Multiplex detection and genotyping of pathogenic bacteria on paper-based biosensor with a novel universal primer mediated asymmetric PCR
Introduction
The microbiological safety of food is a serious concern of consumers and the food industry owing to the significant public health and global economic impact of foodborne diseases (Akhtar et al., 2014). In particular, headache, vomiting, bleeding, meningitis and even abortion can be caused by bacterial pathogens, particularly in developing countries (Torgerson et al., 2014). Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes are three of the most important pathogens that spread through food and water, afflicting people worldwide. Recently, E. coli is listed as the most serious foodborne infection because of its high risk (Elaine et al., 2011). Salmonella strains cause salmonellosis, a zoonotic disease of considerable importance (Feasey et al., 2012). L. monocytogenes can cause listeriosis and serious illness in humans and animals, particularly strains 4b and 1/2a (Cossart and Toledo-Arana, 2008, Lee et al., 2013). Therefore, a method is urgent needed that can quickly and easily detect multiplex pathogens simultaneously, thereby reducing cost.
Traditionally, the detection and enumeration of bacterial pathogens have been based largely on the use of selective culture and standard biochemical methods (Cadnum et al., 2014, Gracias and McKillip, 2004). Such methods suffer from a number of drawbacks. First, pathogenic bacteria that normally occur in low numbers tend to incur large errors in sampling and enumeration. Second, culture-based methods are time-consuming, invariably monospecific, and low-throughput. Third, many pathogenic organisms in the environment, although viable and capable of causing illness, are difficult to culture or even non-culturable (Kong et al., 2002).
Paper-based nucleic acid diagnostics (PBNAD) refers to a rapid detection strategy with wide application in clinical diagnosis, food quality control and environmental monitoring (Yang et al., 2013, Martinez et al., 2010, Parolo and Merkoci, 2013). PBNAD can be used quantitatively or semi-quantitatively to detect amplification products of nucleic acids or many antibody reactivities in 10–15 min. Compared to other technical methods, such as capillary electrophoresis (Frost et al., 2010), electrochemiluminescence detection (Jie et al., 2012) and enzyme-linked immunosorbent assays (Rissin et al., 2010, Lequin, 2005), this method is more simple, more efficient and easier to carry out with inexpensive, biodegradable and renewable materials. PBNAD is usually combined with PCR to achieve high detection efficiency. However, conventional symmetric PCR generates double-stranded amplicons, with a subsequent pre-hybridization for further analysis (Elsholz et al., 2006, Wei et al., 2014). Asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA (ssDNA) amplicons. However, this method requires extensive optimization of the proper primer ratios, the amount of starting material, and the number of amplification cycles to generate a reasonable amount of products for individual template–target combinations (Ang et al., 2012).
In this proof-of-concept study, we developed a paper-based multiplex detection system using an advanced form of multiplex asymmetric PCR. In this asymmetric PCR, a universal primer was introduced to simultaneously detect multiplex genes without the need to optimize the amplification parameters. By using this novel multiplex asymmetric PCR technique, single-stranded products resulting from the amplification of three genes can be detected simultaneously using one gold nanoparticle (AuNP) signal probe without the need for manual addition of probes or a pre-hybridization step. The whole detection procedure on the paper platform can be performed at ambient temperature. The detection limit was 1 pg/μL genomic DNA. This PBNAD method eliminates the need for preheated buffers or additional equipment and greatly simplifies the protocol for sequence-specific PCR amplicon analysis. The method not only can be applied to multi-detection but also can be used for bacterial genotyping widely.
Section snippets
Reagents and apparatus
Bovine serum albumin (BSA), formamide, sodium phosphate, sodium dodecyl sulfate (SDS), Tween-20, phosphate-buffered saline (PBS, pH 7.4, 0.01 M), and sodium chloride-sodium citrate (SSC) buffer (20× concentrate, pH=7) were purchased from Sangon Biotech (Shanghai). Streptavidin and Tris (2-carboxyethyl)-phosphine (TCEP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). HAuCl4·4H2O was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Sodium chloride (NaCl) and sodium
Development of multiplex asymmetric PCR
Single-stranded products have been shown to be very useful for DNA hybridization detection with high efficiency and do not need to be denatured before hybridization (Citartan et al., 2012). Asymmetric PCR aims to maximize the yield of ssDNA targets in a single reaction for hybridization and thereby increase the sensitivity of the PBNAD platform (Hao et al., 2011). However, conventional asymmetric PCR is usually inefficient and difficult to optimize because limiting the concentration of one
Conclusions
We have successfully developed a PBNAD system for the visual detection of pathogenic bacteria with a novel universal primer mediated multiplex asymmetric PCR. With the high efficiency of this amplification method, single or multiplex target genes can be simultaneously detected, and the detection limit was 1 pg/μL genomic DNA. This cost-effective, disposable and robust biosensor poses no health risk to the end user, and the generated signal can be unambiguously read by the naked eye. More
Acknowledgments
This research was supported by the National Basic Research Program of China (2010CB732602), the National Natural Science Foundation of China, China (21475048), the National Science Fund for Distinguished Young Scholars of Guangdong Province (2014A030306008) and the Program of the Pearl River Young Talents of Science and Technology in Guangzhou, China (2013J2200021).
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