Accepted Manuscript

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of

Enterococcus spp. in water

Roland Martzy, Claudia Kolm, Kurt Brunner, Robert L. Mach, Rudolf Krska, Hana
Šinkovec, Regina Sommer, Andreas H. Farnleitner, Georg H. Reischer

PII: S0043-1354(17)30360-3
DOI: 10.1016/j.watres.2017.05.023
Reference: WR 12902

To appear in: Water Research

Received Date: 21 January 2017

Revised Date: 3 May 2017
Accepted Date: 3 May 2017

Please cite this article as: Martzy, R., Kolm, C., Brunner, K., Mach, R.L., Krska, R., Šinkovec, H.,
Sommer, R., Farnleitner, A.H., Reischer, G.H., A loop-mediated isothermal amplification (LAMP)
assay for the rapid detection of Enterococcus spp. in water, Water Research (2017), doi: 10.1016/
j.watres.2017.05.023.

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 ACCEPTED MANUSCRIPT

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1 A loop-mediated isothermal amplification (LAMP) assay for the rapid detection

2 of Enterococcus spp. in water

a,g a,g a b c d
3 Roland Martzy , Claudia Kolm , Kurt Brunner , Robert L. Mach , Rudolf Krska , Hana Šinkovec ,

e,g b,f,g a,b

4 Regina Sommer , Andreas H. Farnleitner , Georg H. Reischer

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a
6 TU Wien, Institute of Chemical, Environmental and Biological Engineering, Molecular Diagnostics

7 Group, Department IFA-Tulln, Konrad-Lorenz-Straße 20, A-3430 Tulln, Austria

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b
8 TU Wien, Institute of Chemical, Environmental and Biological Engineering, Research Group of

9 Environmental Microbiology and Molecular Diagnostics 166/5/4, Gumpendorfer Straße 1a, A-1060

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10 Vienna, Austria
c
11 University of Natural Resources and Life Sciences, Vienna (BOKU), Department IFA-Tulln, Center for

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12 Analytical Chemistry, Konrad-Lorenz-Straße 20, A-3430 Tulln, Austria
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d
13 Medical University Vienna, Center for Medical Statistics, Informatics and Intelligent Systems, Section

14 for Clinical Biometrics, Spitalgasse 23, A-1090 Vienna, Austria

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e
15 Medical University Vienna, Institute for Hygiene and Applied Immunology, Unit Water Hygiene,

16 Kinderspitalgasse 15, A-1090 Vienna, Austria

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17 Karl Landsteiner University of Health Sciences, Research Unit Water Quality and Health, A-3500
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18 Krems, Austria
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19 ICC Interuniversity Cooperation Centre Water & Health, Vienna, Austria (www.waterandhealth.at)

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21 Corresponding author: Georg Reischer

22 E-mail address: georg.reischer@tuwien.ac.at

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24 Running title: LAMP technique for the rapid detection of faecal pollution in water.

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26 Keywords: Enterococcus spp.; faecal indicator bacteria; loop-mediated isothermal amplification;

27 microbiological water quality; low-resource methods; molecular diagnostics

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29 Abstract

30 Faecal pollution of water and the resulting potential presence of human enteric pathogens is a

31 predominant threat to public health. Microbiological water quality can be assessed by the detection of

32 standard faecal indicator bacteria (SFIB) such as E. coli or certain Enterococcus species. In recent

33 years, isothermal amplification methods have become a useful alternative to polymerase chain

34 reaction (PCR), allowing molecular diagnostics with simple or no instrumentation. In this study, a novel

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35 screening method for the molecular detection of Enterococcus spp. by loop-mediated isothermal

36 amplification (LAMP) is described. A set of six specific LAMP primers was designed to amplify a

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37 diagnostic fragment of the Enterococcus 23S rRNA gene, which is present in several enterococcal

38

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species targeted by quantitative PCR (qPCR), which is the standard technique recommended by the

39 US Environmental Protection Agency. Sensitivity and specificity tests were performed using a set of 30

40 Enterococcus and non-target bacterial reference strains. It is shown that LAMP is equally sensitive

41
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and even more specific than the qPCR assay. A dilution series of Enterococcus faecalis DNA revealed
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42 that the LAMP method can reliably detect 130 DNA target copies per reaction within 45 min.

43 Additionally, enterococci isolated from Austrian surface waterbodies, as well as a set of DNA extracts
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44 from environmental waters, were tested. Contingency analysis demonstrated a highly significant

45 correlation between the results of the developed LAMP assay and the reference qPCR method.
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46 Furthermore, a simple staining procedure with a fluorescence dye demonstrated the identification of
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47 amplified products by eye. In conclusion, this method is an important component for the efficient

48 screening and testing of water samples in low-resource settings lacking sophisticated laboratory
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49 equipment and highly trained personnel, requiring only a simple heating block.

50
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51 1. Introduction
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52 Microbial pollution of water resources through faecal input is a threat to public health in developing as

53 well as industrialised countries. According to the WHO, 9% of the world’s population, or 663 million

54 people, are without access to safe drinking water (WHO 2015), which is why the improvement of

55 drinking water quality and sanitation is one of the United Nations Sustainable Development Goals (UN

56 2015). The microbiological quality of water used for drinking, bathing, or irrigation can be determined

57 by the detection of standard faecal indicator bacteria (SFIB) such as Escherichia coli or certain

58 Enterococcus species. The SFIB themselves are natural inhabitants of the gastrointestinal (GI) tract of

59 all mammals and usually do not pose any health risks to humans, but their presence in water suggests
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60 faecal pollution and a possible presence of enteric pathogens as a consequence (Cabelli et al. 1982,

61 Korajkic et al. 2011, Liang et al. 2015, Tallon et al. 2005). In the case of Enterococcus, many species
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62 of this genus are prevalent in the GI tract of mammals at concentrations ranging from 10 to 10 cells

63 per gram faeces (Farnleitner et al. 2010, Layton et al. 2010, Slanetz and Bartley 1957), making them

64 suitable and therefore widely used bacterial indicators for faecal pollution in water.

65 The gold standard for the detection of SFIB in water is their cultivation on agar plates after membrane

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66 filtration of a certain volume of a water sample (ISO 2000, 2005). For instance, various species of the

67 genus Enterococcus can be cultivated on Slanetz-Bartley agar (Slanetz and Bartley 1957), covering

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68 several representatives of intestinal enterococci. This is especially important since it was discovered

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that no single Enterococcus species is prevalent in the faeces of all mammals (Layton et al. 2010).

70 The major shortcomings of cultivation-based methods include the requirement for a microbiological

71 laboratory and that SFIB can only be identified and quantified with certainty after up to 48 hours of

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growth (ISO 2000). The transition of enterococci into the viable but non-culturable (VBNC) state in
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73 aquatic environments may additionally limit the application of cultivation-based methods (Lleò et al.

74 2005).
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75 Therefore, there is a need for more rapid testing methods in the field of microbiological water quality

76 assessment, which can be implemented for monitoring potential faecal input in water resources in
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77 order to prevent or investigate the transmission of waterborne pathogens (Harwood et al. 2005). Ever
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78 since the development of polymerase chain reaction (PCR) for the specific amplification of DNA target

79 genes (Saiki et al. 1988), this technique has been widely used for different applications, including the
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80 detection of pathogenic and non-pathogenic microorganisms. For instance, a quantitative PCR (qPCR)

81 method for the molecular detection of enterococci was implemented by the US Environmental
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82 Protection Agency (USEPA) and is now used for routine water quality monitoring in the US (USEPA
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83 2012). Like the cultivation-based method, this qPCR assay targets diverse Enterococcus species to

84 ensure the detection of faecal input from all warm-blooded animals and to improve the sensitivity of

85 the assay compared to the detection of single species. However, to perform PCR analysis, a

86 molecular biology laboratory with expensive instrumentation, in the form of a thermal cycler, is needed

87 to provide the temperature profile necessary for PCR. Furthermore, the data obtained by qPCR must

88 be analysed using complex computer software and interpreted by trained personnel.

89 To overcome these drawbacks, a group of so-called isothermal amplification methods has emerged

90 recently. The most common isothermal amplification method is termed loop-mediated isothermal
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91 amplification (LAMP) and was developed in 2000 by the group of Notomi (Notomi et al. 2000). The

92 LAMP reaction employs a DNA polymerase with strand-displacement activity and a set of up to three

93 primer pairs that bind to six distinct regions on the target DNA, ensuring high sequence selectivity. The

94 amplification mechanism makes the initial denaturation step obsolete and does not require any

95 temperature changes throughout the reaction, making LAMP a highly specific detection method that

96 can be completely performed at approximately 65 °C (Nagamine et al. 2001). Therefore, a simple

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97 heating block is sufficient to fulfil the requirements for the implementation of a LAMP assay,

98 eliminating the need for costly equipment such as thermal cyclers. Even further, the temperature can

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99 be provided by an exothermal reaction linked to specifically engineered phase-change materials to

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perform the analysis completely instrumentation-free (Sema et al. 2015). Agarose gel electrophoresis

101 can be used to visualise the characteristic ladder-like pattern of the DNA concatemers that are formed

102 in course of the LAMP reaction (Notomi et al. 2000). However, since LAMP generates much higher

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amounts of product DNA than PCR does, the LAMP products can quickly be made visible to the naked
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104 eye by the addition of a DNA-intercalating fluorescence dye (Iwamoto et al. 2003). Additionally, it has

105 been observed that the Bacillus stearothermophilus DNA polymerase (Bst) used in LAMP is much less
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106 susceptible to inhibitory substances than other polymerases such as Taq. For instance, the LAMP

107 method has been shown to tolerate even the presence of stool, urine, or blood in the sample, up to a
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108 certain concentration (Francois et al. 2011). These characteristics highlight the potential for LAMP to
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109 be applied in low-resource settings or directly at the point of use. Based on these properties, many

110 LAMP approaches have been published, especially for the detection of human pathogens in clinical
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111 specimens, e.g., African trypanosomes (Kuboki et al. 2003), Ebola virus (Kurosaki et al. 2007), or HIV-

112 1 (Curtis et al. 2008). In 2016, the WHO recommended the use of a commercial LAMP kit for the
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113 detection of pulmonary tuberculosis (TB) (Loopamp MTBC Detection Kit; Eiken Chemical Company
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114 Ltd; Tokyo, Japan) as a replacement for the use of microscopy to diagnose pulmonary TB in

115 developing countries (WHO 2016). Moreover, several LAMP assays have been developed with

116 respect to the detection of waterborne pathogens, e.g., Giardia (Plutzer and Karanis 2009),

117 Cryptosporidium (Karanis et al. 2007), Toxoplasma gondii (Mahmoudi et al. 2015, Sotiriadou and

118 Karanis 2008), Naegleria fowleri (Mahittikorn et al. 2015), Acanthamoeba (Mahmoudi et al. 2015) or

119 Enterococcus faecalis (Ahmad et al. 2017, Wang et al. 2017, Xu et al. 2014). Additionally, it was

120 demonstrated that LAMP can be combined with microfluidic chips for improved practicability in point-

121 of-care diagnostics (Asiello and Baeumner 2011, Fang et al. 2010, Safavieh et al. 2016).
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122 The aim of this study was to transfer this promising analytical tool into the field of water quality

123 analysis by developing a LAMP assay for the molecular detection of several Enterococcus species

124 serving as indicators for faecal pollution of water. This tool is meant to be a screening method for the

125 rapid assessment of microbiological water quality in regions with little laboratory infrastructure and to

126 complement existing techniques. The diagnostic fragment of the Enterococcus spp. 23S rRNA gene

127 that we used corresponds to that of the widely applied qPCR method of the USEPA (USEPA 2012)

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128 and targets a broad range of Enterococcus species. Our LAMP assay was evaluated and compared to

129 the reference qPCR with respect to sensitivity, specificity, and detection limits. In addition, enterococci

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130 isolated from surface water as well as a set of environmental water samples were analysed, and a

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simple staining procedure of the finished LAMP reaction demonstrated the easy visualisation of

132 amplified products by eye.

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134 2. Materials and Methods

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135 2.1. Bacterial reference strains

136 Important assay parameters (specificity, sensitivity, limit of detection) were tested by analysing the
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137 isolated genomic DNA of pure cultures of different bacterial type strains, denoted as “target strains”

138 (from the genus Enterococcus) and “non-target” strains (closely as well as distantly related species).
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139 The 15 enterococcal target strains used in this study were selected due to the available 23S rRNA
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140 gene sequence information of Enterococcus species in the NCBI database (Behr et al. 2000), and the

141 existence of previous reports of Enterococcus species distributions among human and animal hosts
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142 (Layton et al. 2010). The 15 non-enterococcal strains were selected with a focus on the environmental

143 application of the developed method and the reference qPCR method, ranging from bacteria closely
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144 related to enterococci to distantly related species, all of which may naturally occur in water and are
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145 therefore relevant for assay application.

146 To evaluate the assay sensitivity and specificity, the genomic DNA of the 30 selected bacterial

147 reference strains was analysed with LAMP and qPCR at three different concentrations (1 ng, 10 pg,

148 and 0.1 pg per reaction).

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150 Target strains: Enterococcus asini (DSM-11492), Enterococcus avium (DSM-20679), Enterococcus

151 casseliflavus (DSM-20680), Enterococcus cecorum (DSM-20682), Enterococcus durans (DSM-

152 20633), Enterococcus faecium (DSM-20477), Enterococcus faecalis (DSM-20478), Enterococcus

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153 gallinarum (DSM-20628), Enterococcus hirae (DSM-20160), Enterococcus solitarius (DSM-5634),

154 Enterococcus sulfureus (DSM-6905), Enterococcus columbae (DSM-7374), Enterococcus mundtii

155 (DSM-4838), Enterococcus dispar (DSM-6630), and Enterococcus raffinosus (DSM-5633).

156 Non-target strains: Lactococcus garvieae (DSM-6783), Lactococcus lactis subsp. lactis (DSM-

157 20481), Staphylococcus aureus subsp. aureus (DSM-20232), Streptococcus bovis (DSM-20480),

158 Streptococcus salivarius subsp. salivarius (DSM-20560), Tetragenococcus halophilus subsp.

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159 halophilus (DSM-20339), Vagococcus fluvialis (DSM-5731), Melissococcus plutonius (DSM-29964),

160 Serratia marcescens subsp. marcescens (DSM-30121), Citrobacter freundii (DSM-30039), Bacillus

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161 subtilis (ATCC 6633), Escherichia coli (NCTC 9001), Klebsiella aerogenes (NCTC 9528), Providencia

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rettgeri (NCTC 7475), and Pseudomonas aeruginosa (NCTC 10662).

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164 2.2. Environmental Enterococcus isolates

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These samples comprised 48 Enterococcus isolates from four sampling locations in Austrian surface
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166 waterbodies investigated throughout all four seasons. The enterococci were cultivated according to the

167 ISO standard (ISO 2000), and single colonies were picked from the agar plates for the successive
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168 DNA extraction. The species were subsequently identified as belonging to six different Enterococcus

169 species (E. faecalis, E. faecium, E. mundtii, E. gallinarum, E. casseliflavus, E. hirae) by 16S rRNA
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170 gene sequencing.

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171

172 2.3. Environmental water samples

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173 A set of environmental water DNA samples derived from Austrian waterbodies was selected from

174 samples collected in previous studies (Reischer et al. 2011, Savio et al. 2015). Water samples of
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175 between 10 mL and 4.2 L were filtered through 0.2 µm polycarbonate filters, followed by the isolation
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176 of genomic DNA using a bead-beating and phenol/chloroform extraction protocol (Mayer et al. 2016,

177 Reischer et al. 2008).

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179 2.4. DNA extraction

180 The extraction of genomic DNA from pure cultures of the bacterial reference strains as well as from

181 the Enterococcus isolates was performed by using the peqGOLD Bacterial DNA Kit (Peqlab, Erlangen,

182 Germany) following the manufacturer’s protocol. The isolated DNA was stored at -20 °C until further

183 use.
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185 2.5. Primers and probes

186 All oligonucleotides used in this study are listed in Table 1. Primers and the probe for the qPCR

187 reactions were used as described in USEPA method 1611 (USEPA 2012). LAMP primers were

188 designed using PrimerExplorer V4 software (Eiken Chemical Co., Ltd.; Tokyo, Japan) based on an

189 alignment of the 23S rRNA gene sequences from different Enterococcus species, GenBank acc. no.

190 AJ295298 - AJ295316 (Haugland, 2015, personal communication). These sequences have been the

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191 basis for previously developed and adapted assays for the detection of the Enterococcus 23S rRNA

192 gene (Behr et al. 2000, Ludwig and Schleifer 2000, USEPA 2012). All oligonucleotide primers and the

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193 probe were synthesised by Eurofins MWG (Ebersberg, Germany).

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195 Table 1

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Oligonucleotide Sequence (5‘ 3‘) References
ENT-PCR_fw GAG AAA TTC CAA ACG AAC TTG (21) USEPA (2012)
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qPCR ENT-PCR_rev CAG TGC TCT ACC TCC ATC ATT (21) USEPA (2012)

ENT-PCR_pr TGG TTC TCT CCG AAA TAG CTT TAG GGC TA (29) USEPA (2012)
AAC GTA CGT GGG TTC GGT CCT TCT ACC CAT
ENT-LAMP_FIP This study
GTC CAG GTT GA (41)
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GAT GAG GTG TGG GTA GCG GAG ACG AGG CTA
ENT-LAMP_BIP This study
GCC CTA AAG CT (41)

LAMP ENT-LAMP_F3 CGT AGA CCC GAA ACC ATG TG (20) This study

ENT-LAMP_B3 ACA GTG CTC TAC CTC CAT CA (20) This study
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ENT-LAMP_LoopF GTG CGT TTT ACC GCA CCT (18) This study

ENT-LAMP_LoopB AAT TCC AAA CGA ACT TGG AGA TAG C (25) This study
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197 Table 1: Oligonucleotides used for the qPCR and LAMP reactions targeting the 23S rRNA gene of
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198 various Enterococcus species.

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200
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2.6. LAMP reaction

201 The optimised LAMP reactions were carried out in a total reaction volume of 15 µl containing 1.6 µM

202 each FIP and BIP, 0.2 µM each F3 and B3, 1.2 µM each LoopF and LoopB (Table 1), 0.7 mM of each

203 dNTP (Peqlab, Erlangen, Germany), 0.6 M betaine (Sigma-Aldrich, St. Louis, USA), 4.8 U Bst 2.0

204 DNA polymerase large fragment (New England Biolabs, Ipswich, USA), 0.4 µM Syto® 9 Green

205 Fluorescent Nucleic Acid Stain for real-time monitoring of the reaction (Thermo Fisher Scientific,

206 Waltham, USA), 20 mM Tris-HCl (pH 8.5), 10 mM KCl, 10 mM (NH4)2SO4, 5 mM MgSO4, 0.1% Triton

207 X-100 (Sigma-Aldrich, Ipswich, USA), and 2.5 µl DNA sample. For the assay development and

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208 evaluation, the reactions were performed on a 7500 Fast Real-Time PCR System (Applied

209 Biosystems, New York, USA) according to the following protocol: 60 cycles of 1 min at 64 °C. In

210 course of the evaluation experiments it was shown that the assay can be stopped after a total runtime

211 of 45 minutes (corresponding to 45 cycles of 1 min) without any loss in performance. For the reaction

212 on a heating block, the reaction mixtures were incubated on a Peqlab Digital Block Heater HX-1

213 (Peqlab, Erlangen, Germany) for 45 min at 64 °C, followed by 10 min at 80 °C to stop the reaction. To

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214 visualise the results, 1 µl of the product from the finished LAMP reaction was analysed on 1.5%

215 agarose gel, or 1 µl of a 1000x SYBR® Green I Nucleic Acid Stain (Thermo Fisher Scientific,

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216 Waltham, USA) solution was added to 9 µl aliquots of the products from the finished LAMP reactions

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for detection by eye. Unless noted otherwise, LAMP reactions were carried out in triplicate.

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219 2.7. Quantitative PCR reaction

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The quantitative PCR reactions were carried out with reagent concentrations as described in USEPA
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221 method 1611 (USEPA 2012). The total reaction volume of 15 µl contained 1 µM of each primer, 80 nM
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222 of the probe (Table 1), 7.5 µl Kapa Probe® Fast mix (Peqlab, Erlangen, Germany), and 2.5 µl DNA
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223 extract. The reactions were performed on a 7500 Fast Real-Time PCR System (Applied Biosystems,

224 New York, USA) according to the following protocol: 5 min at 95 °C, followed by 45 cycles of 15 s at
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225 95 °C and 1 min at 60 °C. Unless noted otherwise, qPCR reactions were carried out in triplicate. The
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226 calibration line was generated using a dilution series of plasmid DNA containing the diagnostic

227 fragment of the Enterococcus faecalis 23S rRNA gene that is targeted by the assay.
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228

229 2.8. Statistical analysis

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230 Contingency analysis was performed in two by two contingency tables and by performing chi-square
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231 (χ²) tests using IBM SPSS Statistics 24 software (IBM, 2016). For that purpose, each triplicate of the

232 LAMP and qPCR analysis results was clustered in such a way that zero positive reactions out of three

233 replicates was rated as a negative result and that one, two, or three positives out of the three

234 replicates was rated as a positive result.

235 The calculation of the limit of detection (LOD) was done using R (R Development Core Team, 2008)

236 package MASS after fitting the logistic regression model. This calculation was conducted for qPCR

237 and LAMP.

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239 3. Results and discussion

240 3.1. LAMP assay development and optimisation

241 For the LAMP assay to be comparable to the USEPA qPCR assay, the 23S rRNA gene of

242 Enterococcus spp. was selected as the target gene for the amplification reaction. All Enterococcus

243 spp. 23S rRNA gene sequences available from the NCBI database (Behr et al. 2000) were aligned in

244 NCBI Sequence Viewer 3.17.0. Following primer design, the LAMP assay was initially performed using

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245 a total reaction volume of 25 µl and the reagent concentrations as described in the original publication

246 (Notomi et al. 2000). To optimise the protocol for detecting the Enterococcus spp. 23S rRNA gene,

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247 step-by-step variations of the concentrations of the FIP/BIP (0.8 – 2 µM), LoopF/LoopB (0.2 – 1.6 µM),

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and F3/B3 (0.1 – 0.6 µM) oligonucleotides as well as of the concentrations of dNTPs (0.1 – 0.9 mM),

249 betaine (0.4 – 1.2 M), polymerase (1.2 – 9.6 U per reaction) and magnesium in the buffer (3 – 8 mM)

250 were conducted (see Figures S1 and S2 for exemplary raw data). In the end, the total reaction volume

251
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was decreased from 25 to 15 µl for a better comparison with the reference qPCR method and for
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252 economic reasons. The reagent concentrations in the final reaction volume were not changed

253 compared to the 25 µl reaction mix and no changes in the performance of the LAMP assay could be
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254 observed.

255
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256 3.2. Specificity and sensitivity of the LAMP primers

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257 The assay sensitivity illustrates the true positive rate, whereas the assay specificity indicates the true

258 negative rate. To evaluate these parameters, the genomic DNA of the 30 selected bacterial reference
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259 strains was analysed with LAMP and qPCR at three different concentrations, representing high,

260 medium, and low faecal pollution, respectively (see Figures S3 and S4 for exemplary amplification
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261 curves). It was previously reported that Enterococcus faecalis carries four copies of the 23S rRNA
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262 gene (Marshall et al. 2002). When considering Enterococcus faecalis has a genome size of

263 approximately 3.22 million base pairs (Paulsen et al. 2003), the analysed concentrations are
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264 equivalent to 10 , 10 , and 10 DNA target copies per reaction for this species. Unfortunately, the

265 genome size and 23S rRNA operon number are not known for all species used in this study, which is

266 why the decision was made to report the DNA concentrations as ng per reaction and not as genome

267 equivalents. The results are listed in Table 2.

268 Lemmon and Gardner published mathematical terms predicting sensitivity and specificity of qPCR

269 assays (Lemmon and Gardner 2008):

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270

=1− = 1 −
+

=1− = 1 −
+

271 Following the formulae, the sensitivity and specificity values for the LAMP and qPCR assay were

272 calculated and are reported in Table 2.

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273

274 Table 2

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LAMP assay qPCR assay
1 ng 10 pg 0.1 pg 1 ng 10 pg 0.1 pg
Enterococcus asini 3/3 3/3 3/3 3/3 3/3 3/3

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Enterococcus avium 3/3 3/3 3/3 3/3 3/3 3/3
Enterococcus casseliflavus 3/3 3/3 3/3 3/3 3/3 3/3
Enterococcus cecorum 3/3 3/3 3/3 3/3 3/3 3/3
Enterococcus durans 3/3 3/3 3/3 3/3 3/3 3/3
Target strains

Enterococcus faecium 3/3 3/3 3/3 3/3 3/3 3/3

Enterococcus faecalis 3/3 3/3 3/3 3/3 3/3 3/3

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Enterococcus hirae 3/3 3/3 3/3 3/3 3/3 3/3
Enterococcus gallinarum 3/3 3/3 3/3 3/3 3/3 3/3
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Enterococcus solitarius 3/3 3/3 3/3 3/3 3/3 3/3
Enterococcus sulfureus 3/3 3/3 3/3 3/3 3/3 3/3
Enterococcus columbae 3/3 3/3 3/3 3/3 3/3 3/3
Enterococcus mundtii 3/3 3/3 3/3 3/3 3/3 3/3
Enterococcus dispar 3/3 3/3 3/3 3/3 3/3 3/3
Enterococcus raffinosus 3/3 3/3 3/3 3/3 3/3 3/3
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Sensitivity 100% 100% 100% 100% 100% 100%

Lactococcus garvieae 3/3 0/3 0/3 3/3 0/3 1/3

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Lactococcus lactis 3/3 0/3 0/3 3/3 0/3 0/3

Staphylococcus aureus 3/3 1/3 1/3 3/3 1/3 1/3
Streptococcus bovis 3/3 1/3 0/3 3/3 1/3 0/3
Non-target strains

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Streptococcus salivarius 3/3 1/3 0/3 3/3 1/3 0/3

Tetragenococcus halophilus 3/3 3/3 3/3 3/3 3/3 3/3
Vagococcus fluvialis 3/3 3/3 3/3 3/3 3/3 3/3
Melissococcus plutonius 3/3 3/3 3/3 3/3 3/3 3/3
Serratia marcescens 1/3 0/3 0/3 0/3 0/3 0/3
Citrobacter freundii 0/3 0/3 0/3 3/3 1/3 0/3
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Bacillus subtilis 3/3 0/3 0/3 0/3 0/3 0/3

Escherichia coli 0/3 0/3 0/3 3/3 0/3 0/3
Klebsiella aerogenes 0/3 0/3 0/3 2/3 0/3 0/3
Providencia rettgeri 0/3 0/3 0/3 2/3 0/3 0/3
Pseudomonas aeruginosa 0/3 0/3 0/3 1/3 0/3 0/3
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Specificity 38% 73% 78% 22% 71% 76%

275 Shading scheme: dark grey (3/3); light grey (2/3) and (1/3); white (0/3)
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276 Table 2: Results of the sensitivity and specificity tests performed with LAMP and qPCR. Three

277 different concentrations (1 ng, 10 pg, and 0.1 pg per reaction) of the genomic DNA (gDNA) of the

278 target strains (Enterococcus species) and non-target strains (non-Enterococcus species) were

279 analysed in triplicate using both methods.

280

281 The data suggest that the assays perform reliably with regard to sensitivity, which is promising with

282 respect to the broad range of Enterococcus species that can be detected. Unlike previously published

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283 LAMP assays for the detection of Enterococcus faecalis (Ahmad et al. 2017, Wang et al. 2017, Xu et

284 al. 2014), the assay developed in this study can be applied for the detection of faecal input from any

285 warm-blooded animal. On the other hand, several non-target species are co-detected by both the

286 qPCR and LAMP. This might be due to the high sequence similarity between the different species in

287 the gene fragment that is amplified. For instance, the segment of the 23S rRNA gene that is targeted

288 by the qPCR primers is identical in Tetragenococcus halophilus, Vagococcus fluvialis, Melissococcus

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289 plutonius and Enterococcus faecalis, which is why all three of the non-target organisms are co-

290 detected by qPCR as well as LAMP, even at low concentrations (0.1 pg DNA per reaction). In the case

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291 of high DNA concentrations (1 ng per reaction), additional non-Enterococcus species are co-detected

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when analysed with qPCR and LAMP, probably due to their nearly identical target sequences, with

293 differences in only a few bases. However, the presence of the addressed non-target organisms at

294 such high concentrations reflects an unrealistic, worst-case scenario since those species represent

295
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only a small part of the overall number of microorganisms that might occur in faecally polluted water.
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296 The medium and low DNA concentrations tested are certainly the more realistic levels of target and

297 non-target gene content in application settings.

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298 The results of the sensitivity and specificity experiments were characterised by contingency analysis to

299 assess if the performance of the LAMP assay is comparable to that of the qPCR assay. As described
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300 in the methods section, the replicates were clustered in a way that only a score of zero out of three
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301 replicates was rated as negative. The reason behind this handling of the results is that the developed

302 LAMP assay is regarded as an initial screening method for water samples and that a further
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303 investigation of the samples is required as soon as one out of three replicates shows a positive signal.

304 It could be shown that there is no correlation in the performance of the two methods in case of high
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305 DNA concentrations (1 ng per reaction) (χ²-test; Phi=-0.12; p=0.51), whereas there is a highly
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306 significant and strong correlation in cases of medium (10 pg) (Phi=0.92; p<0.001) and low DNA

307 concentrations (0.1 pg) (Phi=0.93; p<0.001).

308

309 3.3. Limit of detection (LOD) experiments

310 In contrast to qPCR, the LAMP method is not capable of quantifying the number of target genes in the

311 analysed sample, which is why it is of particular importance to determine the limit of detection of the

312 assay. To this end, the isolated genomic DNA of Enterococcus faecalis DSM-20478 was quantified via

313 qPCR, and a subsequent dilution series ranging from 210 to 0.1 DNA target molecules per reaction
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314 (with regard to the 23S rRNA gene) was analysed with LAMP and qPCR, with 20 replicates each

315 (Table S1). To statistically determine the threshold where 95% of the replicates show a positive signal

316 (LOD95), the results from both methods were fitted in a logistic regression model using R. The model

317 and the corresponding values are depicted in Figure 1. The modelled LOD95 for the developed LAMP

318 assay and the reference qPCR were 130 and 7 DNA target copies per reaction, respectively.

319 However, as shown in Table S1, all replicates with 100 target copies showed a positive result with

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320 LAMP. The same result could be observed from the sensitivity tests (Table 2), where the analyte could

321 be detected in all samples containing the lowest DNA concentration (0.1 pg per reaction,

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322 corresponding to 100 DNA target copies). Unlike qPCR, the LAMP reaction is dependent on the

323

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binding of four to six primers and therefore relies upon a longer target gene fragment and,

324 consequently, upon more binding sites within this fragment. Using the optimum predefined values for

325 length, melting temperature and GC content, no specific primer set was found to distinguish target

326
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from non-target species. Hence, the conditions had to be slightly relaxed, which might have decreased
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327 the efficiency of the amplification.

328 The higher LOD for LAMP could influence the reliability of the method when samples with very low
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329 levels of contamination are analysed. Nevertheless, when applied to environmental samples, LAMP

330 was shown to be capable of detecting one out of three replicates in concentrations of approximately
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331 20 target copies per reaction (Table 3).

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332

333 Figure 1
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334

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335 Figure 1: Graph showing the results for qPCR and LAMP obtained from the analysis of the

336 Enterococcus faecalis gDNA dilution series. The horizontal line with the filled symbols indicates the

337 limit of detection where 95% of the replicates are positive (LOD95).

338

339 3.4. Analysis of Enterococcus isolates derived from Austrian waterbodies

340 To investigate the assay sensitivity to Enterococcus strains that have adapted to their environment,

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341 the genomic DNA of enterococci isolated from Austrian waterbodies was analysed with LAMP and

342 qPCR using a concentration of 10 pg per reaction. Because both methods detected 100% of the

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343 isolates (see Table S2 and Figure S5 for representative raw data), it can be assumed that potential

344

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mutations in environmental strains do not influence the sensitivity of the assays or that the adaptation

345 of enterococci to their environment does not necessarily lead to alterations in the targeted 23S rRNA

346 gene sequence. Therefore, both methods are capable of reliably detecting Enterococcus strains that

347
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naturally occur in the gastrointestinal tract and, consequently, in water.
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349 3.5. Analysis of environmental water samples

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350 The presence of enterococci in environmental water samples is potentially accompanied by highly

351 abundant autochthonous and allochthonous microflora, which increases the variety of DNA sequences
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352 that might influence the assay performance. Furthermore, molecular detection methods are often
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353 susceptible to inhibitory substances that are present, especially in highly polluted water samples, and

354 that are co-extracted with the DNA, which leads to false-negative results. For this reason, 30
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355 environmental water samples were selected to represent a gradient regarding the extent of faecal

356 pollution. The results of the qPCR and LAMP analyses of the respective water DNA are illustrated in
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357 Table 3 (also see Figure S6 for exemplary amplification curves).

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358 The differences in the results between the two methods when analysing the moderately polluted

359 samples (surface water and polluted spring water) can be explained by the higher LOD of the LAMP

360 assay compared to the reference qPCR. As seen in Table 3, all samples but one from this category

361 contain less than 100 DNA target copies per 2.5 µl, which indicates why the results obtained by LAMP

362 do not match those obtained by qPCR. Three of the samples were detected by neither of the assays,

363 probably resulting from the complete absence of the target gene. The analysis results of the

364 environmental samples were submitted to contingency analysis to assess if the performance of the

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365 LAMP assay is comparable to that of the qPCR assay. It could be shown that there is a highly

366 significant and strong correlation in the performance of the two methods (χ²-test; Phi=0.80; p<0.001).

367 To exclude false-negative results due to potential inhibitory effects, all samples were spiked with 0.1

368 pg Enterococcus faecalis genomic DNA per reaction (corresponding to 100 copies of the 23S rRNA

369 gene). A subsequent analysis with LAMP and qPCR showed 100% positive results for all samples,

370 demonstrating that the amplification reaction at the previous analysis of the environmental samples

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371 with both methods was not inhibited.

372

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373 Table 3
Mean copy number
Sample
LAMP qPCR ± standard deviation

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no.
determined by qPCR
1 0/3 0/3 n.d.
2 0/3 0/3 n.d.
3 0/3 0/3 n.d.
Spring/ground 4 0/3 0/3 n.d.

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water samples 5 0/3 0/3 n.d.
6 0/3 0/3 n.d.
(unpolluted) 7 0/3 0/3 n.d.
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8 0/3 0/3 n.d.
9 0/3 0/3 n.d.
10 0/3 0/3 n.d.

Detection rate 0% 0%
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11 0/3 0/3 n.d.

12 0/3 3/3 6±2
Surface water/ 13 2/3 3/3 29 ± 4
polluted spring 14 2/3 3/3 37 ± 9
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water samples 15 1/3 3/3 19 ± 5

16 0/3 0/3 n.d.
(moderately 17 3/3 3/3 156 ± 16
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polluted) 18 2/3 3/3 71 ± 10

19 0/3 0/3 n.d.
20 0/3 3/3 12 ± 6

Detection rate 43% 70%

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21 3/3 3/3 10,666 ± 730

22 3/3 3/3 3,369 ± 510
23 3/3 3/3 1,541 ± 172
Waste water 24 3/3 3/3 2,531 ± 207
samples 25 3/3 3/3 5,803 ± 580
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26 3/3 3/3 53,085 ± 581

(highly polluted) 27 3/3 3/3 45,690 ± 4,073
28 3/3 3/3 41,370 ± 1,147
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29 3/3 3/3 138,544 ± 9,201

30 3/3 3/3 37,803 ± 3,354

Detection rate 100% 100%

n.d.: not detected
Shading scheme: dark grey (3/3); light grey (2/3) and (1/3); white (0/3)

374 Table 3: The 23S rRNA target gene in all samples was quantified with qPCR, and a subsequent

375 analysis with LAMP allowed for a direct comparison to the reference assay.

376

377 3.6. Visualisation of the LAMP products

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378 Using agarose gel electrophoresis, we determined that the LAMP products show a characteristic

379 ladder-like pattern (see Figure S7) that is attributed to the complex reaction mechanism that forms

380 concatemers with varying numbers of inverted repeats originating from the DNA target sequence

381 (Notomi et al. 2000). Unfortunately, agarose gel electrophoresis requires expensive laboratory

382 infrastructure and generally toxic reagents, such as ethidium bromide, making this procedure

383 impracticable for applications in low-resource settings. Alternatively, the LAMP products can be

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384 visualised by using a simple staining procedure that allows for detection by eye within one minute. For

385 this reason, three target strains (Enterococcus faecalis, E. faecium, E. durans) as well as two non-

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386 target strains (Lactococcus garvieae, Streptococcus bovis) and a no-template control (NTC; DNA

387

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replaced with water) were analysed using three replicates each and submitted to gel electrophoresis

388 and fluorescence staining in parallel. Figure S7 shows the agarose gel and the corresponding samples

389 stained with SYBR Green I. In case of a positive LAMP reaction, the orange-coloured stain can be

390
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observed to turn into an intense light green by its intercalation in the double-stranded DNA products
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391 and a consequent change in the emission wavelength in the visible spectrum. These findings suggest

392 that the results from agarose gel electrophoresis are in perfect accordance with those obtained from
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393 the fluorescence staining procedure, which is a promising result with respect to the intended

394 application of the assay.

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395
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396 Since LAMP generates such vast amounts of DNA products, there is a certain risk of carryover

397 contamination in the course of opening the reaction tube for the subsequent visualisation procedure.
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398 Especially in low-resource settings a strict separation of areas for the handling of samples with and

399 without DNA is often not feasible. For on-site applications, preparing agar dye capsules (Karthik et al.
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400 2014) could be considered to eliminate the need for opening the reaction tubes after the amplification
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401 reaction. Another strategy to perform visual detection would involve the use of pH-sensitive dyes

402 (Tanner et al. 2015). However, this would include a major change in the reaction conditions and would

403 supposedly require a new assay evaluation.

404

405 4. Conclusions

406 • The developed LAMP assay for the detection of enterococci is intended to be used as an

407 initial screening tool for the rapid assessment of microbiological water quality in low-resource

408 settings.
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409 • The Enterococcus LAMP assay is an important component for an entire workflow for water

410 quality monitoring in regions with little or no laboratory infrastructure (e.g., developing

411 countries). The workflow would also include a simple DNA extraction protocol for DNA from

412 water, which is currently under development in our lab.

413 • For implementation of the method in low-resource settings, freeze-dried reagents could be

414 provided in ready-to-use test tubes.

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415 • Beyond that application, the LAMP assay has great potential to be combined with lab-on-a-

416 chip systems in a portable, battery-powered device.

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417

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418 5. Acknowledgements

419 This study was part of the Life Science Call 2013 project LSC13-020 funded by the

420 Niederösterreichische Forschungs- und Bildungsgesellschaft (NFB) and supported by the Austrian

421
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Science Fund (FWF) project P23900 granted to Andreas H. Farnleitner. This study was a collaboration
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422 with the Interuniversity Cooperation Centre Water & Health (http://www.waterandhealth.at).

423
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- A rapid method for the molecular detection of enterococci in water is presented.
- The developed LAMP assay is equally sensitive and specific as the reference qPCR.
- The reaction takes place at a constant temperature provided by a heating block.
- Results can be visualised within one minute.

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