scholarly journals Development of a multiplex qPCR assay for the simultaneous detection of Mycoplasma bovis, Mycoplasma species, and Acholeplasma laidlawii in milk

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11881
Author(s):  
Kanika Chauhan ◽  
Sharif S. Aly ◽  
Terry W. Lehenbauer ◽  
Karen H. Tonooka ◽  
Kathy Glenn ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Multiplex Assay ◽  
Qpcr Assay ◽  
23S Rrna ◽  
Mycoplasma Bovis ◽  
Amplification Efficiency ◽  
Rrna Gene ◽  
Multiplex Qpcr ◽  
Acholeplasma Laidlawii

Contagious bovine mastitis caused by Mycoplasma bovis and other Mycoplasma species including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma canadense is an economical obstacle affecting many dairy herds throughout California and elsewhere. Routine bacteriological culture-based assays for the pathogens are slow and subject to false-positive results due to the presence of the related, non-pathogenic species Acholeplasma laidlawii. To address the need for rapid and accurate detection methods, a new TaqMan multiplex, quantitative real-time PCR (qPCR) assay was developed that targets the 16S rRNA gene of Mycoplasma, rpoB gene of M. bovis, and the 16S to 23S rRNA intergenic transcribed spacer (ITS) region of A. laidlawii. qPCR amplification efficiency and range of detection were similar for individual assays in multiplex as when performed separately. The multiplex assay was able to distinguish between M. bovis and A. laidlawii as well as detect Mycoplasma spp. collectively, including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma canadense, Mycoplasma arginini and Mycoplasma alkalescens. In milk, the lower limit of detection of M. bovis, M. californicum, and A. laidlawii with the multiplex assay was between 120 to 250 colony forming units (CFU) per mL. The assay was also able to simultaneously detect both M. bovis and A. laidlawii in milk when present in moderate (103 to 104 CFU/mL) to high (106 to 107 CFU/mL) quantities. Compared to laboratory culture-based methods, the multiplex qPCR diagnostic specificity (Sp) was 100% (95% CI [86.8–100]; n = 26) and diagnostic sensitivity (Se) was 92.3% (95% CI [74.9–99.1]; n = 26) for Mycoplasma species in milk samples collected from California dairy farms. Similarly, the Sp was 100% (95% CI [90.5–100]; n = 37) and Se was 93.3% (95% CI [68.1–99.8]; n = 15) for M. bovis. Our assay can detect and distinguish among M. bovis, other prevalent Mycoplasma spp., and non-pathogenic Acholeplasma laidlawii for effective identification and control of mycoplasma mastitis, ultimately supporting dairy cattle health and high-quality dairy products in California.

scholarly journals Rapid Detection, by PCR and Reverse Hybridization, of Mutations in the Helicobacter pylori 23S rRNA Gene, Associated with Macrolide Resistance

1999 ◽  
Vol 43 (7) ◽  
pp. 1779-1782 ◽  
Author(s):  
Leen-Jan van Doorn ◽  
Yvette J. Debets-Ossenkopp ◽  
Armelle Marais ◽  
Ricardo Sanna ◽  
Francis Mégraud ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Fragment Length Polymorphism ◽  
Simultaneous Detection ◽  
Point Mutations ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Reverse Hybridization ◽  
23S Rrna Gene ◽  
H Pylori

ABSTRACT A PCR-based reverse hybridization system (research prototype kit INNO-LiPA for H. pylori resistance) was developed and evaluated for simultaneous detection of 23S ribosomal DNA point mutations, associated with macrolide resistance in Helicobacter pylori. Fifty-seven H. pylori strains (51 natural, 6 laboratory-derived artificial, 52 resistant, and 5 susceptible strains) were tested by PCR-LiPA (detecting mutations A2115→G, G2141→A, A2142→G, A2142→C, A2143→G, A2143→C, and A2143→T), DNA sequencing, restriction fragment length polymorphism, and/or hybridization to oligonucleotide probes. Results were highly concordant, but PCR-LiPA appears to be more sensitive for the simultaneous detection of multiple mutants.

scholarly journals Multiplex qPCR Assay for Direct Detection and Quantification of Colletotrichum truncatum, Corynespora cassiicola, and Sclerotinia sclerotiorum in Soybean Seeds

Plant Disease ◽  
2020 ◽  
Vol 104 (11) ◽  
pp. 3002-3009
Author(s):  
Maísa Ciampi-Guillardi ◽  
Juliana Ramiro ◽  
Maria Heloisa Duarte de Moraes ◽  
Marina Coan Goldoni Barbieri ◽  
Nelson S. Massola
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Internal Control ◽  
Direct Detection ◽  
Simultaneous Detection ◽  
Plant Diseases ◽  
Qpcr Assay ◽  
Soybean Seeds ◽  
Corynespora Cassiicola ◽  
Colletotrichum Truncatum ◽  
Multiplex Qpcr

Precise diagnosis of plant diseases is one of the most effective tools to minimize yield losses. Colletotrichum truncatum, Corynespora cassiicola, and Sclerotinia sclerotiorum are common soilborne pathogens that affect soybeans all over the world. We developed a multiplex quantitative real-time polymerase chain reaction (qPCR) assay to simultaneously detect and quantify the three pathogens in soybean seeds and to survey their occurrence in the main soybean production areas in Brazil. Species-specific primers and probes for C. truncatum and C. cassiicola were designed based on GAPDH and TEF1 genes, respectively, to be combined with qPCR detection of S. sclerotiorum previously reported. The multiplex qPCR assay was successful in the simultaneous detection of C. truncatum, C. cassiicola, and S. sclerotiorum, along with a host internal control. The four pathogens were detected and quantified in artificially and naturally infested soybean seeds, even in the lowest incidence level tested of 0.0625% or 1 infected seed out of 1,599 healthy ones. From 81 seed samples tested, C. truncatum was the most frequently detected pathogen and with higher incidence levels (0.25 to 0.125%), followed by S. sclerotiorum and C. cassiicola, both with lower incidence levels (0.125 to 0.0625%). Together, the results evidenced the high sensitivity of the multiplex qPCR assay, indicating its usefulness for a quick and reliable diagnosis of soybean diseases in seeds.

scholarly journals Prospective Evaluation of ResistancePlus MG, a New Multiplex Quantitative PCR Assay for Detection of Mycoplasma genitalium and Macrolide Resistance

2017 ◽  
Vol 55 (6) ◽  
pp. 1915-1919 ◽  
Author(s):  
S. N. Tabrizi ◽  
J. Su ◽  
C. S. Bradshaw ◽  
C. K. Fairley ◽  
S. Walker ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Clinical Performance ◽  
Simultaneous Detection ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Rrna Gene ◽  
Resistance Mutations ◽  
Content Type ◽  
Conventional Culture

ABSTRACT Mycoplasma genitalium is a significant pathogen for which first-line treatment is becoming less effective due to increased resistance to macrolides. As conventional culture and antimicrobial susceptibility testing is not feasible for routine detection of this pathogen, molecular markers such as detection of mutations in the 23S rRNA gene have been described to predict resistance. Recently, a novel multiplex quantitative PCR (qPCR) assay, ResistancePlus MG, has been described for the simultaneous detection of Mycoplasma genitalium and macrolide resistance. In the current study, the clinical performance of the assay was evaluated on 1,089 consecutive urine and anogenital swab samples in symptomatic and asymptomatic male and female patients. Overall, 6.0% were positive for M. genitalium , with 63.1% having macrolide resistance-associated mutations. Compared to the laboratory-validated qPCR method targeting the 16S rRNA gene and Sanger sequencing to determine 23S rRNA mutations, the sensitivity and specificity of M. genitalium detection were 98.5% and 100% and for detection of macrolide resistance mutations were 100.0% and 96.2%, respectively. This assay offers a considerable advantage in clinical settings for M. genitalium testing by making the results of macrolide resistance and mutation analyses simultaneously available, which is increasingly important with escalating macrolide resistance.

scholarly journals Investigation of Macrolide Resistance Genotypes in Mycoplasma bovis Isolates from Canadian Feedlot Cattle

Pathogens ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 622 ◽  
Author(s):  
Andrea Kinnear ◽  
Tim A. McAllister ◽  
Rahat Zaheer ◽  
Matthew Waldner ◽  
Antonio C. Ruzzini ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Feedlot Cattle ◽  
23S Rrna ◽  
Dilution Method ◽  
Mycoplasma Bovis ◽  
Rrna Gene ◽  
Single Mutation ◽  
E Coli ◽  
23S Rrna Gene ◽  
Chronic Pneumonia

Mycoplasma bovis is associated with bovine respiratory disease (BRD) and chronic pneumonia and polyarthritis syndrome (CPPS) in feedlot cattle. No efficacious vaccines for M. bovis exist; hence, macrolides are commonly used to control mycoplasmosis. Whole genome sequences of 126 M. bovis isolates, derived from 96 feedlot cattle over 12 production years, were determined. Antimicrobial susceptibility testing (AST) of five macrolides (gamithromycin, tildipirosin, tilmicosin, tulathromycin, tylosin) was conducted using a microbroth dilution method. The AST phenotypes were compared to the genotypes generated for 23S rRNA and the L4 and L22 ribosomal proteins. Mutations in domains II (nucleotide 748; E. coli numbering) and V (nucleotide 2059 and 2060) of the 23S rRNA (rrl) gene alleles were associated with resistance. All isolates with a single mutation at Δ748 were susceptible to tulathromycin, but resistant to tilmicosin and tildipirosin. Isolates with mutations in both domain II and V (Δ748Δ2059 or Δ748Δ2060) were resistant to all five macrolides. However, >99% of isolates were resistant to tildipirosin and tilmicosin, regardless of the number and positions of the mutations. Isolates with a Δ748 mutation in the 23S rRNA gene and mutations in L4 and L22 were resistant to all macrolides except for tulathromycin.

scholarly journals Use of a DNA Microarray for Detection and Identification of Bacterial Pathogens Associated with Fishery Products

2011 ◽  
Vol 77 (23) ◽  
pp. 8219-8225 ◽  
Author(s):  
Boyang Cao ◽  
Rongrong Li ◽  
Songjin Xiong ◽  
Fangfang Yao ◽  
Xiangqian Liu ◽  
...  
Keyword(s):  
Dna Microarray ◽  
Bacterial Species ◽  
Simultaneous Detection ◽  
Detection Sensitivity ◽  
23S Rrna ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Content Type ◽  
Detection And Identification ◽  
Fishery Products

ABSTRACTWe established a microarray for the simultaneous detection and identification of diverse putative pathogens often associated with fishery products by targeting specific genes ofListeria monocytogenes,Salmonella,Shigella,Staphylococcus aureus,Streptococcus pyogenes,Vibrio cholerae,Vibrio parahaemolyticus,Vibrio vulnificus, andYersinia enterocoliticaand the 16S-23S rRNA gene internal transcribed spacer (ITS) region ofProteus mirabilisandProteus vulgaris. The microarray contained 26 specific probes and was tested against a total of 123 target bacterial strains that included 55 representative strains, 68 clinical isolates, and 45 strains of other bacterial species that belonged to 8 genera and 34 species, and it was shown to be specific and reproducible. A detection sensitivity of 10 ng DNA or 10 CFU/ml for pure cultures of each target organism demonstrated that the assay was highly sensitive and reproducible. Mock and real fishery product samples were tested by the microarray, and the accuracy was 100%. The DNA microarray method described in this communication is specific, sensitive, and reliable and has several advantages over traditional methods of bacterial culture and antiserum agglutination assays.

scholarly journals Panel of 23S rRNA Gene-Based Real-Time PCR Assays for Improved Universal and Group-Specific Detection of Phytoplasmas

2009 ◽  
Vol 75 (9) ◽  
pp. 2945-2950 ◽  
Author(s):  
Jennifer Hodgetts ◽  
Neil Boonham ◽  
Rick Mumford ◽  
Matthew Dickinson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Multiplex Assay ◽  
The Other ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Detection ◽  
23S Rrna Gene ◽  
Pcr Assays

ABSTRACT Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups. Assays for the 16SrII, 16SrIV, and 16SrXII groups have also been developed to confirm that the 23S rRNA gene can be used to design group-specific assays.

scholarly journals Relationship between Antimicrobial Susceptibility and Multilocus Sequence Type of Mycoplasma bovis Isolates and Development of a Method for Rapid Detection of Point Mutations Involved in Decreased Susceptibility to Macrolides, Lincosamides, Tetracyclines, and Spectinomycin

2019 ◽  
Vol 85 (13) ◽  
Author(s):  
Eiji Hata ◽  
Takehiro Harada ◽  
Megumi Itoh
Keyword(s):  
Antimicrobial Susceptibility ◽  
Antimicrobial Agents ◽  
Point Mutations ◽  
Melting Curve ◽  
Sequence Type ◽  
23S Rrna ◽  
Mycoplasma Bovis ◽  
Rrna Gene ◽  
Content Type ◽  
Transition Mutation

ABSTRACT Mycoplasma bovis isolates belonging to the sequence type 5 (ST5) group, the dominant group in Japan since 1999, were low susceptible to 16-membered macrolides and tetracyclines and were confirmed to have a guanine-to-adenine transition mutation at position 748 in the 23S rRNA gene (rrl) and adenine-to-thymine transversion mutations at positions 965 and 967 in the 16S rRNA gene (rrs) (Escherichia coli numbering). Moreover, isolates of ST93 and ST155, members of the ST5 group, were low susceptible to lincosamides and azithromycin and showed an adenine-to-guanine transition mutation at position 2059 of rrl. Isolates of ST93 were additionally low susceptible to spectinomycin and showed a cytosine-to-adenine transversion mutation at position 1192 of rrs. Strains of the ST5 group seem to spread to Japan and Europe from North America with imported cows, while strains of ST93 and ST155 originated in Japan. Melting curve analysis using hybridization probes revealed the existence of point mutations involved in decreased susceptibility to macrolides, lincosamides, and spectinomycin, as demonstrated by changes in the melting curve shape and/or decreases in the melting peak temperature, so the susceptibility to these antimicrobials can be assessed on the same day. For decreased susceptibility to fluoroquinolones to exist, nonsynonymous mutations in the DNA gyrase gene (gyrA) and topoisomerase IV gene (parC) had to coexist. The combination of amino acid substitutions of serine at position 83 in gyrA and serine at position 80 in parC resulted in particularly low susceptibility to fluoroquinolones. IMPORTANCE Mycoplasma bovis is the main causal species of bovine mycoplasmal disease and leads to significant economic losses because of its severe symptoms, strong infectivity, and refractoriness. As for mastitis, culling cows with intramammary infections is a general countermeasure to prevent spreading. The conventional antimicrobial susceptibility test for mycoplasma is time-consuming and troublesome, but no quick and easy method for grasping the antimicrobial susceptibility of the causal strain exists at present. Treatment without antimicrobial susceptibility information may be one reason why M. bovis infection is refractory. Detecting a mutation involved in decreased susceptibility to antimicrobial agents of the causal strain makes it possible to easily select suitable antimicrobials for treatment, and this technique will help improve the cure rate and prevent the overuse of ineffective antimicrobial agents. In this study, we developed a technique to quickly and easily assess antimicrobial susceptibility based on the genetic characteristics of M. bovis strains in Japan.

scholarly journals Clinical Evaluation of Three Commercial PCR Assays for the Detection of Macrolide Resistance in Mycoplasma genitalium

2019 ◽  
Vol 58 (2) ◽  
Author(s):  
Chloé Le Roy ◽  
Cécile Bébéar ◽  
Sabine Pereyre
Keyword(s):  
Sensitivity And Specificity ◽  
Internal Control ◽  
Simultaneous Detection ◽  
Mycoplasma Genitalium ◽  
Macrolide Resistance ◽  
University Hospital ◽  
23S Rrna ◽  
Rrna Gene ◽  
Content Type ◽  
Commercial Kits

ABSTRACT As macrolide resistance in Mycoplasma genitalium is increasing worldwide, macrolide resistance-associated mutations should be assessed in M. genitalium-positive specimens. New commercial kits are available for detection of macrolide resistance concurrently with M. genitalium. We prospectively evaluated the handling and clinical performances of three commercial kits for detection of macrolide resistance in M. genitalium. Between August and December 2018, remnants of all urogenital specimens determined to be M. genitalium positive using an in-house real-time PCR assay were prospectively collected at the French National Reference Center for Bacterial Sexually Transmitted Infections, Bordeaux University Hospital, Bordeaux, France. The internal control of each kit was added to the primary specimen before DNA extraction, and the absence of amplification inhibition associated with the addition of the three internal controls was assessed. Specimens were evaluated with four assays: the ResistancePlus MG assay (SpeeDx), the S-DiaMGRes assay (Diagenode), the RealAccurate TVMGres assay (PathoFinder), and amplification and sequencing of the 23S rRNA gene (the reference assay). Overall, 195 M. genitalium-positive specimens were assessed. The positive agreement of M. genitalium detection for each kit ranged between 94.8% and 96.4%. Among 154 specimens with M. genitalium positivity as detected by the three commercial kits and 23S rRNA sequencing data, the clinical sensitivity and specificity ranges of the three commercial kits for detecting macrolide resistance-associated mutations were 95 to 100% and 94.6 to 97.3%, respectively. The sensitivity and specificity values were similar among the kits. The launch of three easy-to-use sensitive and specific commercial kits for simultaneous detection of M. genitalium and macrolide resistance will be useful for resistance-guided therapy.

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