Abstract
The technique of loop-mediated isothermal amplification (LAMP) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with potential for greater specificity than two primer methods such as polymerase chain reaction. In this study, a set of LAMP primers was designed targeting the 16S–23S ribosomal RNA intergenic spacer region of Staphylococcus aureus, and tested for sensitivity and specificity in real-time LAMP reactions. Under optimized conditions, the detection limit of our developed assay was 10 fg DNA template of S. aureus per reaction, with no detectable false-positive response, which was 103-fold more sensitive than previously reported LAMP assay. The assay was shown to detect two strains of S. aureus, and did not detect 24 non-S. aureus strains, providing improved specificity over previously reported LAMP assays.
La técnica de amplificación isotérmica mediada por bucles (LAMP) utiliza 4 (o 6) cebadores centrándose en 6 (o 8) regiones en un segmento muy pequeño de un genoma para su amplificación, el cual tiene potencial para conseguir una especificidad superior a 2 métodos cebadores como la PCR. En este estudio, un conjunto de cebadores LAMP fueron diseñados para centrarse en la región intergénica separadora 16S-23S rRNA de Staphylococcus aureus como objetivo, además fueron examinados para estudiar su sensibilidad y especificidad en las reacciones de LAMP a tiempo real. Bajo condiciones óptimas el límite de detección del ensayo que hemos desarrollado fue de 10 fg del molde de ADN de S. aureus por reacción, sin detectar ninguna respuesta falsa-positiva, lo cual resultó 103 veces más sensible que en los resultados del anterior ensayo LAMP. Este ensayo mostró la detección de 2 cepas de S. aureus, además de no detectar 24 cepas que no eran S. aureus, así proporcionó una especificidad mejorada frente a los resultados de los anteriores ensayos LAMP.
Introduction
Loop-mediated isothermal amplification (LAMP), developed and reported by Notomi et al. (Citation2000), can specifically, sensitively and rapidly amplify nucleic acids by utilizing a DNA polymerase enzyme with high strand displacement activity and two pairs of primers recognizing six independent sequences of a target gene under isothermal conditions. Moreover, Nagamine, Hase and Notomi have advanced the method by putting forward loop primers that accelerate the LAMP reaction in Citation2002. Based on the cost effectiveness and sensitivity of LAMP, there has been significant interest in its application toward basic research in medicine and environmental testing, as well as point-of-care testing and diagnosis of infectious diseases in clinical settings (Md. Fakruddin, Citation2011). LAMP has also been broadly applied in pathogen detection, and has successfully detected Escherichia coli O157:H7 (Maruyama, Kenzaka, Yamaguchi, Tani, & Nasu, Citation2003), Actinobacillus actinomycetemcomitans (Osawa et al., Citation2007), Mycobacterium tuberculosis (Iwamoto, Sonobe, & Hayashi, Citation2003), Streptococcus pneumonia (Seki et al., Citation2005), and Listeria monocytogenes (Tang et al., Citation2011). More recently, LAMP has been used to successfully detect S. aureus (Lim, Ju Teh, & Thong, Citation2013).
S. aureus is pathogenic bacterial strain that can be found in a number of different food varieties, including mixed foods (pasta dishes, salads), meat and meat products, egg and egg products, vegetables, baked goods, and cheeses (Zeleny et al., Citation2015). It produces a variety of virulence factors (Hwang et al., Citation2007), leads to serious infections in humans, including endocarditis, deep-seated abscesses and osteomyelitis (Del Rio, Cervera, Moreno, Moreillon, & Miró, Citation2009), and accounts for 2.57% of illnesses in 9,388,075 foodborne illnesses per year in US (Scallan et al., Citation2011). Recently, many countries modified the restriction standards of S. aureus in food, e.g., restriction standard for Chinese frozen pastry products (GB19295-2011) has defined the maximum number of S. aureus for 102–103 CFU/mL, based on the recommendation of the Codex Alimentarius Commission and International Commission on Microbiological Specifications for Food (Chen et al., Citation2014). Therefore, the methods that can detect extremely low levels of S. aureus are necessary in the area of food safety to prevent consumers from exposure to unqualified products.
We describe here our efforts to improve upon existing methods for the sensitive and selective detection of foodborne S. aureus. In this study, a set of LAMP primers was designed targeting the 16S–23S ribosomal RNA (rRNA) intergenic spacer region of S. aureus, and tested over a range of reaction conditions and incubation temperatures for increased sensitivity and specificity in real-time LAMP reactions.
Materials and methods
Primer design
Targeting the 16S–23S rRNA intergenic spacer region (GenBank Locus: U39769.1) of S. aureus, a set of LAMP primers were designed and selected with PrimerExplorer 4 and Oligo 7 according to the reported methodology (Li, Zhang, Wang, Kuang, & Xu, Citation2009), and are listed in .
Table 1. Primers for detecting 16S–23S rRNA intergenic spacer region of Staphylococcus aureus with LAMP.
Tabla 1. Cebadores para la detección de la region intergénica separadora 16S-23S rRNA de Staphylococcus aureus con LAMP.
Selection of reaction temperature for LAMP
Real-time LAMP was performed in a 10 μL reaction mixture containing 0.8 mM each of forward inner primer (FIP) and backward inner primer (BIP), 0.2 mM each of forward outer primer (F3) and backward outer primer (B3), 0.4 mM backward loop primer (Luria–Bertani (LB)), 1.0 mM deoxy-ribonucleoside triphosphate, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 6 mM MgSO4, 0.1% Triton X-100, 7.5% DMSO (Frackman, Kobs, Simpson, & Storts, Citation1998), 1× EvaGreen, 1× Rox, 1 pg S. aureus DNA template and 3.2 U Bst 2.0 WarmStart DNA polymerase (New England Biolabs, Beverly, MA., USA.) (Misawa et al., Citation2007; Tang et al., Citation2011). The reaction mixture was heated at 61°C, 59°C, 57°C, 55°C, or 53°C for 60 min (30 sec per cycle), and a melt curve was obtained using a StepOneTM System (Applied Biosystems, Foster City, CA, USA).
Sensitivity determination of real-time LAMP assay
The real-time LAMP reaction mixtures as described earlier were combined with serial dilutions of S. aureus DNA template ranging from 1 to 10,000 fg, and the reaction mixtures were heated at 57°C for 60 min in a StepOneTM System (Applied Biosystems, Foster City, CA, USA) and the detection limit of the real-time LAMP was determined (Notomi et al., Citation2000).
For comparison, the detection limit of the reported method by Lim et al. (Citation2013) was determined by carrying out reactions according to the conditions specified in their publication.
Specificity determination of real-time LAMP assay
Two strains of S. aureus and 24 non-S. aureus strains were used for the specificity study (). Listeria strains were cultured overnight at 37°C in DifcoTM Buffered Listeria Enrichment Broth Base (Becton, Dickinson and Company, San Jose, CA, USA) and the others in LB broth. DNA from these pure cultures was extracted according to the manufacturer’s handbook of DNeasy® Blood & Tissue Kit (QIAGEN LTD, North Manchester, UK), and these DNA templates were used for determining the specificity of our developed real-time LAMP assay and the Lim assay. The amount of DNA template used was 10 pg per reaction.
Results and analysis
Selection of reaction temperature for real-time LAMP
The real-time LAMP reaction with the newly designed primers was carried out at varying temperatures for 60 min, as shown in . Carrying out the reaction at 57°C resulted in amplification of the positive controls with the shortest threshold for along with negative results for the negative control reactions. Reactions carried out at higher or lower temperatures also resulted in amplification for the positive controls and negative results for the negative controls, but the threshold (30 sec per cycle) was significantly higher. Therefore, 57°C was chosen as the most suitable reaction temperature.
Table 2. Threshold of LAMP reactions at varying temperatures.
Tabla 2. Umbral de reacciones de LAMP a diferentes temperaturas.
Detection limit comparison
The real-time LAMP mixtures with the newly designed primers were used to detect a serial dilution of S. aureus DNA template with heating at 57°C for 60 min. The detection limit of the optimized reaction mixture with the newly designed primers was found to be 10 fg/uL S. aureus DNA template, with no detectable false-positive response, equivalent to 2 copies/reaction, while the detection limits reported by Zhao et al. (Citation2013) and Su et al. (Citation2014) were 30 copies/reaction and 10 copies/reaction, respectively.
For comparison, the detection limit of the original reported LAMP method by Lim et al. (Citation2013) was determined using a serial dilution of S. aureus DNA template as well, the detection limit was determined to be 10 pg/uL S. aureus DNA template, only one of four repeated reactions was positive, which was significantly lower than the detection limit, 2.5 ng/uL, reported by Lim et al. (Citation2013). Moreover, one of four negative controls showed nonspecific amplification.
The reason for such difference in sensitivity may be that the target gene of our developed assay was 16S–23S rRNA intergenic spacer region, which had multiple copies in S. aureus, while the target gene of Lim assay was carsbamate kinase gene (arcC) of S. aureus, which only had one copy in S. aureus.
Assaying selectivity
The optimized real-time LAMP assay with newly designed primer sets was tested with two S. aureus strains and as shown in , both strains were successfully detected. The assay was also tested with 24 non-S. aureus strains, all three repeated reactions of any non-S. aureus strain were negative.
Table 3. Specificity determination with different LAMP methods.
Tabla 3. Determinación de la especificidad con diferentes métodos LAMP.
While the LAMP assay carried out using the primers and conditions reported by Lim et al. can favorably differentiate S. aureus from non-S. aureus strains, the detection time is much higher than with the new primers. The amplification time originally reported by Lim et al. (Citation2013) was 52 min; however, even when the amplification time of Lim LAMP assay was extended to 60 min, one of three repeated reactions for detection of S. aureus ATCC 25923 and two of three repeated reactions for detecting S. aureus ATCC 13565 were negative, as shown in . Extending the amplification time further to 80 min led to nonspecific amplification of negative controls.
Therefore, the newly designed primers combined with the optimized real-time LAMP assay presented here can detect two strains of S. aureus selectively while not detecting non-S. aureus strains, and had some advantages over the original assay reported by Lim et al. (Citation2013) in specificity.
Inclusivity of developed LAMP assay
Because our lab was short of S. aureus strains isolated from food or other sources, the inclusivity of developed LAMP assay was evaluated via sequence alignment in GenBank. Upon multiple alignment, the amplicon region (199 bp) had 100% identities with corresponding sequences of 51 S. aureus strains including 5 methicillin-resistant strains and 99% identities (198/199) with corresponding sequences of 7 S. aureus strains; however, the mutant site was not on the primers, therefore, the developed assay can detect 58 S. aureus strains found at NCBI, so the method was highly inclusive and suitable for analysis of foodborne S. aureus as well as S. aureus from other sources.
Conclusion
In this study, a set of LAMP primers targeting the 16S–23S rRNA intergenic spacer region of S. aureus was designed for use in real-time LAMP. Carrying out the reactions at 57°C allowed for the detection of 100 fg consistently without needing to extend the amplification times and avoiding false-positive results. Reaction optimization using these primers allowed for the sensitivity of this reaction to be extended further to 10 fg S. aureus DNA template, which is 103-fold better than that of previously reported methods (Lim et al., Citation2013). These primers have also been shown to be highly selective specifically for S. aureus and do not detect non-S. aureus. Future work in this area will focus on the design and application of primers for the detection of other foodborne pathogens to provide safer foods for wide consumption.
Disclosure statement
No potential conflict of interest was reported by the author.
Additional information
Funding
References
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