Integrated polymerase chain reaction-based procedures for the detection and identification of species and subspecies of the Gram-positive bacterial genus Lactococcus

Pu, Zhao-Yan; Dobos, Marian; Limsowtin, G. K. Y.; Powell, Ian B.

doi:10.1046/j.1365-2672.2002.01688.x

Cited by 92 publications

(80 citation statements)

References 31 publications

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“…PCR-based assays for Lactococcus garvieae include amplification of the 16S ribosomal RNA gene (Zlotkin et al 1998, Pu et al 2002 and dihydropteroate synthase gene (Aoki et al 2000). However, conventional PCR requires using expensive equipment and other costly materials; electrophoresis must also be performed to detect the amplicon.…”

Section: Discussionmentioning

confidence: 99%

“…However, similar clinical signs are also found in Streptococcus sp. infections (Eldar & Ghittino 1999).Molecular detection methods of Lactococcus garvieae have been developed, including polymerase chain reaction (PCR) (Zlotkin et al 1998, Aoki et al 2000, Pu et al 2002 and 16S rRNA and sodA sequence analysis (Fihman et al 2006). However, although considered rapid and specific methods for detection, these require expensive equipment and other costly materials (Gunimaladevi et al 2005).…”

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confidence: 99%

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Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Tsai¹,

Wang²,

Yoshida³

et al. 2013

Dis. Aquat. Org.

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Based on use of a loop-mediated isothermal amplification (LAMP) identification protocol, this study attempted to detect Lactococcus garvieae in fish by using primer sets designed from an L. garvieae alpha/beta fold family hydrolase gene. Reaction time and temperatures were optimized for 60 min at 60°C with the resulting amplicons visualized by adding SYBR Green I to the reaction tube. The assay specificity was assessed using 45 different bacterial strains. Positive results were observed in all 30 L. garvieae isolates from various aquatic animals. No false-positive results were observed in 15 non-L. garvieae strains. The detection limit of the LAMP assay was 10-fold more sensitive than the conventional polymerase chain reaction (PCR) targeting 16S rDNA when using purified L. garvieae DNA. The detection limit of the LAMP assay was approximately 300 colony-forming units (CFU) using crude bacterial lysates, 100-fold more sensitive than PCR. Furthermore, L. garvieae in spleen, kidney and brain of experimentally challenged tilapia and grey mullet were detected using this optimized LAMP assay. Results of this study demonstrate the effectiveness of LAMP in providing a rapid yet simple test for detecting L. garvieae in fish.KEY WORDS: Lactococcosis · Alpha/beta fold family · Hydrolase gene · Loop-mediated isothermal amplification · Polymerase chain reaction · SYBR Green I Resale or republication not permitted without written consent of the publisherDis Aquat Org 102: [225][226][227][228][229][230][231][232][233][234][235] 2013 swimming. However, similar clinical signs are also found in Streptococcus sp. infections (Eldar & Ghittino 1999).Molecular detection methods of Lactococcus garvieae have been developed, including polymerase chain reaction (PCR) (Zlotkin et al. 1998, Aoki et al. 2000, Pu et al. 2002 and 16S rRNA and sodA sequence analysis (Fihman et al. 2006). However, although considered rapid and specific methods for detection, these require expensive equipment and other costly materials (Gunimaladevi et al. 2005).A method of amplifying nucleic acid sequences with high specificity, sensitivity and rapidity under isothermal conditions (60 to 65°C), loop-mediated isothermal amplification (LAMP) has been developed more recently (Notomi et al. 2000) and applied to fish pathogen detection (Caipang et al. 2004, Savan et al. 2004, Yeh et al. 2005, Itano et al. 2006. The assay uses a set of 4 or 6 primers that create a targetspecific stem loop DNA structure during initial amplification steps; subsequent LAMP auto-cycling is performed by the Bst DNA polymerase large fragment (Notomi et al. 2000). Since it does not require complicated equipment, the LAMP method provides a cost-effective, simple, and rapid (producing a result within 60 min) DNA amplification method.Recently, a fosmid library of Lactococcus garvieae from grey mullet has been constructed and many new genes of L. garvieae were cloned (M. A. Tsai et al. unpubl. data). The clone flg08-6 contained an alpha/beta fold family hydrolase gene that is also...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Tsai¹,

Wang²,

Yoshida³

et al. 2013

Dis. Aquat. Org.

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“…cremoris and L. lactis subsp. lactis have been discriminated before by using the sequence of the 16S rRNA gene (23,36). These authors used a restriction enzyme to visually recognize the differences.…”

Section: Vol 76 2010 Identification Of Lactococcus By Maldi-tof Ms mentioning

confidence: 99%

Identification and Typing of Lactococcus lactis by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Tanigawa

Kawabata

Watanabe

2010

Appl Environ Microbiol

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Currently, the genus Lactococcus is classified into six species: Lactococcus chungangensis, L. garvieae, L. lactis, L. piscium, L. plantarum, and L. raffinolactis. Among these six species, L. lactis is especially important because of its use in the manufacture of probiotic dairy products. L. lactis consists of three subspecies: L. lactis subsp. cremoris, L. lactis subsp. hordniae, and L. lactis subsp. lactis. However, these subspecies have not yet been reliably discriminated. To date, mainly phenotypic identification has been used, with a few genotypic identifications. We discriminated species or subspecies in the genus Lactococcus not only by proteomics identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) but also by phenotypic and genotypic identification. The proteomics identification using differences in the mass spectra of ribosomal proteins was nearly identical to that by genotypic identification (i.e., by analyses of 16S rRNA and recA gene sequences and amplified fragment length polymorphism). The three ribosomal subunits 30S/L31, 50S/L31, and 50S/L35 were the best markers for discriminating L. lactis subsp. cremoris from L. lactis subsp. lactis. Proteomics identification using MALDI-TOF MS was therefore a powerful method for discriminating and identifying these bacteria. In addition, this method was faster and more reliable than others that we examined.Lactococci are lactic acid bacteria (LAB) that are important contributors to the production of fermented dairy products, and some species produce antimicrobial compounds. Most species in the genus Lactococcus have been isolated from foodrelated sources and plants and are generally regarded as safe. Probiotic foods use these LAB, and there have been various studies of the relationship between these foods and the maintenance of human intestinal health (32). Lactococcus was first established as a genus distinct from the genus Streptococcus in 1985 (29).Currently, six species and three subspecies in the genus Lactococcus have been validated. Lactococcus plantarum has been isolated mainly from plants; L. garvieae has been isolated from fish, animals, and milk, and L. piscium has been isolated from salmon. Lactococcus lactis is most commonly found in raw milk, cheese, and other dairy products; L. raffinolactis has been found in raw milk and cheese, and L. chunagangensis has been isolated from wastewater. Among the six species, L. lactis is considered one of the most important in food production because it is used to manufacture fermented milk, butter, and cheese. Because of this importance, the whole genomes of three strains of L. lactis-L. lactis subsp. cremoris SK11 (10) lactis. However, this classification was based on only a few phenotypic characteristics and is considered imperfect because of its inherent disadvantages of sensitivity to culture conditions or bacterial growth phase. Discriminating between L. lactis subsp. cremoris and L. lactis subsp. lactis is particularly difficult but is very imp...

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“…During the last decades, lots of approaches have been introduced under culture independent molecular methods which are frequently used in the identification and characterization purposes of L. lactis such as Culture-Independent Polymerase Chain Reaction (Salbi et al, 2014;) 16S rDNA Sequencing (Aquilanti et al, 2007;Khemariya et al, 2013b), PCR-Denaturing Gradient Gel Electrophoresis (PDGE) (Mrkonjić Fuka et al, 2010;Pogačić et al, 2010;Marui et al, 2015;), PCR-Temporal Temperature Gradient Electrophoresis (TTGE) (ElBaradei et al, 2008), Single-Strand Conformation Polymorphism-PCR (SSCP-PCR) (Saubusse et al, 2007), Real Time PCR (qPCR) (Grattepanche et al, 2005;Ruggirello et al, 2014), Fluorescence in situ Hybridization (FISH) (Miks-Krajnik and Babuchowski, 2014), Amplified Ribosomal DNA Restriction Analysis (Partial ARDRA) (Delgado and Mayo, 2003), Length Heterogeneity PCR (LH-PCR) (Brusetti et al, 2006), Multilocus Sequence Analysis (MLSA) (Rademarker et al, 2007;Khemariya et al, 2012) (GTG) 5 -PCR Fingerprinting (Rademarker et al, 2007;Khemariya et al, 2014), PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) (Deveau et al, 2003;Khemariya et al, 2013b), Multiple Locus Minisatelite Typing (MLVA) (Que'ne'e et al, 2005), RAPD-PCR (Randomly Amplified Polymorphic DNA-PCR) (Samaržija et al, 2002), Pulse Field Gel Electrophoresis (PFGE) (Campo et al, 2002), Repetitive Element Sequence based PCR (Rep-PCR) (Fernandez et al, 2010), Comparative Genomic Hybridization (CGH) (Taibi et al, 2010), Nested PCR (Khemariya et al, 2013c), Multiplex PCR (Pu et al, 2002) and Sau-PCR (Corich et al, 2005).…”

Section: Enumeration Of L Lactismentioning

confidence: 99%

Probiotic Lactococcus lactis: A Review

Khemariya

Singh

Nath

et al. 2017

Turkish JAF Sci.Tech.

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Lactococcus lactis plays a critical role in food, dairy and health sectors. In food and dairy industries, it is found in production processes of various fermented products such as sausages, pickled vegetables, beverages such as beer and wine, breads, soymilk kefir, sour milk, butter, cream, fresh cheese and different types of cheeses, like Cheddar, Colby, Cottage cheese, Camembert, cream cheese, Roquefort and Brie. Additionally, there is an increasing interest towards the possible health benefits of the probiotic activity of this organism which generally is species and strain specific and depends upon the survival in gastrointestinal tract with sufficient number. Certain strains have the ability to produce antimicrobial peptide called nisin which exhibits preservative potential. Therefore, application of bacteriocinogenic Lactococcus lactis in food and dairy sectors to preserve foods as a natural way and contributing health promoting attributes due to probiotic activity would definitely fulfil today's consumer demands. This paper aimed to review the adaptation, antibiotic resistance, therapeutic and preservation potential of bacteriocinogenic and probiotic Lactococcus lactis.

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Integrated polymerase chain reaction-based procedures for the detection and identification of species and subspecies of the Gram-positive bacterial genus Lactococcus

Cited by 92 publications

References 31 publications

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Identification and Typing of Lactococcus lactis by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Probiotic Lactococcus lactis: A Review

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