Prevalence and phylogenetic analysis of Mycoplasma synoviae strains isolated from Polish chicken layer flocks

AbstractIntroduction:Mycoplasma synoviae(MS) is a chicken pathogen of major economic importance.Material and Methods:Between 2010 and 2016, 906 commercial layer chicken flocks in Poland were examined for MS, and the phylogenetic relationship among the strains was established. Regionally dispersed samples were collected and tested with the use of real-time PCR to detect the 16S–23S intergenic spacer region. Positive samples were also tested with LAMP and conventional PCR to detect thevlhAgene.Results:MS genetic material was detected in 265 (29%) of the tested flocks by real-time PCR, in 227 by the LAMP method and in 202 (22%) by conventional PCR. The by-year percentage of positive samples began at 34% in 2010, rose to 44% in 2012, and declined to 29% in 2016. A phylogenetic analysis of PolishM. synoviaestrains using a partial sequence of thevlhAgene showed nine genotypes (A–I), the most frequently occurring being F and C. Pathogenic Polish MS field isolates (n = 27) collected from chickens with clinical signs of infection were grouped for their characteristic symptoms: respiratory for genotypes C, E, F, and I (n = 13), EAA and a drop in laying for genotypes F, E, and C (n = 12), and synovitis for genotype A (n = 2).Conclusion:These data showed the country’s isolate diversity. The high prevalence suggests the need to introduce appropriate control programmes. This is the first report of molecular epidemiological data onM. synoviaeinfection in layer chickens in Poland.

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Characterization of a novel Mycoplasma cynos real-time PCR assay

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638719890858 ◽

2019 ◽

Vol 32 (6) ◽

pp. 793-801 ◽

Cited By ~ 1

Author(s):

Rebecca L. Tallmadge ◽

Renee Anderson ◽

Patrick K. Mitchell ◽

Zachary C. Forbes ◽

Brenda Werner ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Intergenic Spacer ◽

High Specificity ◽

Mycoplasma Species ◽

Limit Of Quantification ◽

Clinical Specimens ◽

Intergenic Spacer Region ◽

Tuf Gene ◽

Gene Assay

Mycoplasma cynos is recognized as an emerging causative pathogen of canine infectious respiratory disease (CIRD) worldwide. We developed a new open-source real-time PCR (rtPCR) assay for M. cynos that performs well under standard rtPCR conditions. Primers and probes were designed to target the M. cynos tuf gene. Reaction efficiencies for the M. cynos tuf gene assay on 2 platforms were based on amplification of standard curves spanning 8 orders of magnitude: ABI 7500 platform, 94.3–97.9% ( r2 ≥ 0.9935); QuantStudio OpenArray platform, 119.1–122.5% ( r2 = 0.9784). The assay performed very well over a range of template input, from 109 copies to the lower limit of quantification at 4 copies of the M. cynos genome on the ABI 7500 platform. Diagnostic performance was estimated by comparison with an in-house legacy assay on clinical specimens as well as testing isolates that were characterized previously by intergenic spacer region (ISR) sequencing. Exclusivity was established by testing 12 other Mycoplasma species. To substantiate the high specificity of the M. cynos tuf gene assay, sequence confirmation was performed on ISR PCR amplicons obtained from clinical specimens. One ISR amplicon sequence revealed M. mucosicanis rather than M. cynos. The complete protocol of the newly developed M. cynos tuf assay is provided to facilitate assay harmonization.

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Clinical spectrum and diagnostic yields of Mycoplasma pneumoniae as a causative agent of community-acquired pneumonia

Journal of Laboratory Physicians ◽

10.4103/jlp.jlp_62_17 ◽

2018 ◽

Vol 10 (01) ◽

pp. 044-049 ◽

Cited By ~ 2

Author(s):

Saroj Dash ◽

Rama Chaudhry ◽

Benu Dhawan ◽

Aparajit Ballav Dey ◽

Sushil Kumar Kabra ◽

...

Keyword(s):

Real Time ◽

Mycoplasma Pneumoniae ◽

Real Time Pcr ◽

Clinical Signs ◽

Signs And Symptoms ◽

Community Acquired Pneumonia ◽

Diagnostic Methods ◽

Conventional Pcr ◽

Clinical Signs And Symptoms ◽

Tract Infections

Abstract INTRODUCTION: Infection with Mycoplasma pneumoniae (M. pneumonia) occurs worldwide which accounts for 15%–20% of cases of community-acquired pneumonia and indistinguishable clinically from other infectious causes of pneumonia. AIM: The aim of this study was to evaluate the real-time polymerase chain reaction (PCR) and to correlate it with other diagnostic methods such as culture, serology (ELISA), and conventional PCR along with the clinical signs and symptoms produced by M. pneumonia. MATERIALS AND METHODS: A total of 130 patients of all age groups presenting with clinical features of lower respiratory tract infections were enrolled over a period of 1 year and 2 months in a tertiary care hospital in Delhi. M. pneumoniae in throat swab samples was detected by real-time PCR, compared with culture, serology, conventional PCR, and clinical signs and symptoms. Univariate analyses were conducted to determine the association of M. pneumoniae infection among different categories of patients. RESULTS: Out of a total of 130 patients, 18 patients (14%) were positive for M. pneumoniae by any test; culture was positive in nine patients (50%), serology (IgM) in eight patients (44.4%), PCR in five patients (27.7%), and real-time PCR was positive in six patients (33.3%). Clinical signs and symptoms were higher in incidence in M. pneumoniae-positive patients. Age-matched healthy controls (30) were included in the study, and all were negative for any diagnostic test performed (P = 0.026). CONCLUSION: It was concluded that combination of M. pneumoniae-specific testing modalities is required for the diagnosis of this etiological agent rather than a single diagnostic method.

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New PCR-based assay for the identification of Pectobacterium carotovorum causing potato soft rot

Plant Disease ◽

10.1094/pdis-08-21-1676-re ◽

2021 ◽

Author(s):

M. Belén Suárez ◽

Marta Diego ◽

F. J. Feria ◽

M J Martín-Robles ◽

Sergio Moreno ◽

...

Keyword(s):

Real Time ◽

Dna Sequences ◽

Real Time Pcr ◽

Bacterial Species ◽

Intergenic Spacer ◽

Soft Rot ◽

Pcr Analysis ◽

Rrna Gene ◽

Accurate Identification ◽

Intergenic Spacer Region

Soft rot on potato tuber is a destructive disease caused by pathogenic bacterial species of the genera Pectobacterium and Dickeya. Accurate identification of the causal agent is necessary to ensure adequate disease management, since different species may have distinct levels of aggressiveness and host range. One of the most important potato pathogens is P. carotovorum, a highly heterogeneous species capable of infecting multiple hosts. The complexity of this species, until recently divided into several subspecies, has made it difficult to develop precise diagnostic tests. This study proposes a PCR assay based on the new pair of primers Pcar1F/R to facilitate the identification of potato isolates of P. carotovorum according to the most recent taxonomic description of this species. The new primers were designed on a variable segment of the 16S rRNA gene and the intergenic spacer region (ITS) of available DNA sequences from classical and recently established species in the genus Pectobacterium. The results of the PCR analysis of genomic DNA from 32 Pectobacterium and Dickeya strains confirmed that the Pcar1F/R primers have sufficient nucleotide differences to discriminate between P. carotovorum and other Pectobacterium species associated with damage to potato crops, with the exception of P. versatile, which improves the specificity of the currently available primers. The proposed assay was originally developed as a conventional PCR but was later adapted to the real-time PCR format for application in combination with the existing real-time PCR test for the potato-specific pathogen P. parmentieri. This should be useful for the routine diagnosis of potato soft rot.

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Application of Real-Time PCR for Quantification of Microcystin Genotypes in a Population of the Toxic Cyanobacterium Microcystis sp

Applied and Environmental Microbiology ◽

10.1128/aem.69.11.6723-6730.2003 ◽

2003 ◽

Vol 69 (11) ◽

pp. 6723-6730 ◽

Cited By ~ 175

Author(s):

Rainer Kurmayer ◽

Thomas Kutzenberger

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Total Population ◽

Intergenic Spacer ◽

Threshold Level ◽

Logarithmic Scale ◽

Cycle Number ◽

Intergenic Spacer Region ◽

Cell Numbers ◽

The Mean

ABSTRACT The cyanobacterium Microcystis sp. frequently develops water blooms consisting of organisms with different genotypes that either produce or lack the hepatotoxin microcystin. In order to monitor the development of microcystin (mcy) genotypes during the seasonal cycle of the total population, mcy genotypes were quantified by means of real-time PCR in Lake Wannsee (Berlin, Germany) from June 1999 to October 2000. Standard curves were established by relating cell concentrations to the threshold cycle (the PCR cycle number at which the fluorescence passes a set threshold level) determined by the Taq nuclease assay (TNA) for two gene regions, the intergenic spacer region within the phycocyanin (PC) operon to quantify the total population and the mcyB gene, which is indicative of microcystin synthesis. In laboratory batch cultures, the cell numbers inferred from the standard curve by TNA correlated significantly with the microscopically determined cell numbers on a logarithmic scale. The TNA analysis of 10 strains revealed identical amplification efficiencies for both genes. In the field, the proportion of mcy genotypes made up the smaller part of the PC genotypes, ranging from 1 to 38%. The number of mcyB genotypes was one-to-one related to the number of PC genotypes, and parallel relationships between cell numbers estimated via the inverted microscope technique and TNA were found for both genes. It is concluded that the mean proportion of microcystin genotypes is stable from winter to summer and that Microcystis cell numbers could be used to infer the mean proportion of mcy genotypes in Lake Wannsee.

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Conventional and real-time PCR assays for specific detection and quantification of Fusarium oxysporum f. sp. ciceris in plants using intergenic spacer region-based marker

Biologia ◽

10.1515/biolog-2015-0041 ◽

2015 ◽

Vol 70 (3) ◽

Cited By ~ 1

Author(s):

Kumari Priyanka ◽

Sunil C. Dubey ◽

Arun K. Singh

Keyword(s):

Fusarium Oxysporum ◽

Host Plant ◽

Real Time ◽

Real Time Pcr ◽

Infected Plant ◽

Intergenic Spacer ◽

Infected Host ◽

Pcr Assay ◽

Intergenic Spacer Region ◽

Plant Samples

AbstractFusarium wilt of chickpea, caused by Fusarium oxysporum f. sp. ciceris (Foc) is one of the most important fungal diseases worldwide. The detection of the pathogen at reasonable time period is of great importance, which requires rapid and sensitive detection methods. The intraspecific divergence sequences found in the intergenic spacer region (IGS) were selected and utilized with the aim to develop a molecular marker specifically to identify the Foc. A marker set, ISR52 F1 and R1 developed, was tested for their specificity as well as sensitivity using conventional as well as real-time polymerase chain reaction (PCR). The specificity of the marker was tested against Foc, other Fusarium species which are closely related to Foc as well as with artificially infected host plant samples. The detection limits of conventional PCR assay was up to 100 pg of infected plant DNA. It proved possible to amplify the IGS region in different portion of a Foc infected host plant by this PCR method. Furthermore, the real-time assay showed more sensitivity and was able to detect the pathogen in infected chickpea plant samples at the DNA concentration of 5 pg. A single melting peak obtained at 87.5°C showed the specificity of the marker towards Foc. Thus, real-time PCR assay proved their potentiality for same-day diagnosis of fungal infection and can be used as a rapid and effective procedure for routine detection and identification of Foc in chickpea samples.

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A Comparison of Ribosomal Gene and Transcript Abundance during High and Low Nitrite Oxidizing Activity Using a Newly Designed Real-Time PCR Detection System Targeting theNitrobacterspp. 16S–23S Intergenic Spacer Region

Environmental Engineering Science ◽

10.1089/ees.2006.23.521 ◽

2006 ◽

Vol 23 (3) ◽

pp. 521-532 ◽

Cited By ~ 8

Author(s):

Shawn A. Hawkins ◽

Kevin G. Robinson ◽

Alice C. Layton ◽

Gary S. Sayler

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Detection System ◽

Intergenic Spacer ◽

Transcript Abundance ◽

Ribosomal Gene ◽

Pcr Detection ◽

Intergenic Spacer Region

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Development of a real-time PCR for detection of the oyster pathogen Nocardia crassostreae based on its homogeneous 16S–23S rRNA intergenic spacer region

Journal of Invertebrate Pathology ◽

10.1016/j.jip.2013.07.002 ◽

2013 ◽

Vol 114 (2) ◽

pp. 120-127 ◽

Cited By ~ 7

Author(s):

Noèlia Carrasco ◽

Ineke Roozenburg ◽

Michal Voorbergen-Laarman ◽

Naoki Itoh ◽

Marc Y. Engelsma

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Intergenic Spacer ◽

23S Rrna ◽

Intergenic Spacer Region

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Porcine Gammaherpesviruses in Italian Commercial Swine Population: Frequent but Harmless

Pathogens ◽

10.3390/pathogens10010047 ◽

2021 ◽

Vol 10 (1) ◽

pp. 47

Author(s):

Giovanni Franzo ◽

Michele Drigo ◽

Matteo Legnardi ◽

Laura Grassi ◽

Maria Luisa Menandro ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clinical Signs ◽

Northern Italy ◽

Porcine Circovirus ◽

Swine Industry ◽

Positive Interaction ◽

High Prevalence ◽

Swine Population ◽

Clinical Conditions

Differently from alpha- and betaherpesviruses affecting swine, interest in the recently discovered Suid gammaherpesvirus 3, Suid gammaherpesvirus 4, and Suid gammaherpesvirus 5, also known as porcine lymphotropic herpesviruses (PLHV-1, PLHV-2, and PLHV-3), has largely focused on their role as potential zoonotic agents in cases of xenotransplantation. However, their role as primary pathogens of swine or as co-factors for other lymphotropic infections has essentially been neglected. The present study aims at filling this gap, evaluating the association between PLHVs infection and different clinical conditions and/or porcine circovirus (PCV) co-infection. One hundred seventy-six samples were obtained from different animals located in a high-density pig area of Northern Italy in the period 2017–2020. The presence of PLHVs and PCVs was tested and quantified by specific real-time PCR: PLHVs were widespread among pigs (PLHV-1, PLHV-2, and PLHV-3 prevalence was 28.97%, 10.79%, and 4.54%, respectively) and detected in all considered tissues and clinical conditions. Frequent co-infections were also observed among PLHVs and with PCVs, although a significant association was not detected with the exception of a positive interaction between PLHV-1 and PLHV-3, and a negative one between PLHV-2 and PCV-2. Significantly, no association between PLHVs, alone or in co-infection, emerged with any of the considered clinical signs, their frequency being comparable between healthy and diseased animals. Based on these pieces of evidence and despite their high prevalence, PLHVs’ relevance for the swine industry appears negligible, either as primary pathogens or as predisposing factors for circovirus-induced diseases.

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