP was obtained from Thermo Scientific (USA). DNA polymerase, dNTPs, and DNA ladder were purchased from TaKaRa Bio Inc. (Dalian, China). Colloidal AuNPs was bought from Hualan Chemical Co., Ltd. (Shanghai, China) and SA was obtained from Sangon Biotech. (Shanghai, China). Bovine serum albumin (BSA)-biotin and anti-Fam antibody were purchased from Ruiqi Biotech
Optimization of the length of biotinylated DNA RPs
To enhance their identification capacity, the length of the biotinylated DNA RPs was optimized in this study. Biotinylated DNA RPs with different molecular weights (101, 333, 526, 732, 1,035, 1,515, and 2,187 bp) were synthesized by PCR (Fig. 2A). As shown in Fig. 2B, the signal value increased with the increasing segment length of the biotinylated DNA RP, reaching the maximum at almost 700 bp and then decreasing slowly with the increasing segment length. Hence, 700 bp was chosen as the optimal
Conclusion
In this study, based on 16S rRNA conjugation with the prepared multiple biotin-labeled DNA probes and LFNAB, a simple and low cost POCT of S. aureus was developed. The results showed that this method was suitable for the detection of pathogens in real samples. The visual test images captured using a smartphone were transferred to a computer based on Bluetooth or Wi-Fi for further quantitatively processing by ImageJ software. The LOD of this method was 103 CFU/mL, and the sensitivity was as low
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
This work was financially supported by the National Key Research and Development Program of China (No. 2016YFD0501001).
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