Loop-mediated Isothermal Amplification of vanA Gene Enables Rapid and Naked-eye Detection of Vancomycin-Resistant Enterococci Infection Hye Jin Kim, Yu Jin Kim, Dong Eun Yong, Jae Myun Lee, Sang Sun Yoon PII: DOI: Reference:

S0167-7012(14)00151-1 doi: 10.1016/j.mimet.2014.05.021 MIMET 4391

To appear in:

Journal of Microbiological Methods

Received date: Revised date: Accepted date:

22 April 2014 29 May 2014 29 May 2014

Please cite this article as: Kim, Hye Jin, Kim, Yu Jin, Yong, Dong Eun, Lee, Jae Myun, Yoon, Sang Sun, Loop-mediated Isothermal Amplification of vanA Gene Enables Rapid and Naked-eye Detection of Vancomycin-Resistant Enterococci Infection, Journal of Microbiological Methods (2014), doi: 10.1016/j.mimet.2014.05.021

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“REVISED”

Loop-mediated Isothermal Amplification of vanA Gene Enables Rapid and

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Naked-eye Detection of Vancomycin-Resistant Enterococci Infection

Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, b

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Hye Jin Kima#, Yu Jin Kima#, Dong Eun Yongb, Jae Myun Leea,c* and Sang Sun Yoona,c*

Department of Laboratory Medicine, c Institute for Immunology and Immunological Diseases, Yonsei

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University College of Medicine, Seoul, Korea.

# These authors contribute equally *

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Corresponding author: Sang Sun Yoon, Ph.D. Department of Microbiology and Immunology Yonsei University College of Medicine 250 Seongsanno, Seodaemun-gu Seoul, 120-752, Korea Tel: +82-2-2228-1824 Fax: +82-2-392-7088 E-mail: [email protected] Jae Myun Lee, M.D., Ph.D. Department of Microbiology and Immunology Yonsei University College of Medicine 250 Seongsanno, Seodaemun-gu Seoul, 120-752, Korea Tel: +82-2-2228-1822 Fax: +82-2-392-7088 E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infection at

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intensive care unit (ICU). Rapid and sensitive detection of VRE infection is in high demand for timely and suitable antibiotic treatment. Here, we optimized a distinct DNA-based diagnostic technique, loop-

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mediated isothermal amplification (LAMP) for rapid detection of the presence of vanA gene, a critical

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component of the gene cluster required for vancomycin resistance. Amplification efficiency was optimal at 62 ˚C and with 2 mM MgSO4. The detection limit of the DNA template was 80 pg and LAMP

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amplicons were detected within 40 min; thereby suggesting a potential applicability of LAMP as a

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sensitive and urgent diagnostic method. Furthermore, positive LAMP reaction was directly detected with the naked-eye by monitoring the formation of a white precipitate or the color change induced by hydroxyl naphtol blue (HNB) dye. Finally, 56 clinical isolates were successfully tested for the presence

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of vanA gene by LAMP, which was determined to be more sensitive than PCR. Together, our results

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clearly demonstrate the usefulness of LAMP for the diagnosis of VRE infection.

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IntroductionVancomycin-resistant enterococci (VRE) are opportunistic pathogens that have

become important clinical concern around the globe (Patel, 2003, Tripathi et al., 2013). Vancomycin-

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resistant E. faecalis and E. faecium clinical isolates were first documented in Europe in the late 1980s (Leclercq et al., 1988, Uttley et al., 1988). VRE infection is particularly fatal to long-staying patients at

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intensive care unit (ICU) and people with diabetes or chronic kidney failure (Bonten et al., 2001,

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d'Azevedo et al., 2009, Ostrowsky et al., 1999, Patel, 2003, Warren et al., 2003). Acquisition of resistance to vancomycin, a glycopeptide antibiotic is associated with modifications in 6 different

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vancomycin-resistant (van) gene operons (Mak et al., 2009, Tripathi, et al., 2013). VRE typically

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possess new DNA in the form of plasmids or transposons that contain genes that confer vancomycin resistance (Dahl et al., 2007). The acquired vancomycin resistance differs from the inherent resistance of some enterococcal species, such as E. gallinarum and E. casseliflavus (Courvalin, 2006).

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Vancomycin resistance is primarily mediated by the acquisition of two gene clusters, vanA and vanB (Gin and Zhanel, 1996). Among these two clusters, vanA is more easily transmittable than vanB and

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thus nosocomial transmission of vanA genotype is more frequently observed (Dahl, et al., 2007). Thus, identification of vanA gene is of importance for the timely detection of VRE infection, which will help clinicians treat already infected patients. Loop-mediated isothermal amplification (LAMP) is an innovative PCR-based amplification assay to determine whether or not the target genes are present in biological specimen (Lim et al., 2013, Notomi et al., 2000). When compared with the other conventionally used diagnostic methods, LAMP possesses following advantages. First, amplification of target genes can be achieved at a constant temperature and therefore no thermal cycler is required for reaction (Ushikubo, 2004). Bst DNA polymerase used in LAMP harbors a strand displacement activity due to the lack of 5’ to 3’ exonuclease activity and is capable of extending the primers at annealing temperature of ~60 ˚C. Second, a positive LAMP reaction can be immediately identified by the formation of a white precipitate, Magnesium Pyrophosphate, Mg2P2O7 (Tomita et al., 2008). Such a rapid diagnosis will be of crucial help, when deciding a subsequent treatment strategy. Currently, several diagnostic tools for the detection of VRE 3

ACCEPTED MANUSCRIPT infection are available that include (i) phenotype testing (Cuzon et al., 2008, d'Azevedo, et al., 2009, Delmas et al., 2007), (ii) conventional PCR (Dutka-Malen et al., 1995) and (iii) quantitative real time

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PCR (Palladino et al., 2003). But whether LAMP can be applied for swift and convenient detection of VRE from clinical samples have never been explored before. For a successful LAMP reaction, primer

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pairs should be designed to appropriately generate an initial dumbbell-like structure, which is used as a

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template for subsequent amplification. In this study, we adopted LAMP assay with a view to come up with an effective and rapid diagnostic tool for detecting VRE in clinical specimen. We chose vanA as a

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target gene for diagnosis. We designed the vanA VRE-LAMP primer set by considering the distance

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between primer regions and annealing temperature. The vanA gene was successfully amplified for as short as 40 minutes and the specificities of the designed primer pairs were confirmed using clinical specimen. The suitable condition for vanA VRE-LAMP has also been optimized. Together, our

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customized and optimized LAMP protocols possess high potential for diagnosis of VRE-borne infection

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in time and cost effective manner.

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ACCEPTED MANUSCRIPT 2.

Materials and Methods

2.1. Bacterial strains and DNA extraction

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All the bacteria strains used in this study were clinical isolates obtained from Department of

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Laboratory Medicine, Yonsei University College of Medicine. Plasmid DNA from laboratory stocks of

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VRE was extracted using AccuPrep® plasmid Mini extraction kit (BIONEER Corp., Korea). DNA from stool isolates (n=57) were recovered using PowerSoil® DNA isolation kit (MO BIO Laboratories Inc.,

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Carlsbad, CA) according to manufacturer’s instructions. After stool samples were collected, specimens were incubated in Enterococcosel broth with 6 μg/ml vancomycin for overnight and presence of vanA

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and vanB were tested with Seeplex® VRE ACE detection kit (Seegene Inc., Seoul, Korea). 2.2. PCR assay for vanA and vanB genePCR amplification of vanA gene was conducted using custom-

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designed primer sets vanA (forward), 5’-GCAATACTGTTTGGGGGTT-3’ and vanA (reverse), 5’CCGGCTTAACAAAAACAGGA-3’ by using the Primer3 online software (http://bioinfo.ut.ee/primer3/).

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Sequences to amplify the vanB gene were described elsewhere (Dutka-Malen, et al., 1995) The vanB primers were 5’-ATGGGAAGCC GATAGTC-3’ (forward) and 5’-GATTTCGTTCCTCG ACC-3’ (reverse). 2.3. Primer design for vanA LAMP

Primers for vanA-LAMP were designed based on the NCBI database sequence of vanA gene (1,032 bp, GenBank number: JN982328-1). In order to maximize hybridization stability, both ends of each inner primer (6 bp) and 3’ end of outer primers (6 bp) were designed to have >50 % GC content (Table 1) (Parida et al., 2008). Tm of each region (i.e., F1, F2, F3, B1, B2 and B3) was calculated based on the equation described previously (Howley et al., 1979). In addition, following considerations were made to achieve optimal amplification. First, distance between 3’ end of F3 (or B3) and 5’ end of F2 (or B2) should be less than 20 bp. Second, distance between 3’ end of F2 (or B2) and 5’ end of F1 (or B1) should be 40-60 bp. Third, the length of starting dumbbell-like structure amplified with LAMP primer should be between 130~250 bp (Notomi, et al., 2000). 5

ACCEPTED MANUSCRIPT 2.4. The optimization of vanA LAMP conditions To establish the most appropriate condition for vanA LAMP, a range of reaction temperatures from

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58 ˚C to 70 ˚C was tested. To determinate the optimal concentration of MgSO4, we made 10X LAMP

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buffer containing 200 mM Tris-HCl (pH 8.8, Sigma-Aldrich Co. St. Louis, MO), 100 mM (NH4)2SO4 (Sigma-Aldrich) 100 mM KCl (Sigma-Aldrich), 1% Tween 20 (Sigma-Aldrich) and 6 different

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concentrations of MgSO4 (Sigma-Aldrich); 0, 10, 20, 30, 60 and 80 mM. The mixed LAMP buffer was

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diluted to 1X before use. We also sought to determine the lowest template DNA concentration that can yield detectable amplification. Hydroxy naphtol blue (HNB) in concentration of 3 mM was added for

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rapid detection. 2.5. LAMP reaction and detection of products

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The optimized reaction mixture for vanA VRE LAMP as follows.; 20 mM Tris-HCl buffer(pH 8.8), 10

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mM KCl, 2 mM MgSO4, 10 mM (NH4)2SO4, 0.1 % Triton X-100, 1.4 mM each dNTP mix, 5 % DMSO,

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20 pM inner primers (FIP and BIP), 5 pM outer primers (F3 and B3) and 8 units Bst DNA polymerase. Every reaction was incubated at 62 ˚C for 1 hour and heat-inactivated by incubating at 95 ˚C for 3 min. Positive vanA LAMP reaction was confirmed by checking the formation of a white precipitate, magnesium pyrophosphate (Mg2P2O7) after centrifuging PCR tubes at 13,000 rpm for 2 min. Positive reaction was further confirmed by running 1.5 % agarose gel. Each LAMP experiment was performed in triplicate. To prevent cross-contamination between samples, filter tips (USA Scientific, Inc., Ocala, FL) were used when handling reagents.

2.6. DNA sequencing

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ACCEPTED MANUSCRIPT Product of vanA LAMP was confirmed by sequencing analyses. After gel electrophoresis, the shortest band on the agarose gel was purified by QIAquick gel extraction kit (QIAGEN, GmbH, Hilden,

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Results

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Germany) and sent to Macrogen Inc. (Seoul, South Korea) for sequencing.

3.1. Establishment of vanA LAMP and product confirmation

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For a more rapid and specific detection of vanA gene (1,032 bp), LAMP primers were manually designed based on the guideline as outlined in Materials and Methods. Fig. 1A shows nucleotide

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sequences used to design LAMP primers and Fig. 1B displays domains that constitute both forward and backward inner primers. Initial dumbbell-like structure produced by strand displacement is shown

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with each domain color-coded in Fig. 1C. Our primer sets listed in Table 1 amplified 229 bp (82~310)

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target sequence of vanA gene between F2 and B2 region (Fig. 1B).

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LAMP reaction was accomplished using a plasmid DNA that harbors the vanA gene. To determine the optimal reaction temperature, LAMP reaction was performed at a range of temperatures from 58 to 70 ˚C for 1 hr. A distinct ladder-like band pattern, an indication of a successful LAMP amplification, was observed at 60, 61, 62 and 63 ˚C (Fig. 2A). After gel electrophoresis, the lowest band shown with a black arrow was purified and sequenced. The sequenced nucleotide matched to the vanA gene (data not shown) further confirming that the LAMP amplification occurred specifically to the target gene. We then sought to determine the optimal MgSO4 concentration for the reaction. At 62 ˚C, best amplification was achieved with 2 mM concentration, not with any other concentration (Fig. 2B). Together, these results suggested that the optimum reaction condition, using the primers of our choice, was the reaction temperature of 62 ˚C and MgSO4 concentration of 2 mM.

3.2. Sensitivity of LAMP reaction

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ACCEPTED MANUSCRIPT In order to determine the detection limit of LAMP for vanA gene, the LAMP assay was performed using a series of DNA samples prepared from a 5-fold serial dilution of an initial concentration of 10 ng.

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A typical ladder-like amplification was detected using target DNA of as low as 80 pg (Fig. 3A). We also evaluated how fast the LAMP reaction could occur to the level of detection. When using 10 ng DNA,

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by inactivating the Bst polymerase at 95 ˚C for 3 minutes.

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LAMP amplicons were detected within 40 min at 62 ˚C (Fig. 3B). Each LAMP reaction was terminated

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3.3. Naked-eye detection of positive vanA LAMP reaction

Under a condition where urgent care is in demand, it is highly desirable to determine the positive

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reaction as rapid as possible. Positive LAMP reaction was accompanied with the production of a white precipitate (Mori et al., 2001, Yang et al., 2010). This notion was also validated in our LAMP reaction.

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As shown in Fig. 4A, a pelleted Mg2P2O7 precipitate was clearly observed after the LAMP reaction with

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10 ng DNA, but not in a negative control reaction. We next tested the usefulness of hydroxy naphthol

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blue (HNB) dye for a real-time detection of LAMP amplification. As LAMP reaction proceeds, Mg2+ concentration is decreased and the HNB dye changes its color from red to blue (Goto et al., 2009). A clear color change was observed in our vanA LAMP reaction (Fig. 4B). A ladder-like band pattern was confirmed by agarose gel (data not shown). 3.4. Application of vanA LAMP for efficient detection of VRE in clinical specimen The optimized vanA LAMP was evaluated for clinical specimen. Among 56 stool isolates, 25 were determined to be vancomycin-resistant based on their capabilities to grow in Enterococcosel broth media containing 6 μg/ml vancomycin, while the other 31 strains were sensitive to vancomycin (Table 2). Twenty one from 25 VRE strains were positive for the presence of vanA gene by regular PCR. On the other hand, 4 VRE strains were negative for vanA gene. All 21 PCR-positive VRE strains were also positive for the vanA LAMP assay (Table 2). Out of 4 PCR-negative VRE strains, 2 were turned out to possess vanA gene as assessed by LAMP. This result further demonstrated a higher sensitivity of

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ACCEPTED MANUSCRIPT LAMP. Out of 2 VRE strains that were negative for both vanA PCR and LAMP, one strain was determined to possess vanB gene (data not shown). Three strains among 31 VSE strains were positive

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for vanA PCR suggesting that the presence of vanA gene would not always result in vancomycin resistance. Twenty seven from 28 PCR-negative VSE strains did not produce vanA LAMP amplicons

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(Table 2). Overall, results from vanA PCR and LAMP were consistent with each other, while the LAMP

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assay produced more sensitive results in diagnosing the presence of vanA gene.

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4. Discussion

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Phenotype testing that involves bacterial growth in selective media, such as VREBAC® and C-ID (Chrom-ID) has been widely used to diagnose VRE infection (Cuzon, et al., 2008, d'Azevedo, et al.,

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2009). Although culture-dependent diagnosis can easily be performed, it is time-consuming and often

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requires additional confirmation (Tripathi, et al., 2013). PCR-based diagnosis has also been used due to its specific amplification of target genes (Dutka-Malen, et al., 1995). However, quantitative measure

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of whether or not the target gene is present is not always straightforward in the PCR analysis and amplified products are confirmed by gel electrophoresis (Mak, et al., 2009, Sloan et al., 2004). Quantitative real time PCR (qRT-PCR) was suggested as an alternative method for a rapid and specific diagnosis (Palladino, et al., 2003). The strongest advantage of qRP-PCR is real-time observation of the amplified DNA. The wide use of qRT-PCR, however, has been hampered because of the need of expensive equipment. In this study, we demonstrated the usefulness of LAMP for detecting VRE infection in clinical specimen. The vanA gene has been targeted, the presence of which is critical for vancomycin resistance. Due to the sensitive and robust nature of Bst DNA polymerase (Zhang et al., 2001), protocols for LAMP reaction vary significantly between laboratories and we also made considerable efforts to optimize the best condition. Based on our results, optimal amplification was achieved using 2 mM MgSO4. This finding is in contrast with a previous report, in which 8 mM MgSO4 was used (Song et

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ACCEPTED MANUSCRIPT al., 2012). Magnesium concentration affects several steps in LAMP, such as primer annealing, strand displacement and formation of primer-dimer structure (Innis, 1990). It will be of interest to examine

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whether the optimal MgSO4 concentration varies depending on shapes of the dumbbell-like structure. We sought to determine the lowest amount of DNA template that can yield a positive reaction. This

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notion is of importance when dealing with clinical specimen, especially under a condition where fast on-

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site diagnosis is necessary. The minimum concentration of DNA for detection was found to be 0.08 ng and amplification was observed within 40 min in a reaction using 10 ng DNA. This result clearly differs

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from a previous report, in which a detection limit of 62.5 ng DNA was suggested (Chan et al., 2012).

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Real-time detection of vanA amplification was achieved by simply monitoring the formation of Mg2P2O7 precipitate. Shown as a ladder pattern on the agarose gel, LAMP amplicons are produced in a remarkably large quantity yielding a high amount of pyrophosphate ion, which then forms a complex

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with Mg2+ ion. Therefore, the detection of the precipitate can be used as an indicator of positive LAMP amplification. Naked eye detection was further facilitated by the addition of HNB dye in the LAMP

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reaction. Because the presence of HNB dye does not interfere with LAMP reaction, the dye can be added in the initial LAMP reaction mixture, a feature that can be of significant help for a high-throughput application.

During the study, we often experienced false-positive results. This is likely due to the highly sensitive nature of LAMP reaction. To prevent such undesirable results, it is strongly recommended to keep the amount of DNA template as low as possible. We also noticed that cross-contamination was significantly minimized by the use of filter tips. In conclusion, we developed a diagnostic method for a rapid and inexpensive detection of VRE infection. When coupled with a fast and reliable DNA extraction technique, LAMP can provide an unmatched protocol for a timely detection of drug-resistant genes from clinical specimens. Early detection of vanA gene can help clinicians choose a better antibiotic treatment regimen, which will eventually reduce social costs caused by VRE infection.

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ACCEPTED MANUSCRIPT Acknowledgements This work was also supported by a grant from the Korea Healthcare Technology R&D Project, Ministry

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for Health, Welfare and Family Affairs, HI12C1251-00013. The authors have declared no conflicts of

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interest.

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ACCEPTED MANUSCRIPT References

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Table 1. LAMP primers used in this study.

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Table 2. Summary of PCR and LAMP assay using clinical specimen

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Positions of vanA LAMP primers and structure of initial dumbbell-like amplicon. (A)

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Nucleotide sequence of vanA gene and regions of sequence for LAMP primers. (B) Schematic diagram

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showing the positions at which each primer anneals. (C) Structure of initial dumbbell-like amplicon of

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vanA LAMP reaction.

Figure 2. Optimization of vanA LAMP reaction. (A) Determination of optimal reaction temperature for

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vanA LAMP. Plasmid DNA (10 ng) was used as a template and the reaction was performed for 1 hr at temperatures indicated at the top. “DW” means distilled water used in replace of the template DNA as a

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negative control. The band shown as black arrow was purified for sequencing analysis. Reaction products were loaded onto 1.5 % agarose gel for analysis. (B) Determination of optimal MgSO4

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concentration for vanA LAMP. LAMP reaction was performed at 62 ˚C for 1 hr using a range of MgSO4

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as a negative control.

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concentrations indicated above. Again, “DW” means distilled water used in replace of the template DNA

Figure 3. Determination of the detection limit of template DNA and minimum reaction time. (A) Template DNA in amounts of 0 pg, 3.2 pg, 16 pg, 80 pg, 400 pg, 2 ng or 10 ng was used for LAMP reaction. Amplification was performed with 2 mM MgSO4 at 62 ˚C for 1 hr. Reaction products were loaded onto 1.5 % agarose gel for analysis. (B) The LAMP reaction was performed for 30, 40, 50 or 60 min. At desired reaction time, LAMP reaction was terminated by inactivating Bst DNA polymerase at 95 ˚C for 3 min. Figure 4. Rapid detection of positive LAMP reaction. (A) Images of test tubes after 1 hr LAMP reaction. “DW” means distilled water used in replace of the template DNA as a negative control. Insoluble Mg2P2O7 precipitate was clearly observed after 2 min centrifugation at 13,000 rpm. (B) Color change of positive LAMP reaction induced by HNB dye.

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ACCEPTED MANUSCRIPT Highlights

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LAMP reaction with manually designed primers was optimized for detection of vanA gene. Positive LAMP reaction was confirmed by naked-eye detection of Mg2P2O7 precipitate. The LAMP reaction was successfully applied for detection of Vancomycin-Resistant Enterococci infection.

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Loop-mediated isothermal amplification of vanA gene enables a rapid and naked-eye detection of vancomycin-resistant enterococci infection.

Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infection at intensive care unit (ICU). A rapid and sensitive detec...
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