Molecular and Cellular Probes xxx (2014) 1e3
Contents lists available at ScienceDirect
Molecular and Cellular Probes journal homepage: www.elsevier.com/locate/ymcpr
Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus Chien-Hsun Huang a, Mu-Tzu Chang b, *, Lina Huang a a b
Bioresource Collection and Research Center, Food Industry Research and Development Institute, 331 Shih-Pin Road, Hsinchu 30062, Taiwan National Health Research Institutes, Institute of Biotechnology and Pharmaceutical Research, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 October 2013 Accepted 13 March 2014 Available online xxx
Identifying Lactobacillus species using only phenotypic and genotypic (16S rDNA sequence analysis) techniques yields inaccurate results. The objective of this study was to develop species-specific primers based on randomly amplified polymorphic DNA (RAPD) fingerprinting to distinguish species within the closely related Lactobacillus plantarum group. One of these primers, OPD-3, produced a species-specific band that was found only in the tested Lactobacillus pentosus. This specific fragment was isolated from agarose gel and ligated into a vector for DNA sequencing. A pair of primers, SpOPD3Lpen-F1/R1, that were highly specific sequence-characterized-amplified-regions (SCARs) were designed according to the nucleotide sequences of the specific RAPD marker. These primers were used for PCR analysis of the template DNA of the Lactobacillus strains, and a single 542 bp species-specific band was found only in L. pentosus. Using PCR, a novel species-specific primer pair is shown to rapidly, accurately and effectively distinguish L. pentosus from other species in the L. plantarum group of probiotic bacteria. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: RAPD fingerprinting Species identification Species-specific PCR Lactobacillus pentosus
The current taxonomy of the Lactobacillus plantarum group comprises five closely related species: L. plantarum (subsp. plantarum and subsp. argentoratensis), Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus fabifermentans and Lactobacillus xiangfangensis [1]. Some strains of L. plantarum and L. pentosus have been shown to be probiotic [2e5] and are widely used in commercially fermented products. The conventional methods of distinguishing Lactobacillus strains depend on phenotypic tests such as morphological and biochemical analyses. Many researchers have relied on API 50 CH carbohydrate fermentation strips to identify Lactobacillus strains [6e8]. However, this commercial identification kit may not always reliably distinguish closely related Lactobacillus species [9]. A comparative analysis of 16S ribosomal RNA sequences is the most commonly used genotypic method for bacterial identification [10]. Generally, strains that show at least 97% sequence similarity in their 16S rRNA sequences are considered the same species [11,12]. Unfortunately, a high degree of similarity (reaching 98.9e99.9%) has been observed for the 16S rRNA gene sequences among the L. plantarum group strains. Therefore, different genotypic technology with rapidity, accuracy and low cost
* Corresponding author. E-mail address: [email protected] (M.-T. Chang).
is required for differentiating L. plantarum group bacteria. Speciesspecific primers are effective for PCR-based identification involving a small number of species or a particular species. This method uses specific primers that target highly variable regions of universal genes. The species-specific PCR (SS-PCR) technique has been applied for the rapid and accurate identification of species within the L. plantarum group [13]. However, the species-specific primers mainly target rDNA genes or intergenic spacer regions; primers specific for a select species can only be developed if there are known gene sequences that contain sufficient species-specific nucleotide variation. In this study, we describe novel speciesspecific DNA sequences cloned from L. pentosus by RAPD fingerprinting analysis and a pair of primers that we developed based on cloned sequences that can be used for identifying species within the L. plantarum group. One hundred random primers from RAPD 10mer kits of OPA, OPB, OPC, OPD, OPE and OPF were used (Operon Alameda, CA, USA) in PCR amplifications that were performed in 15 mL volumes containing 100 mM TriseHCl pH 8.0, 1.5 mM MgCl2, 50 mM KCl, 10 mM dNTPs, 0.14 mM of primer, 100 ng of template DNA, 0.5 U DyNAzyme (Finnzymes Oy., Keilaranta, Espoo, Finland) and sterile deionized water. The thermal protocol was performed as described previously [14]. The species-specific fragments were recovered from an agarose gel using the QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, CA, USA) and then cloned into the pCR2.1-TOPO
http://dx.doi.org/10.1016/j.mcp.2014.03.003 0890-8508/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Huang C-H, et al., Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus, Molecular and Cellular Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.03.003
2
C.-H. Huang et al. / Molecular and Cellular Probes xxx (2014) 1e3
Fig. 1. RAPD fingerprints of Lactobacillus plantarum group strains. (A) Genomic DNA samples were amplified with random primers (OPD-3). The L. pentosus specific fragment is indicated by the white frame. Lanes: M, 100 bp DNA ladder markers; 1, L. pentosus BCRC 11053T; 2, L. pentosus BCRC 12250; 3, L. pentosus BCRC 12944; 4, L. plantarum subsp. plantarum BCRC 10069T; 5, L. plantarum subsp. argentoratensis.
vector using the TOPO Cloning Kit (Invitrogen, San Diego, CA, USA). The cloned DNA fragment was then sequenced using a BigDye Terminator v3.1 cycle-sequencing kit on a 3730xl DNA Analyzer (Applied Biosystems and Hitachi, Foster City, CA, USA). The DNA sequences were assembled using the commercial computer program Vector NTI Version 9.0 (Invitrogen, USA). A pair of speciesspecific primers, SpOPD3Lpen-F1/R1, were designed from the cloned sequence using GCG sequence analysis software (Genetic Computer Group, WI, USA). The thermal protocol of above primer pair using the following condition: initial strand denaturation at 94 C for 5 min; then 20 cycles of 94 C for 1 min, 65 C for 1 min and 72 C for 1.5 min; and a final extension step at 72 C for 7 min. The RAPD process uses PCR with random oligonucleotide primers to amplify genomic DNA and analyze sequence polymorphisms. RAPD is easy to perform, is cost-effective, does not require prior sequence knowledge and requires only a small amount of template genomic DNA [15]. The RAPD method has become widely accepted as a valid taxonomic and phylogenetic tool for a large range of organisms, including lactobacilli [14,16e18].
Fig. 2. Species-specific DNA sequence of Lactobacillus pentosus. The species-specific primers (shaded) were designed according to the cloned sequence.
Fig. 3. PCR products amplified from genomic DNA of L. plantarum group strains. Template DNA was amplified with the species-specific primer pair (542 bp). Lanes: M, 100 bp DNA ladder markers; 1, L. pentosus BCRC 11053T; 2, L. pentosus BCRC 12250; 3, L. pentosus BCRC 12944; 4, L. plantarum subsp. plantarum BCRC 10069T; 5, L. plantarum subsp. argentoratensis.
However, RAPD reproducibility is variable and the process is sensitive to reaction conditions, including template DNA quality, ramping speed and the type of instrument used. Therefore, it is more efficient to clone the RAPD markers and convert them into sequence characterized amplified regions (SCARs). Long complementary primers permit a more stringent annealing temperature than that used with the RAPD analysis and typically results in the amplification of a single locus [19].The template DNA of the Lactobacillus strains was amplified with the OPD-3 primer (50 Table 1 Strains used in this study and their detection using primer pair. No.
Species
BCRC
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 29 30 31 Others
10069T 10357 12251 14059 15478 17639 18858 80222 80577 80578 80580 80581 Silage Lactobacillus plantarum 17638T subsp. argentoratensis 17640 12327 Lactobacillus paraplantarum 17178T 17971 17970 Lactobacillus pentosus 11053T 12250 12944 15317 17972 17987 17989 17973 80017 80118 Lactobacillus fabifermentans 18841T Lactobacillus xiangfangensis 80512T Lactobacillus casei BCRC 10697T, Lactobacillus paracasei BCRC 12248T, Lactobacillus rhamnosus BCRC 10940T, Lactobacillus sakei BCRC 14622T, Lactobacillus curvatus BCRC 12189T, Lactobacillus graminis BCRC 17898T, Lactobacillus fuchuensis BCRC 80305T
SS-PCR SpOPD3Lpen-F1/R1
Lactobacillus plantarum subsp. plantarum
þ þ þ þ þ þ þ þ þ þ
BCRC, Bioresource Collection and Research Center at Food Industry Research and Development Institute, Taiwan, ROC. þ, PCR amplicons with each primer pair detected; , PCR amplicons with each primer pair not detected.
Please cite this article in press as: Huang C-H, et al., Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus, Molecular and Cellular Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.03.003
C.-H. Huang et al. / Molecular and Cellular Probes xxx (2014) 1e3
GTCGCCGTCA-30 ) by RAPD-PCR. This amplification produced a species-specific band specific for L. pentosus (Fig. 1). This specific fragment was then isolated and ligated into the pCR2.1-TOPO vector for nucleotide sequencing. The cloned DNA was sequenced (Fig. 2), entered into the GenBank database and aligned to homologs using the BLAST analysis. These sequences showed high congruence with the ABC transporter, an ATP-binding and permease protein of L. pentosus, which is known to play a critical role in multidrug resistance (MDR). From this, we deduced that these cloned sequences likely exhibit high variation, which would make them ideal templates for designing specific primers to discriminate among species within the L. plantarum group. A primer pair, SpOPD3Lpen-F1/R1, was designed from the cloned sequences, and these primers successfully generated a single species-specific band when used in PCR reactions with L. pentosus DNA (Fig. 3). Annealing temperatures may influence PCR specificity, and determining the optimal annealing temperature is a time consuming process [20]. Lower annealing temperatures and additional PCR amplification cycles may lead to non-specific PCR products. Thus, either a high annealing temperature or a short annealing time should be used. In the present study, we found that the most appropriate annealing temperature for SpOPD3Lpen-F1/ R1was 65 C, with 20 cycles of PCR amplification. Moreover, to confirm the species specificity of this set of primers, 34 Lactobacillus strains were tested and demonstrated accuracies reaching 100% (Table 1). In conclusion, we have developed a pair of species-specific primers based on RAPD fingerprinting. These novel speciesspecific primers can be a rapid, cost-effective, accurate and reliable way to distinguish the L. pentosus species from the L. plantarum group. Acknowledgments This research was supported by National Science Council, ROC by Ministry of Economic Affairs, ROC (project no. 102-EC-17-A-0104-0525). References [1] Gu CT, Wang F, Li CY, Liu F, Huo GC. Lactobacillus xiangfangensis sp. nov., isolated from Chinese pickle. Int J Syst Evol Microbiol 2012;62:860e3.
3
[2] Merry RJ, Dhanoa MS, Theodorou MK. Use of freshly cultured lactic acid bacteria as silage inoculants. Grass For Sci 1995;50:112e23. [3] Oneca M, Irigoyen A, Ortigosa M, Torre P. PCR and RAPD identification of L. plantarum strains isolated from ovine milk and cheese. Geographical distribution of strains. FEMS Microbiol Lett 2003;227:271e7. [4] Ruiz-Barba JL, Cathcart DP, Warner PJ, Jimenez-Diaz R. Use of Lactobacillus plantarum LPCO10, a bacteriocin producer, as a starter culture in Spanish-style green olive fermentations. Appl Environ Microbiol 1994;60:2059e64. [5] Urlings HA, Bijker PG, van Logtestijn JG. Fermentation of raw poultry byproducts for animal nutrition. J Anim Sci 1993;71:2420e6. [6] Boyd MA, Antonio MAD, Hillier SL. Comparison of API 50 CH strips to wholechromosomal DNA probes for identification of Lactobacillus species. J Clin Microbiol 2005;43:5309e11. [7] Bringel F, Castioni A, Olukoya DK, Felis GE, Torriani S, Dellaglio F. Lactobacillus plantarum subsp. argentoratensis subsp. nov., isolated from vegetable matrices. Int J Syst Evol Microbiol 2005;55:1629e34. [8] De Bruyne K, Camu N, De Vuyst L, Vandamme P. Lactobacillus fabifermentans sp. nov. and Lactobacillus cacaonum sp. nov., isolated from Ghanaian cocoa fermentations. Int J Syst Evol Microbiol 2009;59:7e12. [9] Johansson ML, Quednau M, Ahrne S, Molin G. Classification of lactobacillus plantarum by restriction endonuclease analysis of total chromosomal DNA using conventional agarose gel electrophoresis. Int J Syst Bacteriol 1995;45: 670e5. [10] Petti CA. Detection and identification of microorganisms by gene amplification and sequencing. Clin Infect Dis 2007;44:1108e14. [11] McCartney AL. Application of molecular biological methods for studying probiotics and the gut flora. Br J Nutr 2002;88:S29e37. [12] Vandamme P, Pot B, Gillis M, Devos P, Kersters K, Swings J. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 1996;60:407e38. [13] Torriani S, Felis GE, Dellaglio F. Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers. Appl Environ Microbiol 2001;67: 3450e4. [14] Huang CC, Lee FL. Development of novel species-specific primers for species identification of the Lactobacillus casei group based on the RAPD fingerprints. J Sci Food Agric 2009;89:1831e7. [15] Williams JG, Hanafey MK, Rafalski JA, Tingey SV. Genetic analysis using random amplified polymorphic DNA markers. Meth Enzymol 1993;218:704e 40. [16] Huang CH, Lee FL. The dnaK gene as a molecular marker for the classification and discrimination of the Lactobacillus casei group. Antonie Van Leeuwenhoek 2011;99:319e27. [17] Huang CC, Lee FL, Liou JS. Rapid discrimination and classification of the Lactobacillus plantarum group based on a partial dnaK sequence and DNA fingerprinting techniques. Antonie Van Leeuwenhoek 2010;97:289e96. [18] Schillinger U, Yousif NM, Sesar L, Franz CM. Use of group-specific and RAPDPCR analyses for rapid differentiation of Lactobacillus strains from probiotic yogurts. Curr Microbiol 2003;47:453e6. [19] Cushwa WT, Medrano JF. Applications of the random amplified polymorphic DNA (RAPD) assay for genetic analysis of livestock species. Anim Biotechnol 1996;7:11e31. [20] Rychlik W, Spencer WJ, Rhoads RE. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res 1990;18:6409e12.
Please cite this article in press as: Huang C-H, et al., Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus, Molecular and Cellular Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.03.003
Contents lists available at ScienceDirect
Molecular and Cellular Probes journal homepage: www.elsevier.com/locate/ymcpr
Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus Chien-Hsun Huang a, Mu-Tzu Chang b, *, Lina Huang a a b
Bioresource Collection and Research Center, Food Industry Research and Development Institute, 331 Shih-Pin Road, Hsinchu 30062, Taiwan National Health Research Institutes, Institute of Biotechnology and Pharmaceutical Research, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 October 2013 Accepted 13 March 2014 Available online xxx
Identifying Lactobacillus species using only phenotypic and genotypic (16S rDNA sequence analysis) techniques yields inaccurate results. The objective of this study was to develop species-specific primers based on randomly amplified polymorphic DNA (RAPD) fingerprinting to distinguish species within the closely related Lactobacillus plantarum group. One of these primers, OPD-3, produced a species-specific band that was found only in the tested Lactobacillus pentosus. This specific fragment was isolated from agarose gel and ligated into a vector for DNA sequencing. A pair of primers, SpOPD3Lpen-F1/R1, that were highly specific sequence-characterized-amplified-regions (SCARs) were designed according to the nucleotide sequences of the specific RAPD marker. These primers were used for PCR analysis of the template DNA of the Lactobacillus strains, and a single 542 bp species-specific band was found only in L. pentosus. Using PCR, a novel species-specific primer pair is shown to rapidly, accurately and effectively distinguish L. pentosus from other species in the L. plantarum group of probiotic bacteria. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: RAPD fingerprinting Species identification Species-specific PCR Lactobacillus pentosus
The current taxonomy of the Lactobacillus plantarum group comprises five closely related species: L. plantarum (subsp. plantarum and subsp. argentoratensis), Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus fabifermentans and Lactobacillus xiangfangensis [1]. Some strains of L. plantarum and L. pentosus have been shown to be probiotic [2e5] and are widely used in commercially fermented products. The conventional methods of distinguishing Lactobacillus strains depend on phenotypic tests such as morphological and biochemical analyses. Many researchers have relied on API 50 CH carbohydrate fermentation strips to identify Lactobacillus strains [6e8]. However, this commercial identification kit may not always reliably distinguish closely related Lactobacillus species [9]. A comparative analysis of 16S ribosomal RNA sequences is the most commonly used genotypic method for bacterial identification [10]. Generally, strains that show at least 97% sequence similarity in their 16S rRNA sequences are considered the same species [11,12]. Unfortunately, a high degree of similarity (reaching 98.9e99.9%) has been observed for the 16S rRNA gene sequences among the L. plantarum group strains. Therefore, different genotypic technology with rapidity, accuracy and low cost
* Corresponding author. E-mail address: [email protected] (M.-T. Chang).
is required for differentiating L. plantarum group bacteria. Speciesspecific primers are effective for PCR-based identification involving a small number of species or a particular species. This method uses specific primers that target highly variable regions of universal genes. The species-specific PCR (SS-PCR) technique has been applied for the rapid and accurate identification of species within the L. plantarum group [13]. However, the species-specific primers mainly target rDNA genes or intergenic spacer regions; primers specific for a select species can only be developed if there are known gene sequences that contain sufficient species-specific nucleotide variation. In this study, we describe novel speciesspecific DNA sequences cloned from L. pentosus by RAPD fingerprinting analysis and a pair of primers that we developed based on cloned sequences that can be used for identifying species within the L. plantarum group. One hundred random primers from RAPD 10mer kits of OPA, OPB, OPC, OPD, OPE and OPF were used (Operon Alameda, CA, USA) in PCR amplifications that were performed in 15 mL volumes containing 100 mM TriseHCl pH 8.0, 1.5 mM MgCl2, 50 mM KCl, 10 mM dNTPs, 0.14 mM of primer, 100 ng of template DNA, 0.5 U DyNAzyme (Finnzymes Oy., Keilaranta, Espoo, Finland) and sterile deionized water. The thermal protocol was performed as described previously [14]. The species-specific fragments were recovered from an agarose gel using the QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, CA, USA) and then cloned into the pCR2.1-TOPO
http://dx.doi.org/10.1016/j.mcp.2014.03.003 0890-8508/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Huang C-H, et al., Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus, Molecular and Cellular Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.03.003
2
C.-H. Huang et al. / Molecular and Cellular Probes xxx (2014) 1e3
Fig. 1. RAPD fingerprints of Lactobacillus plantarum group strains. (A) Genomic DNA samples were amplified with random primers (OPD-3). The L. pentosus specific fragment is indicated by the white frame. Lanes: M, 100 bp DNA ladder markers; 1, L. pentosus BCRC 11053T; 2, L. pentosus BCRC 12250; 3, L. pentosus BCRC 12944; 4, L. plantarum subsp. plantarum BCRC 10069T; 5, L. plantarum subsp. argentoratensis.
vector using the TOPO Cloning Kit (Invitrogen, San Diego, CA, USA). The cloned DNA fragment was then sequenced using a BigDye Terminator v3.1 cycle-sequencing kit on a 3730xl DNA Analyzer (Applied Biosystems and Hitachi, Foster City, CA, USA). The DNA sequences were assembled using the commercial computer program Vector NTI Version 9.0 (Invitrogen, USA). A pair of speciesspecific primers, SpOPD3Lpen-F1/R1, were designed from the cloned sequence using GCG sequence analysis software (Genetic Computer Group, WI, USA). The thermal protocol of above primer pair using the following condition: initial strand denaturation at 94 C for 5 min; then 20 cycles of 94 C for 1 min, 65 C for 1 min and 72 C for 1.5 min; and a final extension step at 72 C for 7 min. The RAPD process uses PCR with random oligonucleotide primers to amplify genomic DNA and analyze sequence polymorphisms. RAPD is easy to perform, is cost-effective, does not require prior sequence knowledge and requires only a small amount of template genomic DNA [15]. The RAPD method has become widely accepted as a valid taxonomic and phylogenetic tool for a large range of organisms, including lactobacilli [14,16e18].
Fig. 2. Species-specific DNA sequence of Lactobacillus pentosus. The species-specific primers (shaded) were designed according to the cloned sequence.
Fig. 3. PCR products amplified from genomic DNA of L. plantarum group strains. Template DNA was amplified with the species-specific primer pair (542 bp). Lanes: M, 100 bp DNA ladder markers; 1, L. pentosus BCRC 11053T; 2, L. pentosus BCRC 12250; 3, L. pentosus BCRC 12944; 4, L. plantarum subsp. plantarum BCRC 10069T; 5, L. plantarum subsp. argentoratensis.
However, RAPD reproducibility is variable and the process is sensitive to reaction conditions, including template DNA quality, ramping speed and the type of instrument used. Therefore, it is more efficient to clone the RAPD markers and convert them into sequence characterized amplified regions (SCARs). Long complementary primers permit a more stringent annealing temperature than that used with the RAPD analysis and typically results in the amplification of a single locus [19].The template DNA of the Lactobacillus strains was amplified with the OPD-3 primer (50 Table 1 Strains used in this study and their detection using primer pair. No.
Species
BCRC
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 29 30 31 Others
10069T 10357 12251 14059 15478 17639 18858 80222 80577 80578 80580 80581 Silage Lactobacillus plantarum 17638T subsp. argentoratensis 17640 12327 Lactobacillus paraplantarum 17178T 17971 17970 Lactobacillus pentosus 11053T 12250 12944 15317 17972 17987 17989 17973 80017 80118 Lactobacillus fabifermentans 18841T Lactobacillus xiangfangensis 80512T Lactobacillus casei BCRC 10697T, Lactobacillus paracasei BCRC 12248T, Lactobacillus rhamnosus BCRC 10940T, Lactobacillus sakei BCRC 14622T, Lactobacillus curvatus BCRC 12189T, Lactobacillus graminis BCRC 17898T, Lactobacillus fuchuensis BCRC 80305T
SS-PCR SpOPD3Lpen-F1/R1
Lactobacillus plantarum subsp. plantarum
þ þ þ þ þ þ þ þ þ þ
BCRC, Bioresource Collection and Research Center at Food Industry Research and Development Institute, Taiwan, ROC. þ, PCR amplicons with each primer pair detected; , PCR amplicons with each primer pair not detected.
Please cite this article in press as: Huang C-H, et al., Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus, Molecular and Cellular Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.03.003
C.-H. Huang et al. / Molecular and Cellular Probes xxx (2014) 1e3
GTCGCCGTCA-30 ) by RAPD-PCR. This amplification produced a species-specific band specific for L. pentosus (Fig. 1). This specific fragment was then isolated and ligated into the pCR2.1-TOPO vector for nucleotide sequencing. The cloned DNA was sequenced (Fig. 2), entered into the GenBank database and aligned to homologs using the BLAST analysis. These sequences showed high congruence with the ABC transporter, an ATP-binding and permease protein of L. pentosus, which is known to play a critical role in multidrug resistance (MDR). From this, we deduced that these cloned sequences likely exhibit high variation, which would make them ideal templates for designing specific primers to discriminate among species within the L. plantarum group. A primer pair, SpOPD3Lpen-F1/R1, was designed from the cloned sequences, and these primers successfully generated a single species-specific band when used in PCR reactions with L. pentosus DNA (Fig. 3). Annealing temperatures may influence PCR specificity, and determining the optimal annealing temperature is a time consuming process [20]. Lower annealing temperatures and additional PCR amplification cycles may lead to non-specific PCR products. Thus, either a high annealing temperature or a short annealing time should be used. In the present study, we found that the most appropriate annealing temperature for SpOPD3Lpen-F1/ R1was 65 C, with 20 cycles of PCR amplification. Moreover, to confirm the species specificity of this set of primers, 34 Lactobacillus strains were tested and demonstrated accuracies reaching 100% (Table 1). In conclusion, we have developed a pair of species-specific primers based on RAPD fingerprinting. These novel speciesspecific primers can be a rapid, cost-effective, accurate and reliable way to distinguish the L. pentosus species from the L. plantarum group. Acknowledgments This research was supported by National Science Council, ROC by Ministry of Economic Affairs, ROC (project no. 102-EC-17-A-0104-0525). References [1] Gu CT, Wang F, Li CY, Liu F, Huo GC. Lactobacillus xiangfangensis sp. nov., isolated from Chinese pickle. Int J Syst Evol Microbiol 2012;62:860e3.
3
[2] Merry RJ, Dhanoa MS, Theodorou MK. Use of freshly cultured lactic acid bacteria as silage inoculants. Grass For Sci 1995;50:112e23. [3] Oneca M, Irigoyen A, Ortigosa M, Torre P. PCR and RAPD identification of L. plantarum strains isolated from ovine milk and cheese. Geographical distribution of strains. FEMS Microbiol Lett 2003;227:271e7. [4] Ruiz-Barba JL, Cathcart DP, Warner PJ, Jimenez-Diaz R. Use of Lactobacillus plantarum LPCO10, a bacteriocin producer, as a starter culture in Spanish-style green olive fermentations. Appl Environ Microbiol 1994;60:2059e64. [5] Urlings HA, Bijker PG, van Logtestijn JG. Fermentation of raw poultry byproducts for animal nutrition. J Anim Sci 1993;71:2420e6. [6] Boyd MA, Antonio MAD, Hillier SL. Comparison of API 50 CH strips to wholechromosomal DNA probes for identification of Lactobacillus species. J Clin Microbiol 2005;43:5309e11. [7] Bringel F, Castioni A, Olukoya DK, Felis GE, Torriani S, Dellaglio F. Lactobacillus plantarum subsp. argentoratensis subsp. nov., isolated from vegetable matrices. Int J Syst Evol Microbiol 2005;55:1629e34. [8] De Bruyne K, Camu N, De Vuyst L, Vandamme P. Lactobacillus fabifermentans sp. nov. and Lactobacillus cacaonum sp. nov., isolated from Ghanaian cocoa fermentations. Int J Syst Evol Microbiol 2009;59:7e12. [9] Johansson ML, Quednau M, Ahrne S, Molin G. Classification of lactobacillus plantarum by restriction endonuclease analysis of total chromosomal DNA using conventional agarose gel electrophoresis. Int J Syst Bacteriol 1995;45: 670e5. [10] Petti CA. Detection and identification of microorganisms by gene amplification and sequencing. Clin Infect Dis 2007;44:1108e14. [11] McCartney AL. Application of molecular biological methods for studying probiotics and the gut flora. Br J Nutr 2002;88:S29e37. [12] Vandamme P, Pot B, Gillis M, Devos P, Kersters K, Swings J. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 1996;60:407e38. [13] Torriani S, Felis GE, Dellaglio F. Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers. Appl Environ Microbiol 2001;67: 3450e4. [14] Huang CC, Lee FL. Development of novel species-specific primers for species identification of the Lactobacillus casei group based on the RAPD fingerprints. J Sci Food Agric 2009;89:1831e7. [15] Williams JG, Hanafey MK, Rafalski JA, Tingey SV. Genetic analysis using random amplified polymorphic DNA markers. Meth Enzymol 1993;218:704e 40. [16] Huang CH, Lee FL. The dnaK gene as a molecular marker for the classification and discrimination of the Lactobacillus casei group. Antonie Van Leeuwenhoek 2011;99:319e27. [17] Huang CC, Lee FL, Liou JS. Rapid discrimination and classification of the Lactobacillus plantarum group based on a partial dnaK sequence and DNA fingerprinting techniques. Antonie Van Leeuwenhoek 2010;97:289e96. [18] Schillinger U, Yousif NM, Sesar L, Franz CM. Use of group-specific and RAPDPCR analyses for rapid differentiation of Lactobacillus strains from probiotic yogurts. Curr Microbiol 2003;47:453e6. [19] Cushwa WT, Medrano JF. Applications of the random amplified polymorphic DNA (RAPD) assay for genetic analysis of livestock species. Anim Biotechnol 1996;7:11e31. [20] Rychlik W, Spencer WJ, Rhoads RE. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res 1990;18:6409e12.
Please cite this article in press as: Huang C-H, et al., Cloning of a novel specific SCAR marker for species identification in Lactobacillus pentosus, Molecular and Cellular Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.03.003
