RESEARCH LETTER
Loop-mediated isothermal amplification (LAMP) assays for rapid detection and differentiation of Nosema apis and N. ceranae in honeybees ska1, Grzegorz Borsuk2, Grzegorz Wozniakowski3, Sebastian Gnat4 & Aneta A. Ptaszyn 5 Wanda Małek 1
Department of Botany and Mycology, Faculty of Biology and Biotechnology, Institute of Biology and Biochemistry, Maria Curie-Skłodowska University, Lublin, Poland; 2Department of Biological Basis of Animal Production, Faculty of Biology and Animal Breeding, University of Life Sciences, Lublin, Poland; 3Department of Poultry Viral Diseases, National Veterinary Research Institute, Puławy, Poland; 4Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Institute of Biological Bases of Animal Diseases, University of Life Sciences, Lublin, Poland; and 5 Department of Genetics and Microbiology, Maria Curie-Skłodowska University, Lublin, Poland
ska, Correspondence: Aneta A. Ptaszyn Department of Botany and Mycology, Faculty of Biology and Biotechnology, Institute of Biology and Biochemistry, Maria CurieSkłodowska University, 19 Akademicka St., 20033 Lublin, Poland. Tel.: +48 81 537 50 92; fax: +48 81 537 54 14; e-mail: aneta. [email protected]
MICROBIOLOGY LETTERS
Received 15 May 2014; revised 7 June 2014; accepted 19 June 2014. Final version published online 17 July 2014. DOI: 10.1111/1574-6968.12521 Editor: Michael Bidochka Keywords LAMP; Nosema spp. detection; 16S rDNA gene amplification.
Abstract Nosemosis is a contagious disease of honeybees (Apis mellifera) manifested by increased winter mortality, poor spring build-up and even the total extinction of infected bee colonies. In this paper, loop-mediated isothermal amplifications (LAMP) were used for the first time to identify and differentiate N. apis and N. ceranae, the causative agents of nosemosis. LAMP assays were performed at a constant temperature of 60 °C using two sets of six species-specific primers, recognising eight distinct fragments of 16S rDNA gene and GspSSD polymerase with strand displacement activity. The optimal time for LAMP and its Nosema species sensitivity and specificity were assessed. LAMP only required 30 min for robust identification of the amplicons. Ten-fold serial dilutions of total DNA isolated from bees infected with microsporidia were used to determine the detection limit of N. apis and N. ceranae DNAs by LAMP and standard PCR assays. LAMP appeared to be 103-fold more sensitive than a standard PCR in detecting N. apis and N. ceranae. LAMP methods developed by us are highly Nosema species specific and allow to identify and differentiate N. apis and N. ceranae.
Introduction Nosemosis is a panzootic contagious disease of bees and is caused by two fungal species, Nosema apis and N. ceranae, belonging to phylum Microsporidia. This disease afflicts bees with reduced honey yield, increased winter mortality and poor spring build-up, and consequently, it may lead to the extinction of bee colonies (Fries, 1993). For the first time, nosemosis was described in honeybees (Apis mellifera) 100 years ago (Zander, 1909). Initially, all nosemosis of eastern honeybees was caused by N. apis. However, at the end of the twentieth century, a new microsporidian species, N. ceranae, was found to be infecting honeybees in Asia (Fries et al., 1996). The first infection of Apis mellifera by N. ceranae was recorded in Taiwan in 2005 (Huang et al., 2005). Soon after that, infections of A. mellifera by N. ceranae were ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
described in Europe (Higes et al., 2006; Paxton et al., 2007), United States (Chen et al., 2008), China and Vietnam (Liu et al., 2008). Researchers suggest that N. ceranae also infects bumblebees (Plischuk et al., 2009). Presently, bee infections by N. ceranae have been identified all over the world (Klee et al., 2007). Nosemosis causes many changes at the level of both individual bees and bee colonies and it results in global economic losses. The lifespan of infected bees is reduced by one-third. In colonies infected by Nosema spp., the worker bees become lethargic and unwilling to work (Fries, 1993). Honey yield in colonies decreases by 40%, and wax secretion falls by 25% (Kostecki, 1962). Nosema spp. spores can be observed mainly in the intestinal epithelial cells and in various tissues and glands (Bourgeois et al., 2012; Copley & Jabaji, 2012; Ptaszy nska et al., 2012). FEMS Microbiol Lett 357 (2014) 40–48
LAMP for rapid detection of N. apis and N. ceranae
Several methods have been used to control infections caused by N. apis, for example, heat treatment, fumigation and administration of fumagillin. Unfortunately, most nosemosis in bees today is caused by N. ceranae, which is more virulent than N. apis (Paxton et al., 2007; Huang et al., 2008; Botias et al., 2013). More worrying is that infections caused by N. ceranae are the reason for a significant decrease in bee immunity (Ant unez et al., 2009) and that these infections are more resistant to fumagillin treatment than those caused by N. apis (Huang et al., 2013). Fumagillin should be used with extreme caution and only used when it is really necessary because this antimicrosporidial drug affects the bee physiology and significantly shortens lifespan of queen bees and workers, and furthermore, it is clastogenic and cytotoxic to cultured human lymphocytes (Webster, 1994; Rada et al., 1997; Stevanovic et al., 2008). Therefore, better nosemosis control requires information about the causative agents of the disease as well as rapid and reliable methods for distinguishing N. apis from N. ceranae. The identification of Nosema spp. in honeybees has been performed traditionally by detecting their spores using a light microscope, although differentiation of N. apis and N. ceranae spores by this technique is very difficult (Chen et al., 2013; Fries et al., 2013). Under transmission electron microscopy (TEM), spores of N. apis and N. ceranae can be distinguished based on the number of polar filament coils (Fries et al., 1996; Chen et al., 2009; Fries, 2010). More recently, standard, duplex (Martın-Hernandez et al., 2007) and real-time PCR (Bourgeois et al., 2010; Burgher-MacLellan et al., 2010) assays have been developed for N. apis and N. ceranae diagnostics. However, these techniques are relatively slow or require specialist equipment and they have to be conducted in a specialised laboratory. Therefore, the goal of this study was to develop a new method for rapid and robust distinction of N. apis and N. ceranae directly in apiaries using simple equipment. Fourteen years ago, a new highly specific and efficient method of nucleic acid amplification, termed loop-mediated isothermal amplification (LAMP), was developed by Notomi et al. (2000). In LAMP, a target DNA sequence is multiplied using DNA polymerase with high replication and strand displacement activities and two to three pairs of specially designed primers that recognise six to eight distinct sequences in the template DNA. The LAMP is very sensitive and can amplify even a few copies of target DNA to make up to 109 copies in < 30 min (Mori et al., 2001, 2004; Iwasaki et al., 2003; Tomita et al., 2008). The LAMP products can be detected by the naked eye as white precipitate after the introduction of magnesium to the reaction mixture, thus eliminating the additional gel analysis. FEMS Microbiol Lett 357 (2014) 40–48
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LAMP requires following a set of four to six primers: forward inner primer (FIP), backward inner primer (BIP), forward outer primer (F3), backward outer primer (B3), and additionally forward loop primer (LF) and backward loop primer (LB), which significantly reduces the time of the amplification (Nagamine et al., 2002). The inner primers, containing sequences of the sense and antisense strands of the target DNA, initiate LAMP, and outer primers displace the amplified strands and release singlestranded DNAs with the assistance of Bst or GspSSD DNA polymerase, which have high-strand displacement activities (Notomi et al., 2000; Mori & Notomi, 2009). The singlestranded DNAs form hairpin structures to initiate the loops for cyclic amplification. During elongation, each strand is displaced, and in every cycle, new loops are produced. The final products are the stem-loop DNA fragments with several inverted repeats of a target DNA and the cauliflower-like structures, formed in the process of hybridisation between alternately inverted repeats present in the same strand (Notomi et al., 2000; Nagamine et al., 2002). Because of the high sensitivity and short time of analysis, LAMP is used for the rapid detection of different pathogens including bacteria (e.g. Geojith et al., 2011; Biswas & Sakai, 2014), viruses (e.g. Ren & Li, 2011; Wozniakowski et al., 2012), protozoa (e.g. Poon et al., 2006; Njiru et al., 2008) and fungi (e.g. Inacio et al., 2008; Jez dryczka et al., 2013; Suebsing et al., 2013). The LAMP described in this paper could be an ideal tool for Nosema species identification and differentiation of field conditions, especially in resource-poor countries.
Material and methods Microscopic identification of Nosema spp. spores in honeybees and isolation of total DNA from bees infected and uninfected by N. apis and N. ceranae
Individual Apis mellifera carnica was collected from winter losses from 12 local apiaries in the spring. From each apiary, at least 200 bees from five hives were taken. Bees from a one hive were treated as a one sample. From every sample, we took 10 bees, homogenised in the 10 mL of distilled water and examined under an optical microscope Olympus BX61 for the presence of Nosema spp. spores (Cantwell, 1970; Hornitzky, 2008; Fries et al., 2013). From the Nosema-infected and Nosema-uninfected bee colonies, 120 bees were taken for further studies. The alimentary tracts from each individual were removed, and total DNA was isolated by the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instruction. DNA isolated from individual bees was used as a template ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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for the detection of N. apis- and N. ceranae-specific 16S rDNA gene by standard PCR technique (Martın-Hernandez et al., 2007) and LAMP. Detection of N. apis- and N. ceranae-specific 16S rDNA gene in honeybees using standard PCR
To identify N. apis and N. ceranae in honeybees, a standard PCR was carried out using DNA isolated from bees and primers for both Nosema species’ 16S rDNA genes. The PCR was performed in a 25 lL reaction mixture of the Qiagen Taq PCR Core Kit containing 2.5 lL PCR buffer, 5 lL Q solution, 0.1 mM dNTP mixture, 0.7 U Taq DNA polymerase, 0.2 lM of each forward and reverse primer specific for N. apis and N. ceranae, c. 2 lL (c. 50 ng) of DNA template and ddH2O to a final reaction volume of 25 lL. For DNA amplification, the following PCR cycling conditions were used: 1 min at 94 °C, 1 min at 61.8 °C and 1 min at 72 °C, repeated for 30 cycles and 10 min at 72 °C. The primers used to differentiate both Nosema species’ 16S rDNA gene by standard PCR were as follows: 321-APIS for N. apis (FOR 50 -GGG GGC ATG TCT TTG ACG TAC TAT GTA-30 , REV 50 -GGG GGG CGT TTA AAA TGT GAA ACA ACT ATG-30 ) and 218MITOC for N. ceranae (FOR 50 -CGG CGA CGA TGT GAT ATG AAA ATA TTA A-30 , REV 50 -CCC GGT CAT TCT CAA ACA AAA AAC CG-30 ) (Martın-Hernandez et al., 2007; Hamiduzzaman et al., 2010). The PCR products were analysed using 2% agarose gel electrophoresis, stained with ethidium bromide and visualised under UV light using a CCD camera coupled to a computer with the Scion Image program. The 100-bp and 50-bp DNA ladders
(Fermentas) were used as size markers. Differences in 16S rDNA gene amplicons’ lengths were the criteria to differentiate N. apis and N. ceranae honeybee infections. To confirm the validity of Nosema species identification by standard PCR, some amplicons were sequenced using ABI PRISM 3100 – Avant. Amplified N. apis and N. ceranae 16S rDNA gene sequences were released into GenBank under Accession Number KC707997─KC708007. Oligonucleotide primers for LAMP
The two sets of FIP, BIP, F3 and B3 LAMP primers were designed based on the N. apis and N. ceranae 16S rDNA gene sequences (GenBank Accession Nos. respectively, JQ639306.1 and DQ078785) using the Primer Explorer version 4 (Net Laboratory, Tokyo, Japan). The descriptions and sequences of the designed LAMP primers are presented in the Table 1. The condition of LAMP
LAMP was carried out in a 15 lL mixture containing 5 lL of Isothermal Mastermix (Optigene, Japan), 50 pmol FIP and BIP inner primers, 25 pmol LF and LB loop primers, 10 pmol F3 and B3 outer primers, 2.0 lL template DNA (c. 50 ng) and deionised water to a final volume of 15 lL. The reaction mixture was incubated at 60 °C for different time periods (5, 15, 30 and 60 mins). After incubation, the reaction was terminated by heating at 80 °C for 2 min. LAMP products were monitored under daylight by yellowish green colour of reaction mixtures, under UV
Table 1. LAMP primers designated for Nosema apis and N. ceranae Primer Nosema apis
Nosema ceranae
Sequence 0
apFIP
5 -GTTACCCGTTATTGCCTTGTTAATTTT GTAAGAGTGAGACCTATCAGC-30
apBIP
50 -ACTTTGTAATATTCCGGAGAAGGAG TTTTCCATAGGTCAAGTTTCGCC-30
apF3 apB3 apLF apLB cerFIP
50 -ACTACGTTAAAGTGTAGCTAAC-30 50 -TCCCATAACTGCCTCAGAT-30 50 -CCATTACCTTAACAACTA-30 50 -CCTGAGAGACGGCTACTA-30 50 -TACTGCTGGAACTACAATGGTAATTTTTT GTATGTATTTTTTGAACAAGGACG-30
cerBIP
50 -ACTATGCCGACGATGTGATATGATTTT CATACTACCCCCAGAGCC-30
cerF3 cer B3 cerLF cerLB
50 -AGCGAAAGCTGTACACTT-30 50 -CTTTAATTTTCAATCGTGCGAT-30 50 -ATCTTCGCTCCTCCAGCT-30 50 -AAATATTAATTTGTATTA-30
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Description FIP primer, containing at 50 end the sequence complementary to F1 (24 nt written in italics), TTTT linker and F2 sequence at 30 end (21 nt written in italics). BIP primer, containing at 50 end the B1 sequence (25 nt written in italics), TTTT linker and at 30 end the sequence complementary to B2 (19 nt written in italics). F3 primer, consisting of 22 nucleotides. B3 primer, consisting of 19 nucleotides: Loop primers designed to accelerate the LAMP reaction. FIP primer, containing at 50 end the sequence complementary to F1 (24 nt written in italics), TTTT linker and at 30 end the F2 sequence (20 nt written in italics). BIP primer, containing at 50 end the B1 sequence (23 nt written in italics), TTTT linker and at 30 end the sequence complementary to B2 (18 nt written in italics). F3 primer, consisting of 18 nucleotides B3 primer, consisting of 22 nucleotides Loop primers designed to accelerate the LAMP reaction
FEMS Microbiol Lett 357 (2014) 40–48
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(a)
(b)
I
II
Fig. 1. Optimisation of LAMP reaction time for the detection of Nosema apis (a) and Nosema ceranae (b). LAMP reactions were performed at 60 °C for 5, 15, 30 and 60 mins. The LAMP products were observed under daylight as a green colour of positive reaction (AI, BI), under UV light as a green fluorescence (AII, BII) and electrophoretically on agar gels as characteristic ladder-like patterns (AIII, BIII).
(a)
III
(b)
(c)
Fig. 2. Real-time PCR with F3 and B3 LAMP primers. The melt curves and the melting temperatures of Nosema apis and Nosema ceranae amplicons received in the reactions with DNA deriving from honeybees infected by N. apis (a), N. ceranae (b) and N. apis + N. ceranae (c).
by green fluorescence after the addition of 1 lL of SYBR Green I (Invitrogen) diluted 1 : 10 in DMSO and under their electrophoresis on 2% agarose gel FEMS Microbiol Lett 357 (2014) 40–48
stained with ethidium bromide as a multiple DNA bands of different molecular weights (a ladder-like pattern). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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(a)
(b)
I
II
III
IV Fig. 3. Sensitivity of LAMP and standard PCR assays in the detection of Nosema apis (a) and Nosema ceranae (b). The 10-fold serial dilutions responded to 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg of DNA in the sample. The LAMP products were visualised under daylight as a green colour (AI, BI), under UV as green fluorescence (AII, BII) and as a ladder-like patterns by agarose gel electrophoresis (AIII, BIII). The standard PCR products were documented by electrophoresis in agarose gel based on their sizes (AIV, BIV), (L: 100-bp DNA ladder; N: negative control).
The condition of real-time PCR with the F3 and B3 LAMP primers
To confirm that LAMP amplified the correct targets of DNA, a real-time PCR analyses with F3 and B3 LAMP primers and N. apis and N. ceranae DNAs were performed on Applied Biosystems 7500 using QuantiTect SYBR Green PCR Kit (Qiagen). The reaction was performed in 25 lL of a total mixture containing 12.5 lL of 29 QuantiTect SYBR Green PCR Master Mix (Qiagen), 20 pmol of each primer, 1 lL of DNA template ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
(c. 25 ng) which was used in LAMP and deionised water added to a final volume of 25 lL. The melt curves were received using 1 °C step with a hold of 30 s at each step, from 60 to 95 °C. Sensitivity of LAMP and standard PCR assays
The sensitivity of the LAMP and standard PCR methods, that is, the lowest detection limit of N. apis and N. ceranae DNAs of each test, were determined using 10-fold serial dilutions of the DNA isolated from individual bees FEMS Microbiol Lett 357 (2014) 40–48
LAMP for rapid detection of N. apis and N. ceranae
infected by N. apis and N. ceranae and reaction conditions as described above for standard PCR. LAMP was carried out at 60 °C for 30 min.
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The Nosema species specificity of LAMP methods was assessed by comparing the results of LAMP tests with those from a standard PCR used for the identification and differentiation of N. apis and N. ceranae, based on species-specific sizes of their 16S rDNA gene amplification products (Martın-Hernandez et al., 2007).
amplicons were identified based on their characteristic sizes (Fig. 4). We found 100% agreement in speciesspecific detection of N. apis and N. ceranae DNAs using LAMP and standard PCR. Successful amplification of N. apis 16S rDNA gene was only found with N. apis LAMP-specific primers similar as in the case of N. ceranae 16S rDNA gene, which was amplified only with N. ceranae LAMP-specific primers. LAMP products were not detected in the reactions performed with N. apis DNA and N. ceranae primers and vice versa (Fig. 4). These data support the high Nosema species specificity of presented LAMP assays.
Results
Discussion
Species specificity of LAMP assays
The validation of LAMP in the identification of N. apis and N. ceranae in honeybees
Identification of N. apis and N. ceranae in honeybees by loop-mediated isothermal amplifications (LAMP) was performed at the temperature of 60 °C for 5, 15, 30 and 60 mins. The optimal reaction time was established for 30 min (Fig. 1). The LAMP products were visualised by eye as white precipitate, as green fluorescence under UV and as a ladder-like patterns on agarose gels. To determine whether LAMP amplified the correct target DNA, real-time PCRs with F3 and B3 LAMP primers and total DNA isolated from bees infected solely by N. apis, only with N. ceranae and with both N. apis and N. ceranae, were carried out, and the melting curves of amplification products were analysed. All amplicons from each real-time PCR showed reproducible melt curves with a melting temperature (Tm) of amplified N. apis (c. 84 °C)- and N. ceranae (c. 82 °C)-specific 16S rDNA gene fragments (Fig. 2).
The microsporidian parasites, N. apis and N. ceranae, seriously affect honeybees’ populations and cause considerable economic losses in apicultures. The early diagnosis of the nosemosis and its control is essential for maintaining healthy and productive apiaries. Given a lack
(a)
(b)
The sensitivity of LAMP and PCR assays in detecting N. apis and N. ceranae DNAs
LAMP detected N. apis and N. ceranae target DNA down to the total DNA concentration of 100 fg, whereas the standard PCR detected these DNA samples to the total DNA concentration of 100 pg (Fig. 3). Therefore, LAMP appeared to be 103-fold more sensitive than standard PCR in N. apis and N. ceranae detection. Species specificity of LAMP for N. apis and N. ceranae detection
Nosema species specificity of LAMP methods was evaluated by a comparison of the results of these reactions with those from standard PCR (Martın-Hernandez et al., 2007) where N. apis- and N. ceranae-specific FEMS Microbiol Lett 357 (2014) 40–48
(c)
Fig. 4. Detection of Nosema apis and Nosema ceranae in individual specimens by LAMP using two sets of primers specific for N. apis (a) and N. ceranae (b) and by standard PCR as a control (c) using primers specific for N. apis (321-APIS) and N. ceranae (218-MITOC). L: 50-bp DNA ladder; N: negative control; 1–3: bees infected only by N. apis; 4–6: bees infected only by N. ceranae; 7–9: bees identified as N. apis + N. ceranae infected; 10–12: bees identified as free from N. apis and N. ceranae infection.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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of quick, robust and sensitive methods for the identification of honeybee nosemosis in its early stages, we have designated reliable and sensitive LAMP assays for rapid diagnosis of N. apis and N. ceranae infections in honeybees using GspSSD DNA polymerase, two sets of primers and the multicopy 16S rRNA gene as a target DNA. The presence of six primers (in the one set), recognising eight distinct sequences on the target DNA, accelerates the loop-mediated isothermal amplification reaction and increases its sensitivity (Nagamine et al., 2002). In our LAMP tests, performed at 60 °C, 16S rDNA gene sequences of both Nosema species were successfully amplified during 30 min of reaction (Fig. 1). Amplicons obtained in real-time PCR for both Nosema species showed reproducible melt curves and very similar melting temperatures (Tm) (Fig. 2) corresponding to the nucleotide sequences with the same G+C content. The higher Tm of N. apis 16S rDNA gene amplicons than those of N. ceranae 16S rDNA gene reflects the former’s higher G+C content, that is, 41.12% in comparison with 35.71% in the case of N. ceranae as calculated using program BioEdit 7.0.9 (Hall, 1999). LAMP was 103-fold more sensitive than standard PCR in the detection of N. apis and N. ceranae DNAs (Fig. 3). LAMP, due to the six primers used, was not only a very rapid and sensitive test to diagnose nosemosis in honeybees, but also highly specific in the detection of N. apis and N. ceranae DNAs (Fig. 2). There were no false results and no cross-reaction in LAMP between primers designated for N. apis 16S rDNA gene to N. ceranae DNAs and vice versa (Fig. 4). All DNA samples from honeybees, identified by the loop-mediated isothermal amplification reaction as containing DNA of N. apis and N. ceranae or both these microsporidian DNAs, were positively supported by standard PCR (321 bp and 218–219 bp for N. apis and N. ceranae, respectively) (Fig. 4) (MartınHernandez et al., 2007). It should be emphasised that LAMP described in this paper allows not only to identify but also to differentiate N. apis and N. ceranae infections in bees. In conclusion, the developed LAMP methods offer a good alternative to the standard PCR technique for the rapid and simple identification and differentiation of N. apis and N. ceranae infections in honeybees. It detects microsporidian DNA with high sensitivity and specificity without negative influence on amplification process of nontarget DNA copresence. The low cost of equipment required for isothermal amplification of DNA (only water bath or heating block) enables the use of LAMP for the rapid, cost-effective and accurate identification of microsporidian reservoirs in honeybees even by small field laboratories associated with apiaries. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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Liu F, Wang Q, Dai PL, Wu YY, Song HK & Zhou T (2008) Natural stripe of Microsporidia of honeybee in China. Chin Bull Entomol 45: 963–966. Martın-Hernandez R, Meana A, Prieto L, Martınez Salvador A, Garrido-Bail on E & Higes M (2007) Outcome of colonization of Apis mellifera by Nosema ceranae. Appl Environ Microbiol 73: 6331–6338. Mori Y & Notomi T (2009) Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother 15: 60–69. Mori Y, Nagamine K, Tomita N & Notomi T (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289: 150–154. Mori Y, Kitao M, Tomita N & Notomi T (2004) Real-time turbidimetry of LAMP reaction for quantifying template DNA. J Biochem Biophys Methods 59: 145–157. Nagamine K, Hase T & Notomi T (2002) Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes 16: 223–229. Njiru ZK, Mikosza ASJ, Matovu E, Enyaru JCK, Ouma JO, Kibona SN, Thompson RCA & Ndung’u JM (2008) African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA. Int J Parasitol 38: 589–599. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N & Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28: E63. Paxton RJ, Klee J, Korpela S & Fries I (2007) Nosema ceranae has infected Apis mellifera in Europe since at least 1998 and may be more virulent than Nosema apis. Apidologie 38: 558– 565. Plischuk S, Martın-Hernandez R, Lucıa M, Prieto L, Botıas C, Meana A, Abrahamovich AH, Lange C & Higes M (2009) South American native bumblebees (Hymenoptera: Apidae) infected by Nosema ceranae (Microsporidia), an emerging pathogen of honeybees (Apis mellifera). Environ Microbiol Rep 1: 131–135. Poon LLM, Wong BWY, Ma EHT et al. (2006) Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin Chem 52: 303–306. Ptaszy nska AA, Borsuk G, Anusiewicz M & Mułenko W (2012) Location of Nosema spp. spores within body of honey bee. Med Weter 68: 618–621. Rada V, Machova M, Huk J, Marounek M & Duskova D (1997) Microflora in the honeybee digestive tract: counts, characteristics and sensitivity to veterinary drugs. Apidologie 28: 357–365. Ren X & Li P (2011) Development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus. Virus Genes 42: 229–235.
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Stevanovic J, Stanimirovic Z, Radakovic M & Stojic V (2008) In vitro evaluation of the clastogenicity of fumagillin. Environ Mol Mutagen 49: 594–601. Suebsing R, Prombun P, Srisala J & Kiatpathomchai W (2013) Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp. J Appl Microbiol 114: 1254–1263. Tomita N, Mori Y, Kanda H & Notomi T (2008) Loop-mediated isothermal amplification (LAMP) of gene
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sequences and simple visual detection of products. Nat Protoc 3: 877–882. Webster TC (1994) Fumagillin affects Nosema-apis and honey-bees (Hymonopterai-Apidae). J Econ Entomol 87: 601– 604. Wozniakowski G, Kozdru n W & Samorek-Salamonowicz E (2012) Loop-mediated isothermal amplification for the detection of goose circovirus. Virol J 9: 110. Zander E (1909) Tierische Parasiten als Krankenheitserreger bei der Biene. M€ unchener Bienenzeitung 31: 196–204.
FEMS Microbiol Lett 357 (2014) 40–48
Loop-mediated isothermal amplification (LAMP) assays for rapid detection and differentiation of Nosema apis and N. ceranae in honeybees ska1, Grzegorz Borsuk2, Grzegorz Wozniakowski3, Sebastian Gnat4 & Aneta A. Ptaszyn 5 Wanda Małek 1
Department of Botany and Mycology, Faculty of Biology and Biotechnology, Institute of Biology and Biochemistry, Maria Curie-Skłodowska University, Lublin, Poland; 2Department of Biological Basis of Animal Production, Faculty of Biology and Animal Breeding, University of Life Sciences, Lublin, Poland; 3Department of Poultry Viral Diseases, National Veterinary Research Institute, Puławy, Poland; 4Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Institute of Biological Bases of Animal Diseases, University of Life Sciences, Lublin, Poland; and 5 Department of Genetics and Microbiology, Maria Curie-Skłodowska University, Lublin, Poland
ska, Correspondence: Aneta A. Ptaszyn Department of Botany and Mycology, Faculty of Biology and Biotechnology, Institute of Biology and Biochemistry, Maria CurieSkłodowska University, 19 Akademicka St., 20033 Lublin, Poland. Tel.: +48 81 537 50 92; fax: +48 81 537 54 14; e-mail: aneta. [email protected]
MICROBIOLOGY LETTERS
Received 15 May 2014; revised 7 June 2014; accepted 19 June 2014. Final version published online 17 July 2014. DOI: 10.1111/1574-6968.12521 Editor: Michael Bidochka Keywords LAMP; Nosema spp. detection; 16S rDNA gene amplification.
Abstract Nosemosis is a contagious disease of honeybees (Apis mellifera) manifested by increased winter mortality, poor spring build-up and even the total extinction of infected bee colonies. In this paper, loop-mediated isothermal amplifications (LAMP) were used for the first time to identify and differentiate N. apis and N. ceranae, the causative agents of nosemosis. LAMP assays were performed at a constant temperature of 60 °C using two sets of six species-specific primers, recognising eight distinct fragments of 16S rDNA gene and GspSSD polymerase with strand displacement activity. The optimal time for LAMP and its Nosema species sensitivity and specificity were assessed. LAMP only required 30 min for robust identification of the amplicons. Ten-fold serial dilutions of total DNA isolated from bees infected with microsporidia were used to determine the detection limit of N. apis and N. ceranae DNAs by LAMP and standard PCR assays. LAMP appeared to be 103-fold more sensitive than a standard PCR in detecting N. apis and N. ceranae. LAMP methods developed by us are highly Nosema species specific and allow to identify and differentiate N. apis and N. ceranae.
Introduction Nosemosis is a panzootic contagious disease of bees and is caused by two fungal species, Nosema apis and N. ceranae, belonging to phylum Microsporidia. This disease afflicts bees with reduced honey yield, increased winter mortality and poor spring build-up, and consequently, it may lead to the extinction of bee colonies (Fries, 1993). For the first time, nosemosis was described in honeybees (Apis mellifera) 100 years ago (Zander, 1909). Initially, all nosemosis of eastern honeybees was caused by N. apis. However, at the end of the twentieth century, a new microsporidian species, N. ceranae, was found to be infecting honeybees in Asia (Fries et al., 1996). The first infection of Apis mellifera by N. ceranae was recorded in Taiwan in 2005 (Huang et al., 2005). Soon after that, infections of A. mellifera by N. ceranae were ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
described in Europe (Higes et al., 2006; Paxton et al., 2007), United States (Chen et al., 2008), China and Vietnam (Liu et al., 2008). Researchers suggest that N. ceranae also infects bumblebees (Plischuk et al., 2009). Presently, bee infections by N. ceranae have been identified all over the world (Klee et al., 2007). Nosemosis causes many changes at the level of both individual bees and bee colonies and it results in global economic losses. The lifespan of infected bees is reduced by one-third. In colonies infected by Nosema spp., the worker bees become lethargic and unwilling to work (Fries, 1993). Honey yield in colonies decreases by 40%, and wax secretion falls by 25% (Kostecki, 1962). Nosema spp. spores can be observed mainly in the intestinal epithelial cells and in various tissues and glands (Bourgeois et al., 2012; Copley & Jabaji, 2012; Ptaszy nska et al., 2012). FEMS Microbiol Lett 357 (2014) 40–48
LAMP for rapid detection of N. apis and N. ceranae
Several methods have been used to control infections caused by N. apis, for example, heat treatment, fumigation and administration of fumagillin. Unfortunately, most nosemosis in bees today is caused by N. ceranae, which is more virulent than N. apis (Paxton et al., 2007; Huang et al., 2008; Botias et al., 2013). More worrying is that infections caused by N. ceranae are the reason for a significant decrease in bee immunity (Ant unez et al., 2009) and that these infections are more resistant to fumagillin treatment than those caused by N. apis (Huang et al., 2013). Fumagillin should be used with extreme caution and only used when it is really necessary because this antimicrosporidial drug affects the bee physiology and significantly shortens lifespan of queen bees and workers, and furthermore, it is clastogenic and cytotoxic to cultured human lymphocytes (Webster, 1994; Rada et al., 1997; Stevanovic et al., 2008). Therefore, better nosemosis control requires information about the causative agents of the disease as well as rapid and reliable methods for distinguishing N. apis from N. ceranae. The identification of Nosema spp. in honeybees has been performed traditionally by detecting their spores using a light microscope, although differentiation of N. apis and N. ceranae spores by this technique is very difficult (Chen et al., 2013; Fries et al., 2013). Under transmission electron microscopy (TEM), spores of N. apis and N. ceranae can be distinguished based on the number of polar filament coils (Fries et al., 1996; Chen et al., 2009; Fries, 2010). More recently, standard, duplex (Martın-Hernandez et al., 2007) and real-time PCR (Bourgeois et al., 2010; Burgher-MacLellan et al., 2010) assays have been developed for N. apis and N. ceranae diagnostics. However, these techniques are relatively slow or require specialist equipment and they have to be conducted in a specialised laboratory. Therefore, the goal of this study was to develop a new method for rapid and robust distinction of N. apis and N. ceranae directly in apiaries using simple equipment. Fourteen years ago, a new highly specific and efficient method of nucleic acid amplification, termed loop-mediated isothermal amplification (LAMP), was developed by Notomi et al. (2000). In LAMP, a target DNA sequence is multiplied using DNA polymerase with high replication and strand displacement activities and two to three pairs of specially designed primers that recognise six to eight distinct sequences in the template DNA. The LAMP is very sensitive and can amplify even a few copies of target DNA to make up to 109 copies in < 30 min (Mori et al., 2001, 2004; Iwasaki et al., 2003; Tomita et al., 2008). The LAMP products can be detected by the naked eye as white precipitate after the introduction of magnesium to the reaction mixture, thus eliminating the additional gel analysis. FEMS Microbiol Lett 357 (2014) 40–48
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LAMP requires following a set of four to six primers: forward inner primer (FIP), backward inner primer (BIP), forward outer primer (F3), backward outer primer (B3), and additionally forward loop primer (LF) and backward loop primer (LB), which significantly reduces the time of the amplification (Nagamine et al., 2002). The inner primers, containing sequences of the sense and antisense strands of the target DNA, initiate LAMP, and outer primers displace the amplified strands and release singlestranded DNAs with the assistance of Bst or GspSSD DNA polymerase, which have high-strand displacement activities (Notomi et al., 2000; Mori & Notomi, 2009). The singlestranded DNAs form hairpin structures to initiate the loops for cyclic amplification. During elongation, each strand is displaced, and in every cycle, new loops are produced. The final products are the stem-loop DNA fragments with several inverted repeats of a target DNA and the cauliflower-like structures, formed in the process of hybridisation between alternately inverted repeats present in the same strand (Notomi et al., 2000; Nagamine et al., 2002). Because of the high sensitivity and short time of analysis, LAMP is used for the rapid detection of different pathogens including bacteria (e.g. Geojith et al., 2011; Biswas & Sakai, 2014), viruses (e.g. Ren & Li, 2011; Wozniakowski et al., 2012), protozoa (e.g. Poon et al., 2006; Njiru et al., 2008) and fungi (e.g. Inacio et al., 2008; Jez dryczka et al., 2013; Suebsing et al., 2013). The LAMP described in this paper could be an ideal tool for Nosema species identification and differentiation of field conditions, especially in resource-poor countries.
Material and methods Microscopic identification of Nosema spp. spores in honeybees and isolation of total DNA from bees infected and uninfected by N. apis and N. ceranae
Individual Apis mellifera carnica was collected from winter losses from 12 local apiaries in the spring. From each apiary, at least 200 bees from five hives were taken. Bees from a one hive were treated as a one sample. From every sample, we took 10 bees, homogenised in the 10 mL of distilled water and examined under an optical microscope Olympus BX61 for the presence of Nosema spp. spores (Cantwell, 1970; Hornitzky, 2008; Fries et al., 2013). From the Nosema-infected and Nosema-uninfected bee colonies, 120 bees were taken for further studies. The alimentary tracts from each individual were removed, and total DNA was isolated by the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instruction. DNA isolated from individual bees was used as a template ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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for the detection of N. apis- and N. ceranae-specific 16S rDNA gene by standard PCR technique (Martın-Hernandez et al., 2007) and LAMP. Detection of N. apis- and N. ceranae-specific 16S rDNA gene in honeybees using standard PCR
To identify N. apis and N. ceranae in honeybees, a standard PCR was carried out using DNA isolated from bees and primers for both Nosema species’ 16S rDNA genes. The PCR was performed in a 25 lL reaction mixture of the Qiagen Taq PCR Core Kit containing 2.5 lL PCR buffer, 5 lL Q solution, 0.1 mM dNTP mixture, 0.7 U Taq DNA polymerase, 0.2 lM of each forward and reverse primer specific for N. apis and N. ceranae, c. 2 lL (c. 50 ng) of DNA template and ddH2O to a final reaction volume of 25 lL. For DNA amplification, the following PCR cycling conditions were used: 1 min at 94 °C, 1 min at 61.8 °C and 1 min at 72 °C, repeated for 30 cycles and 10 min at 72 °C. The primers used to differentiate both Nosema species’ 16S rDNA gene by standard PCR were as follows: 321-APIS for N. apis (FOR 50 -GGG GGC ATG TCT TTG ACG TAC TAT GTA-30 , REV 50 -GGG GGG CGT TTA AAA TGT GAA ACA ACT ATG-30 ) and 218MITOC for N. ceranae (FOR 50 -CGG CGA CGA TGT GAT ATG AAA ATA TTA A-30 , REV 50 -CCC GGT CAT TCT CAA ACA AAA AAC CG-30 ) (Martın-Hernandez et al., 2007; Hamiduzzaman et al., 2010). The PCR products were analysed using 2% agarose gel electrophoresis, stained with ethidium bromide and visualised under UV light using a CCD camera coupled to a computer with the Scion Image program. The 100-bp and 50-bp DNA ladders
(Fermentas) were used as size markers. Differences in 16S rDNA gene amplicons’ lengths were the criteria to differentiate N. apis and N. ceranae honeybee infections. To confirm the validity of Nosema species identification by standard PCR, some amplicons were sequenced using ABI PRISM 3100 – Avant. Amplified N. apis and N. ceranae 16S rDNA gene sequences were released into GenBank under Accession Number KC707997─KC708007. Oligonucleotide primers for LAMP
The two sets of FIP, BIP, F3 and B3 LAMP primers were designed based on the N. apis and N. ceranae 16S rDNA gene sequences (GenBank Accession Nos. respectively, JQ639306.1 and DQ078785) using the Primer Explorer version 4 (Net Laboratory, Tokyo, Japan). The descriptions and sequences of the designed LAMP primers are presented in the Table 1. The condition of LAMP
LAMP was carried out in a 15 lL mixture containing 5 lL of Isothermal Mastermix (Optigene, Japan), 50 pmol FIP and BIP inner primers, 25 pmol LF and LB loop primers, 10 pmol F3 and B3 outer primers, 2.0 lL template DNA (c. 50 ng) and deionised water to a final volume of 15 lL. The reaction mixture was incubated at 60 °C for different time periods (5, 15, 30 and 60 mins). After incubation, the reaction was terminated by heating at 80 °C for 2 min. LAMP products were monitored under daylight by yellowish green colour of reaction mixtures, under UV
Table 1. LAMP primers designated for Nosema apis and N. ceranae Primer Nosema apis
Nosema ceranae
Sequence 0
apFIP
5 -GTTACCCGTTATTGCCTTGTTAATTTT GTAAGAGTGAGACCTATCAGC-30
apBIP
50 -ACTTTGTAATATTCCGGAGAAGGAG TTTTCCATAGGTCAAGTTTCGCC-30
apF3 apB3 apLF apLB cerFIP
50 -ACTACGTTAAAGTGTAGCTAAC-30 50 -TCCCATAACTGCCTCAGAT-30 50 -CCATTACCTTAACAACTA-30 50 -CCTGAGAGACGGCTACTA-30 50 -TACTGCTGGAACTACAATGGTAATTTTTT GTATGTATTTTTTGAACAAGGACG-30
cerBIP
50 -ACTATGCCGACGATGTGATATGATTTT CATACTACCCCCAGAGCC-30
cerF3 cer B3 cerLF cerLB
50 -AGCGAAAGCTGTACACTT-30 50 -CTTTAATTTTCAATCGTGCGAT-30 50 -ATCTTCGCTCCTCCAGCT-30 50 -AAATATTAATTTGTATTA-30
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
Description FIP primer, containing at 50 end the sequence complementary to F1 (24 nt written in italics), TTTT linker and F2 sequence at 30 end (21 nt written in italics). BIP primer, containing at 50 end the B1 sequence (25 nt written in italics), TTTT linker and at 30 end the sequence complementary to B2 (19 nt written in italics). F3 primer, consisting of 22 nucleotides. B3 primer, consisting of 19 nucleotides: Loop primers designed to accelerate the LAMP reaction. FIP primer, containing at 50 end the sequence complementary to F1 (24 nt written in italics), TTTT linker and at 30 end the F2 sequence (20 nt written in italics). BIP primer, containing at 50 end the B1 sequence (23 nt written in italics), TTTT linker and at 30 end the sequence complementary to B2 (18 nt written in italics). F3 primer, consisting of 18 nucleotides B3 primer, consisting of 22 nucleotides Loop primers designed to accelerate the LAMP reaction
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(a)
(b)
I
II
Fig. 1. Optimisation of LAMP reaction time for the detection of Nosema apis (a) and Nosema ceranae (b). LAMP reactions were performed at 60 °C for 5, 15, 30 and 60 mins. The LAMP products were observed under daylight as a green colour of positive reaction (AI, BI), under UV light as a green fluorescence (AII, BII) and electrophoretically on agar gels as characteristic ladder-like patterns (AIII, BIII).
(a)
III
(b)
(c)
Fig. 2. Real-time PCR with F3 and B3 LAMP primers. The melt curves and the melting temperatures of Nosema apis and Nosema ceranae amplicons received in the reactions with DNA deriving from honeybees infected by N. apis (a), N. ceranae (b) and N. apis + N. ceranae (c).
by green fluorescence after the addition of 1 lL of SYBR Green I (Invitrogen) diluted 1 : 10 in DMSO and under their electrophoresis on 2% agarose gel FEMS Microbiol Lett 357 (2014) 40–48
stained with ethidium bromide as a multiple DNA bands of different molecular weights (a ladder-like pattern). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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(a)
(b)
I
II
III
IV Fig. 3. Sensitivity of LAMP and standard PCR assays in the detection of Nosema apis (a) and Nosema ceranae (b). The 10-fold serial dilutions responded to 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg of DNA in the sample. The LAMP products were visualised under daylight as a green colour (AI, BI), under UV as green fluorescence (AII, BII) and as a ladder-like patterns by agarose gel electrophoresis (AIII, BIII). The standard PCR products were documented by electrophoresis in agarose gel based on their sizes (AIV, BIV), (L: 100-bp DNA ladder; N: negative control).
The condition of real-time PCR with the F3 and B3 LAMP primers
To confirm that LAMP amplified the correct targets of DNA, a real-time PCR analyses with F3 and B3 LAMP primers and N. apis and N. ceranae DNAs were performed on Applied Biosystems 7500 using QuantiTect SYBR Green PCR Kit (Qiagen). The reaction was performed in 25 lL of a total mixture containing 12.5 lL of 29 QuantiTect SYBR Green PCR Master Mix (Qiagen), 20 pmol of each primer, 1 lL of DNA template ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
(c. 25 ng) which was used in LAMP and deionised water added to a final volume of 25 lL. The melt curves were received using 1 °C step with a hold of 30 s at each step, from 60 to 95 °C. Sensitivity of LAMP and standard PCR assays
The sensitivity of the LAMP and standard PCR methods, that is, the lowest detection limit of N. apis and N. ceranae DNAs of each test, were determined using 10-fold serial dilutions of the DNA isolated from individual bees FEMS Microbiol Lett 357 (2014) 40–48
LAMP for rapid detection of N. apis and N. ceranae
infected by N. apis and N. ceranae and reaction conditions as described above for standard PCR. LAMP was carried out at 60 °C for 30 min.
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The Nosema species specificity of LAMP methods was assessed by comparing the results of LAMP tests with those from a standard PCR used for the identification and differentiation of N. apis and N. ceranae, based on species-specific sizes of their 16S rDNA gene amplification products (Martın-Hernandez et al., 2007).
amplicons were identified based on their characteristic sizes (Fig. 4). We found 100% agreement in speciesspecific detection of N. apis and N. ceranae DNAs using LAMP and standard PCR. Successful amplification of N. apis 16S rDNA gene was only found with N. apis LAMP-specific primers similar as in the case of N. ceranae 16S rDNA gene, which was amplified only with N. ceranae LAMP-specific primers. LAMP products were not detected in the reactions performed with N. apis DNA and N. ceranae primers and vice versa (Fig. 4). These data support the high Nosema species specificity of presented LAMP assays.
Results
Discussion
Species specificity of LAMP assays
The validation of LAMP in the identification of N. apis and N. ceranae in honeybees
Identification of N. apis and N. ceranae in honeybees by loop-mediated isothermal amplifications (LAMP) was performed at the temperature of 60 °C for 5, 15, 30 and 60 mins. The optimal reaction time was established for 30 min (Fig. 1). The LAMP products were visualised by eye as white precipitate, as green fluorescence under UV and as a ladder-like patterns on agarose gels. To determine whether LAMP amplified the correct target DNA, real-time PCRs with F3 and B3 LAMP primers and total DNA isolated from bees infected solely by N. apis, only with N. ceranae and with both N. apis and N. ceranae, were carried out, and the melting curves of amplification products were analysed. All amplicons from each real-time PCR showed reproducible melt curves with a melting temperature (Tm) of amplified N. apis (c. 84 °C)- and N. ceranae (c. 82 °C)-specific 16S rDNA gene fragments (Fig. 2).
The microsporidian parasites, N. apis and N. ceranae, seriously affect honeybees’ populations and cause considerable economic losses in apicultures. The early diagnosis of the nosemosis and its control is essential for maintaining healthy and productive apiaries. Given a lack
(a)
(b)
The sensitivity of LAMP and PCR assays in detecting N. apis and N. ceranae DNAs
LAMP detected N. apis and N. ceranae target DNA down to the total DNA concentration of 100 fg, whereas the standard PCR detected these DNA samples to the total DNA concentration of 100 pg (Fig. 3). Therefore, LAMP appeared to be 103-fold more sensitive than standard PCR in N. apis and N. ceranae detection. Species specificity of LAMP for N. apis and N. ceranae detection
Nosema species specificity of LAMP methods was evaluated by a comparison of the results of these reactions with those from standard PCR (Martın-Hernandez et al., 2007) where N. apis- and N. ceranae-specific FEMS Microbiol Lett 357 (2014) 40–48
(c)
Fig. 4. Detection of Nosema apis and Nosema ceranae in individual specimens by LAMP using two sets of primers specific for N. apis (a) and N. ceranae (b) and by standard PCR as a control (c) using primers specific for N. apis (321-APIS) and N. ceranae (218-MITOC). L: 50-bp DNA ladder; N: negative control; 1–3: bees infected only by N. apis; 4–6: bees infected only by N. ceranae; 7–9: bees identified as N. apis + N. ceranae infected; 10–12: bees identified as free from N. apis and N. ceranae infection.
ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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of quick, robust and sensitive methods for the identification of honeybee nosemosis in its early stages, we have designated reliable and sensitive LAMP assays for rapid diagnosis of N. apis and N. ceranae infections in honeybees using GspSSD DNA polymerase, two sets of primers and the multicopy 16S rRNA gene as a target DNA. The presence of six primers (in the one set), recognising eight distinct sequences on the target DNA, accelerates the loop-mediated isothermal amplification reaction and increases its sensitivity (Nagamine et al., 2002). In our LAMP tests, performed at 60 °C, 16S rDNA gene sequences of both Nosema species were successfully amplified during 30 min of reaction (Fig. 1). Amplicons obtained in real-time PCR for both Nosema species showed reproducible melt curves and very similar melting temperatures (Tm) (Fig. 2) corresponding to the nucleotide sequences with the same G+C content. The higher Tm of N. apis 16S rDNA gene amplicons than those of N. ceranae 16S rDNA gene reflects the former’s higher G+C content, that is, 41.12% in comparison with 35.71% in the case of N. ceranae as calculated using program BioEdit 7.0.9 (Hall, 1999). LAMP was 103-fold more sensitive than standard PCR in the detection of N. apis and N. ceranae DNAs (Fig. 3). LAMP, due to the six primers used, was not only a very rapid and sensitive test to diagnose nosemosis in honeybees, but also highly specific in the detection of N. apis and N. ceranae DNAs (Fig. 2). There were no false results and no cross-reaction in LAMP between primers designated for N. apis 16S rDNA gene to N. ceranae DNAs and vice versa (Fig. 4). All DNA samples from honeybees, identified by the loop-mediated isothermal amplification reaction as containing DNA of N. apis and N. ceranae or both these microsporidian DNAs, were positively supported by standard PCR (321 bp and 218–219 bp for N. apis and N. ceranae, respectively) (Fig. 4) (MartınHernandez et al., 2007). It should be emphasised that LAMP described in this paper allows not only to identify but also to differentiate N. apis and N. ceranae infections in bees. In conclusion, the developed LAMP methods offer a good alternative to the standard PCR technique for the rapid and simple identification and differentiation of N. apis and N. ceranae infections in honeybees. It detects microsporidian DNA with high sensitivity and specificity without negative influence on amplification process of nontarget DNA copresence. The low cost of equipment required for isothermal amplification of DNA (only water bath or heating block) enables the use of LAMP for the rapid, cost-effective and accurate identification of microsporidian reservoirs in honeybees even by small field laboratories associated with apiaries. ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
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