Use of high-resolution melting curve analysis to identify <i>Mycoplasma</i> species commonly isolated from ruminant, avian, and canine samples

Rebelo, Ana Rita; Parker, Lois; Cai, Hugh Y.

doi:10.1177/1040638711416846

Cited by 24 publications

(16 citation statements)

References 11 publications

(34 reference statements)

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“…A method reported by Boonyayatra et al (2012a) used amplification of 16S rDNA by PCR followed by complementary phenotypic assays to distinguish between Mycoplasma and Acholeplasma; however, the method did not further speciate and required additional culture for completion. Rebelo et al (2011) reported a relatively rapid method that employed PCR amplification of ITS followed by high resolution melt analysis; however, this method only identified Mycoplasma and analysis is difficult. Recently, a matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry method for identification of ruminant Mycoplasma (Pereyre et al, 2013) and a rapid DNA microarray assay for identification of both Mycoplasma and Acholeplasma (Schnee et al, 2012) have been reported.…”

Section: Discussionmentioning

confidence: 98%

Validation of a mycoplasma molecular diagnostic test and distribution of mycoplasma species in bovine milk among New York State dairy farms

Gioia

Werner

Nydam

et al. 2016

Journal of Dairy Science

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Mycoplasma mastitis is a contagious and costly disease of dairy cattle that significantly affects animal health and milk productivity. Mycoplasma bovis is the most prevalent and invasive agent of mycoplasma mastitis in dairy cattle, and early detection is critical. Other mycoplasma have been isolated from milk; however, the role and prevalence of these species as mastitis pathogens are poorly understood. Routine screening of milk for mycoplasma by bacteriological culture is an important component of a farm control strategy to minimize a herd mycoplasma outbreak, but phenotypic methods have limited ability to speciate mycoplasma, affecting how farms and practitioners can understand the role and effect of species other than M. bovis in herd health. Fastidious mycoplasma culture can be lengthy and inconclusive, resulting in delayed or false negative reports. We developed and validated a multitarget PCR assay that can in the same day confirm or reject a presumptive positive mycoplasma culture found upon bacteriological testing of clinical specimens, further discriminate between Acholeplasma and Mycoplasma, and identify M. bovis. Coupled with sequence analysis isolates can be further identified as bovine mycoplasma Mycoplasma arginini, Mycoplasma alkalescens, Mycoplasma canadense, Mycoplasma bovirhinis, Mycoplasma bovigenitalium, Mycoplasma californicum, Acholeplasma laidlawii, and Acholeplasma oculi. Assay validation included analysis of 845 mycoplasma representing these species and 30 additional bacterial species obtained from routine milk submissions to the Quality Milk Production Services from New York State farms and veterinary clinics between January 2012 and December 2015. Among 95 herds, we found 8 different Mycoplasma species and 3 different Acholeplasma species, with an overall prevalence of M. bovirhinis of 1%, A. oculi of 2%, M. arginini of 2%, M. californicum of 3%, M. canadense of 10%, M. bovigenitalium of 10%, A. laidlawii of 11%, M. alkalescens of 17%, and M. bovis of 78%. More than one mycoplasma was found in 14% of the herds tested, and both M. bovis and Acholeplasma were found in 6% of the farms. Incorporation of the validated molecular diagnostic assay into routine bacteriological screening as a supportive confirmation and identification tool will lead to an improved assessment of Mycoplasma and Acholeplasma prevalence data, which will facilitate increased knowledge about the role of these mycoplasma in mastitis.

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Section: Discussionmentioning

confidence: 98%

Validation of a mycoplasma molecular diagnostic test and distribution of mycoplasma species in bovine milk among New York State dairy farms

Gioia

Werner

Nydam

et al. 2016

Journal of Dairy Science

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“…Qualitative PCR and amplification of an approximately 500 base-pair (bp) segment of the 16S-23S bacterial intergenic spacer (IGS) region was performed to detect Mycoplasma spp. using a modification of previously published methods (Rebelo et al 2011). Reactions were prepared containing 12.5 mL of Amplitaq Gold 360 Master Mix (Applied Biosystems, Foster City, California, USA), 25 pmol each of a forward F1 (59-ACACCATGGGAGYTGGTAAT-39) and reverse R1 primer (59-CTCCWTCGACT-TYCAGACCCAAGGCAT-39), 10 mL of molecular-grade water, and 2 mL (approximately 7 ng to 38 ng) of nucleic acid extract.…”

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confidence: 99%

AMycoplasmaSpecies of Emydidae Turtles in the Northeastern USA

Ossiboff

Raphael

Ammazzalorso

et al. 2015

Journal of Wildlife Diseases

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ABSTRACT:Mycoplasma infections can cause significant morbidity and mortality in captive and wild chelonians. As part of a health assessment of endangered bog turtles (Glyptemys muhlenbergii) in the northeastern US, choanal and cloacal swabs from these and other sympatric species, including spotted turtles (Clemmys guttata), eastern box turtles (Terrapene carolina carolina), wood turtles (Glyptemys insculpta), and common snapping turtles (Chelydra serpentina) from 10 sampling sites in the states (US) of Delaware, New Jersey, and Pennsylvania, were tested by PCR for Mycoplasma. Of 108 turtles tested, 63 (58.3%) were PCR positive for Mycoplasma including 58 of 83 bog turtles (70%), three of three (100%) eastern box turtles, and two of 11 (18%) spotted turtles; all snapping turtles (n57) and wood turtles (n54) were negative. Sequence analysis of portions of the 16S-23S intergenic spacer region and the 16S ribosomal RNA gene revealed a single, unclassified species of Mycoplasma that has been previously reported in eastern box turtles, ornate box turtles (Terrapene ornata ornata), western pond turtles (Emys marmorata), and red-eared sliders (Trachemys scripta elegans). We document a high incidence of Mycoplasma, in the absence of clinical disease, in wild emydid turtles. These findings, along with wide distribution of the identified Mycoplasma sp. across a broad geographic region, suggest this bacterium is likely a commensal inhabitant of bog turtles, and possibly other species of emydid turtles, in the northeastern US.

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“…This type of PCR has a higher sensitivity in comparison to conventional PCR. It also produces quicker results and can quantify DNA in tested samples [11, 12]. …”

Section: Introductionmentioning

confidence: 99%

Comparison of different NAT assays for the detection of microorganisms belonging to the class Mollicutes

et al. 2017

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BackgroundMollicutes detection can be cumbersome due to their slow growth in vitro. For this reason, the use of DNA based on generic molecular tests represents an alternative for rapid, sensitive and specific detection of these microorganism. For this reason, six previously described nucleic acid testing assays were compared to evaluate their ability to detect microorganisms belonging to the class Mollicutes.MethodsA panel of 61 mollicutes, including representatives from the Mycoplasma, Acholeplasma, Mesoplasma, Spiroplasma and Ureaplasma genus, were selected to evaluate the sensitivity and specificity of these assays. A total of 21 non-mollicutes, including closely related non-mollicutes species, were used to evaluate specificity. Limits of detection were calculated to determine the analytical sensitivity of the assays. The two best performing assays were subsequently adapted into real-time PCR format, followed by melting curve analysis.ResultsBoth assays performed satisfactorily, with a 100% specificity described for both assays. The detection limits were found to be between 10−4 and 10−5 dilutions, equivalent to 15 to 150 genome copies approximately. Based on our work, both van Kuppeveld and Botes real-time PCR assays were found to be the best performing tests in terms of sensitivity and specificity. Furthermore, Botes real-time PCR assay could detect phytoplasmas as well.ConclusionsThese assays can be very useful for the rapid, specific and sensitive screening cell line contaminants, clinical samples as well as detecting non-culturable, unknown species of mollicutes or mollicutes whose growth is slow or difficult.

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Use of high-resolution melting curve analysis to identify Mycoplasma species commonly isolated from ruminant, avian, and canine samples

Cited by 24 publications

References 11 publications

Validation of a mycoplasma molecular diagnostic test and distribution of mycoplasma species in bovine milk among New York State dairy farms

Validation of a mycoplasma molecular diagnostic test and distribution of mycoplasma species in bovine milk among New York State dairy farms

AMycoplasmaSpecies of Emydidae Turtles in the Northeastern USA

Comparison of different NAT assays for the detection of microorganisms belonging to the class Mollicutes

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