Molecular and Cellular Probes 30 (2016) 205e210
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Application of a loop-mediated isothermal amplification (LAMP) assay for molecular identification of Trueperella pyogenes isolated from various origins A. Abdulmawjood a, *, J. Wickhorst b, O. Hashim a, O. Sammra b, A.A. Hassan b, €mmler b, E. Prenger-Berninghoff c, G. Klein a M. Alssahen b, C. La a
Institute of Food Quality and Food Safety, Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany €t Gießen, Schubertstr 81, 35392 Gießen, Germany Institut für Pharmakologie und Toxikologie, Justus-Liebig-Universita c €t, Frankfurterstr. 85-91, 35392 Gießen, Germany Institut für Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universita b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 April 2016 Received in revised form 26 May 2016 Accepted 26 May 2016 Available online 27 May 2016
In the present study 28 Trueperella pyogenes strains isolated from various origins could successfully be identified with a newly designed loop-mediated isothermal amplification (LAMP) assay based on gene cpn60 encoding chaperonin. No cross reaction could be observed with control strains representing four species of genus Trueperella and seven species of closely related genus Arcanobacterium. The present cpn60 LAMP assay might allow a reliable and low cost identification of T. pyogenes also in laboratories with less specified equipment. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Trueperella pyogenes Loop-mediated isothermal amplification LAMP Real-time fluorometer
1. Introduction Trueperella pyogenes is a worldwide known pathogen of domestic ruminants and pigs causing mastitis, abortion and a variety of pyogenic infections [1,2]. This bacterial pathogen is also able to cause diseases in a large number of animal species, including antelopes, bison, camels, chicken, deer, elephants, gazelles, horses, macaws, reindeer, turkeys and wildebeest and also in companion animals such as dogs and cats [3,4]. More recently T. pyogenes was isolated from septicemia of a bearded dragon and a gecko and from lung abscesses of one-humped camels and was further characterized phenotypically and genotypically [5,6]. The identification of T. pyogenes is usually performed by cultural and biochemical properties [7], with high resolving phenotypic techniques such as matrix assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) [8] and Fourier transform infrared spectroscopy
* Corresponding author. Institute of Food Quality and Food Safety, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany. E-mail address:
[email protected] (A. Abdulmawjood). http://dx.doi.org/10.1016/j.mcp.2016.05.003 0890-8508/© 2016 Elsevier Ltd. All rights reserved.
(FT-IR) [9], and genotypically using 16S rDNA, 16Se23S rDNA intergenic spacer region, the pyolysin encoding gene plo and the bsubunit of RNA polymerase encoding gene rpoB as molecular targets [5]. The species identity of T. pyogenes from bovine mastitis and from various other origins could also be confirmed genotypically by PCR-mediated amplification and sequencing of the superoxide dismutase A encoding gene sodA and the subsequent use of T. pyogenes sodA gene specific oligonucleotide primer [7]. Notomi et al., in 2000 [10] reported about a simple, time-saving technology for in vitro nucleic acid amplification, named loop-mediated isothermal amplification (LAMP). It is highly sensitive and specific, amplifying at a constant temperature without the need of expensive equipment like thermocycler. In addition, with less than 30 min, the LAMP reaction is faster in comparison to real-time PCR [10e12]. In the present study the chaperonin encoding gene cpn60 of T. pyogenes was used as novel target to develop a loop-mediated isothermal amplification (LAMP) assay coupled with a real-time fluorometer. This LAMP assay allowed a molecular identification of previously characterized T. pyogenes of various origins.
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Table 1 Oligonucleotide primer sequences used for T. pyogenes LAMP assay. Designation
Sequence
Primer length
Melting temp
T.pyo-F3 T.pyo-B3 T.pyo-BIF T.pyo-BIP T.pyo-LoopF T.pyo-LoopB
50 -CGTTGAGGAGTCCAACAC-30 50 -GCCACGGATCTTGTTGAC-30 50 -TGGACTCGACGAGGAGCACCGTACTTCGTTACCGAC-30 50 -ATCTCTTGCCGCTGCTCGGATGTCTTCCGCGATGATT-30 50 -TCGAGGACGACTTCCTGG-30 50 -AGAAGGTCATGCAGACCG-30
18 18 36 37 18 18
56.0 C 56.0 C 75 C 73.9 C 58.2 C 56.0 C
bp bp bp bp bp bp
2. Materials and methods
2.2. Template preparation
2.1. Bacterial strains
As described previously as rapid and simple processing step [13], a freshly cultivated bacterial colony was picked up, suspended into a tube containing 500 ml HYPLEX® LPTV buffer tube (Amplex Diagnostics, Giessen, Germany) and boiled for 10 min using a heatblock. Subsequently 3 ml of this suspension was used as template.
The strains used in the present study included the reference strains T. pyogenes DSM 20630u and T. pyogenes DSM 20594, other presently available reference strains of genus Trueperella (n ¼ 4) and Arcanobacterium (n ¼ 9) [13] and T. pyogenes field strains (n ¼ 26). The T. pyogenes field strains had been characterized phenotypically and genotypically as described [7]. The bacterial culturing of the T. pyogenes strains was carried out on blood agar base for 48 h at 37 C in a candel jar.
2.3. Design of oligonucleotide primers for LAMP assay The design of the oligonucleotide primers for the LAMP assay
Table 2 Inclusivity and exclusivity test of the T. pyogenes LAMP assay based on cpn60 gene using T. pyogenes and non- T. pyogenes strains. 1. T. pyogenes strains Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.
Strain number
pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes pyogenes
DSM 20630 DSM 20594 1908/07 1947/07 2495/07 280/09 799/09 1440/09 1489/09 P2346/09 1848/07 2089/07 5939/08 6284/08 1070/09 1071/09 1342/09 1870/07 1971/07 5144/08 2074/07 36/03 3186/02 P2847/01 142/09 6122/08 852/09 4984/03
u
Animal
Specimen
Detection time mm:ss
Melting temperature
Pig cow Cow Cow Cow Cow Cow Cow Cow Cow Pig Pig Pig Pig Pig Pig Pig Sheep Sheep Sheep Goat Mountain Reedbuck Horse Dog Dog Cat Rabbit Gecko
e Mammary gland Milk Milk Milk Urine Uterus Nostril abscess Dermoid cyst Cervix Hind leg Lung abscess Cervix Lung Ear abscess Liver Lung Kidney Skin Skin Lung Tonsils Wound Ear Vagina Anal gland Lung Intestine
14:30 14:00 13:30 14:15 21:30 18:45 14:00 21:00 14:30 14:45 13:30 18:15 13:15 19:15 19:4520:45 21:30 16:15 21:45 16:29 13:30 19:15 19:15 15:30 17:10 16:30 23:51 25:25
89.30 89.20 89.24 88.95 88.96 89.10 89.30 88.91 89.14 89.10 89.34 89.10 89.19 88.95 89.10 88.96 89.01 89.01 89.06 89.10 89.34 89.88 89.21 89.25 89.16 89.31 89.05 89.05
2. Non- T. pyogenes strains
1 2 3 4 5 6 7 8 9 10 11 12 13
Species
Strain number
Detection time mm:ss
Melting temperature
T. bonasi T. abortisuis T. bernardiae T. bialowiezensis A. haemolyticum A. hippocoleae A. canis A. phocae A. phocae A. phocisimile A. phocisimile A. pluranimalium A. pinnipediorum
DSM 17163u DSM 19515u DSM 9152u DSM 17162u DSM 20595u DSM 15539u DSM 25104u DSM 10002u DSM 10003 DSM 26142u 4112 DSM 13483u DSM 28752u
e e e e e e e e e e e e e
e e e e e e e e e e e e e
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Fig. 1. Amplification curves of the detection limit (LOD) of the LAMP assay using serial dilutions of the T. pyogenes strain DSM 20594T (top). The anneal reaction curves of the same amplicons (bottom).
was based on the sequence of gene cpn60 of T. pyogenes reference strain DSM 20630u published in the National Center for Biotechnology (NCBI) GenBank under accession no. AY691206. A set of six oligonucleotide primers, including two outer primers (forward primer Pyo-F3 and backward primer Pyo-B3), two inner primers (forward inner primer Pyo-FIP and backward inner primer Pyo-BIP) and two loop primers Pyo-LoopF and Pyo-LoopB, were designed using LAMP Designer software, ver. 1.10 (PREMIER Biosoft, Palo Alto, CA, USA) (Table 1). These primers were selected to be specific for T. pyogenes cpn60 gene. The oligonucleotide primers were synthesized by EuroFins MWG Operon (Ebersberg, Germany). For investigating the specificity the oligonucleotide sequences were submitted to the NCBI GenBank using LAMP Designer software. 2.4. LAMP reaction and amplification condition The LAMP reaction was carried out with a total volume of 25 ml
of the reaction mixture containing 0.5 ml each of Pyo-F3 and Pyo-B3 primer (25 pmol/ml) equivalent to 0.5 mM final concentration, 2.0 ml each of Pyo-FIP and Pyo-BIP primer (25 pmol/ml) equivalent to 2 mM final concentration, 1.0 ml each of Pyo-LoopF and Pyo-LoopB primer (25 pmol/ml) equivalent to 1 mM final concentration and 15 ml Isothermal Master Mix Iso-001 (Optigene, Horsham, UK). Subsequently 3 ml DNA preparation was added as a template. The LAMP assay was run at 72 C for 30 min with a melting curve analysis step (annealing curve 98 to 80 C ramping at 0.05 C per s) in a portable real-time fluorometer (Genie II®, Optigene) according to the manufacturer instructions. The LAMP reaction was also implemented in 1.5 ml tubes using a heatblock at 72 C for 30 min. The presence of LAMP products was subsequently determined by electrophoresis of 10 ml of the reaction products in a 2% agarose gel (Peqlab, Erlangen, Germany), with Trisacetate-electrophoresis buffer (TAE, pH 7.8) and a 100-bp DNA ladder (Roche Diagnostic, Mannheim, Germany) as molecular
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Fig. 2. Typical amplification signal of LAMP products of T. pyogenes field strains and a negative control (top). The anneal reaction curves of the same amplicons (bottom).
marker.
2.5. Analytical sensitivity of the LAMP assay and limit of detection (LOD) For determination of the analytic sensitivity of the LAMP assay a serially diluted DNA isolated from reference strain T. pyogenes DSM 20594u was used with the conditions mentioned above. For this test the DNA was isolated using the DNeasy blood and tissue isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Serial dilutions (101e106) were prepared using Tris buffer (TE, pH 8.0). The amount of DNA ranged from 0.79 ng/ml (101) to 0.079 pg/ml (106) bacterial DNA. The test was run in triplicate. The DNA concentration was estimated by using NanoDrop 2000c photometer (Peqlab). In addition, the LOD was estimated by preparation of serial dilutions (101-106) of reference strain T. pyogenes DSM 20594u and
strain T. pyogenes 1908/07 in HYPLEX® LPTV buffer tube (Amplex Diagnostics, Germany). The cfu/ml was subsequently estimated. The LPTV buffer containing the bacteria was boiled for 10 min using a heatblock, then 3 ml of this suspension was used as template. 2.6. Positive and negative predictive values (PPV and NPV) The PPV was calculated as: (number of true positives)/(number of true positives þ number of false positives) x 100, the NPV was calculated as: (number of true negatives)/(number of true negatives þ number of false negatives) x 100. The accuracy was calculated as: (number of true positives þ number of true negatives)/(total number of samples) 100. 3. Results and discussion A molecular identification of bacteria by using chaperonin 60
A. Abdulmawjood et al. / Molecular and Cellular Probes 30 (2016) 205e210
Fig. 3. Agarose gel electrophoresis of LAMP products of T. pyogenes DSM 20594T (1, 3) and T. pyogenes 1908/07 (2, 4). The positive reaction appeared as ladder-like pattern with the purified DNA using DNeasy tissue isolation kit (lane 1, 2), and DNA isolated with the HYPLEX® LPTV method (lane 3, 4); lane 5 represents a negative strain, lane 6 a non template control; lane M ¼ DNA Marker 50 bp ladder (Biozym Diagnostic, Oldendorf, Germany).
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homogeneity of the LAMP products, which is an important postLAMP reaction step to prove the products specificity. The melting temperature e of the T. pyogenes cpn60 specific amplicon was 89.2 C (sd ± 0.18) (Figs. 1 and 2). These results demonstrated the high specificity of this assay. No cross reaction could be observed with any of the control strains representing four additional species of genus Trueperella or seven species of genus Arcanobacterium (Table 2). The benefit of using the real-time fluorometer equipment of the present study is to avoid a cross contamination by using a closed system. In addition, the positive fluorescent results are presented on a screen and could be followed in real time, a quantitative estimation is possible and the annealing curve signal could be used as a confirmation of the specificity of the positive signals. The use of such a portable, fast and relatively cheap equipment like Genie II® allows the flexible application of this diagnostic assay in the field. However, this is not possible with other methods such as real-time PCR. The LAMP assay of all investigated strains could also be performed by using a heat block, which is available in many laboratories and subsequent separation of the LAMP products by agarose
Table 3 Limit of detection (LOD) of the LAMP assay using serial dilutions of two T. pyogenes strains. Used strains
T. pyogenes DSM 20594T T. pyogenes 1908/07
cfu/ml
7.2 107 1.2 108
Detection time (h:mm:ss) (Annealing temperature C)
LOD
101
102
103
104
105
0:14:45 (89.69) 0:13:30 (89.79)
0:17:15 (89.74) 0:16:30 (89.79)
0:21:45 (89.59) 0:19:15 (89.79)
0:24:30 (89.35) 0:22:30 (89.69)
0:29:30 (89.30) 0:25:45 (89.44)
encoding gene cpn60 has been described for identification of various gram positive bacteria [14,15] leading to the creation of a chaperonin sequence database which represents now one of the largest collections of sequences available for the protein encoding gene cpn60, also including gene cpn60 of T. pyogenes [16]. In the present study gene cpn60 of T. pyogenes was used to design six oligonucleotide primers which can be used in a LAMP assay for detection of this bacterial species. The application of LAMP assays for detection of foodborne bacterial pathogens and toxicants as well as mycotoxin producing food borne fungi has been reviewed by Niessen et al. [17]. This technique has also been used to identify Leptospira spp. [18], Erysipelothrix rhusiopathiae [19], Streptococcus equi subsp. zooepidemicus [20], for identification of ostrich meat [21] and more recently for identification of Arcanobacterium pluranimalium [13]. Zhang et al. [22] used the T. pyogenes pyolysin encoding gene plo, which is well known as a universal target for molecular identification of T. pyogenes [5,7,23,24], for development of a LAMP assay yielding a specific reaction for T. pyogenes. However, these authors used only a limited number of T. pyogenes strains and no control strains of closely related species of genus Trueperella and Arcanobacterium. The cpn60 LAMP assay of the present study provided a rapid and reliable identification of all 26 T. pyogenes field strains of various origins and both T. pyogenes reference strains (Table 2). The PPV, NPV and accuracy values of the assay with the investigated strains were 100%. This could be demonstrated using a real time fluorometer. Melting curve analysis which is termed by Genie II® as anneal curve analysis revealed no significant differences among the different T. pyogenes strains. The melting curve analysis was carried out at the end of each LAMP reaction. The amplicons were denatured at 98 followed by a gradual decrease in temperature up to 80 C. Fluorescence intensity was monitored during this final temperature decrease, resulting in the generation of a melting curve. Analysing of such a curve allows the determination of the
21.6 cfu/reaction 33.6 cfu/reaction
gel electrophoresis. To perform the LAMP assays amplicons, a simple DNA preparation by using HYPLEX® LPTV buffer yielded an amplicon quality comparable to the purified DNA isolated with DNeasy blood and tissue isolation kit (Fig. 3). The analytical sensitivity of the LAMP assay was determined on the basis of a dilution row of the bacterial DNA. The lowest amount of DNA yielding a positive was 0.79 pg/ml (st ±0.095). The LOD for this LAMP assay was 21.6 cfu/reaction (3 ml prepared DNA) for T. pyogenes DSM 20594u reference strain and 33.6 cfu/ reaction for T. pyogenes 1908/07. They were detectable after 29:30 min:s and 25:45 min:s, respectively (Table 3 and Fig. 1). These results demonstrated the high sensitivity of this assay. The melting temperature for all diluted steps was again around 89 C (Table 3 and Fig. 1). Comparable to the previously described LAMP-mediated identification of A. pluranimalium using the species-specific gene pla [13], gene cpn 60 used in the present study appeared to be a species-specific target for LAMP-mediated detection of T. pyogenes of various origins. This might allow an identification of this bacterial pathogen also in less equipped microbiological laboratories. References [1] C. L€ ammler, H. Hartwigk, Actinomyces pyogenes und Arcanobacterium haemolyticum, in: 2nd Auflage, in: H. Blobel (Ed.), T. Schließer Handbuch der bakteriellen Infektionen bei Tieren, Band II/3, Gustav Fischer Verlag, Jena, Stuttgart, Germany, 1995, pp. 196e240. [2] R. Moore, A. Miyoshi, L.G.C. Pacheco, N. Seyffert, V. Azevedo, Corynebacterium and Arcanobacterium, in: C.L. Gyles, J.F. Prescott, G. Songer, C.O. Thoen (Eds.), Pathogenesis of Bacterial Infections of Animals, fourth ed., Iowa State University Press, Ames, IA, 2010, pp. 133e147. [3] B.H. Jost, S.J. Billington, Arcanobacterium pyogenes: molecular pathogensis of an animal opportunist, Antonie Leeuwenhoek 88 (2005) 87e102. [4] S.J. Billington, K.W. Post, B.H. Jost, Isolation of Arcanobacterium (Actinomyces) pyogenes from cases of feline otitis externa and canine cystitis, J. Vet. Diagn. Invest. 14 (2002) 159e162. €mmler, A.A. Hassan, [5] H. Ülbegi-Mohyla, M. Hijazin, J. Alber, C. La
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