New sensitive real-time PCR targeting p28 gene for detection of Ehrlichia canis in blood samples from dogs

ABSTRACT: This study aims to describe a new detection method of a quantitative real-time polymerase chain reaction (qPCR) targeting the 28 kDa outer membrane protein gene (p28) as well as to compare this method with a conventional PCR (cPCR), which targets the same gene, in order to evaluate the performance of the technique designed in this study in detecting Ehrlichia canis (E. canis). Optimum oligonucleotides concentrations were reached, and the analytical sensitivity and specificity of the qPCR were performed. A total of 218 dogs’ whole blood samples were conventionally collected for this study. The DNA was extracted from each sample. Subsequently, the samples were tested by an established cPCR and the new qPCR to compare each technique’s performances. This new qPCR method for the molecular detection of E. canis presented a detection limit of ten copies of the fragment and was considered specific for E. canis according to analytical specificity analyses performed in vitro and in silico. The standard curve revealed 100% efficiency and a coefficient of determination (R2) equivalent to 99.8%. Among the samples examined by qPCR, 24.31% were considered positive, significantly greater than those detected by cPCR (15.13%). The qPCR technique reached a higher sensitivity than the cPCR when targeting the p28 gene in detecting E. canis. The qPCR standardized in this study is an efficient method for confirming canine monocytic ehrlichiosis (CME) diagnosis and might provide the parasitemia monitoring during the disease treatment.

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Evaluation of RealStar® Alpha Herpesvirus PCR Kit for Detection of HSV-1, HSV-2, and VZV in Clinical Specimens

BioMed Research International ◽

10.1155/2019/5715180 ◽

2019 ◽

Vol 2019 ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Cyril C. Y. Yip ◽

Siddharth Sridhar ◽

Kit-Hang Leung ◽

Andrew K. W. Cheng ◽

Kwok-Hung Chan ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Test Performance ◽

Analytical Sensitivity ◽

Pcr Assay ◽

Clinical Specimens ◽

Varicella Zoster ◽

Simplex Virus ◽

Hsv 1

Several commercial PCR kits are available for detection of herpes simplex virus (HSV) and varicella zoster virus (VZV), but the test performance of one CE-marked in vitro diagnostic kit—RealStar® alpha Herpesvirus PCR Kit—has not been well studied. This study evaluated the performance of RealStar® alpha Herpesvirus PCR Kit 1.0 on the LightCycler® 480 Instrument II for detection and differentiation of HSV-1, HSV-2, and VZV in human clinical specimens. We evaluated the analytical sensitivity of the RealStar® and in-house multiplex real-time PCR assays using serial dilutions of nucleic acids extracted from clinical specimens. The analytical sensitivity of the RealStar® assay was 10, 32, and 100 copies/reaction for HSV-1, HSV-2, and VZV, respectively, which was slightly higher than that of the in-house multiplex real-time PCR assay. Reproducibility of the cycle threshold (Cp) values for each viral target was satisfactory with the intra- and interassay coefficient of variation values below 5% for both assays. One-hundred and fifty-three clinical specimens and 15 proficiency testing samples were used to evaluate the diagnostic performance of RealStar® alpha Herpesvirus PCR Kit against the in-house multiplex real-time PCR assay. The RealStar® assay showed 100% sensitivity and specificity when compared to the in-house assay. Cp values of the RealStar® and in-house assays showed excellent correlation. RealStar® alpha Herpesvirus PCR is a sensitive, specific, and reliable assay for the detection of HSV-1, HSV-2, and VZV, with less extensive verification requirements compared to a laboratory developed assay.

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Occurrence of Ehrlichia canis and Anaplasma platys in household dogs from northern Parana

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612012005000009 ◽

2012 ◽

Vol 21 (4) ◽

pp. 379-385 ◽

Cited By ~ 15

Author(s):

Gislaine Cristina Ferreira da Silva ◽

Aline do Nascimento Benitez ◽

Aline Girotto ◽

Alessandra Taroda ◽

Marilda Carlos Vidotto ◽

...

Keyword(s):

Ehrlichia Canis ◽

Differential Diagnoses ◽

Laboratory Evaluation ◽

Blood Collection ◽

Routine Laboratory ◽

Anaplasma Platys ◽

Pcr Assay ◽

Blood Samples ◽

Canine Monocytic Ehrlichiosis ◽

Hematological Alterations

Canine monocytic ehrlichiosis caused primarily by Ehrlichia canis and canine thrombocytic anaplasmosis induced by Anaplasma platys are important emerging zoonotic tick-borne diseases of dogs. There is evidence that these pathogens can also affect humans. This study evaluated the presence of E. canis and A. platys in blood samples collected from 256 domiciled dogs in the municipality of Jataizinho, located in north region of the State of Parana, Brazil, by PCR assay. The occurrence of E. canis and A. platys was 16.4% (42/256) and 19.4% (49/256), respectively; while 5.47% (14/256) of the dogs evaluated were co-infected by these two organisms. The presence of E. canis and A. platys was not significantly associated with the variables evaluated (sex, age, outdoor access, and presence of ticks during blood collection). Infection of dogs by E. canis was associated with anemia and thrombocytopenia, while infection induced by A. platys was related only to thrombocytopenia. Canine monocytic ehrlichiosis and canine thrombocytic anaplasmosis should be included in the differential diagnoses when these hematological alterations are observed during routine laboratory evaluation of dogs.

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A SYBR green I real-time polymerase chain reaction (PCR) assay for detection and quantification of Trichomonas gallinae

Parasitology Research ◽

10.1007/s00436-020-06887-x ◽

2020 ◽

Vol 119 (11) ◽

pp. 3909-3913

Author(s):

Zaida Rentería-Solís ◽

Tran Nguyen-Ho-Bao ◽

Shahinaz Taha ◽

Arwid Daugschies

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Small Subunit ◽

Sybr Green ◽

Pcr Assay ◽

Standard Curve ◽

Trichomonas Gallinae ◽

Host Interaction

Abstract Trichomonas gallinae are parasitic flagellates of importance in wild and domestic birds. The parasite is worldwide distributed, and Columbine birds are its main host. Current research focuses mostly on epidemiological and phylogenetic studies. However, there is still a lack of knowledge regarding parasite-host interaction or therapy development. Real-time PCR is a useful tool for diagnostic and quantification of gene copies in a determined sample. By amplification of a 113-bp region of the 18S small subunit ribosomal RNA gene, a SYBR green-based real-time PCR assay was developed. A standard curve was prepared for quantification analysis. Assay efficiency, linearity, and dissociation analysis were successfully performed. Specificity, sensibility, and reproducibility analysis were tested. This assay could be a useful tool not only for diagnostic purposes but also for future in vivo and in vitro T. gallinae studies.

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Efficacy of a Doxycycline Treatment Regimen Initiated during Three Different Phases of Experimental Ehrlichiosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01622-09 ◽

2010 ◽

Vol 54 (12) ◽

pp. 5012-5020 ◽

Cited By ~ 26

Author(s):

Jennifer C. McClure ◽

Michelle L. Crothers ◽

John J. Schaefer ◽

Patrick D. Stanley ◽

Glen R. Needham ◽

...

Keyword(s):

Antibiotic Therapy ◽

Treatment Regimen ◽

Rhipicephalus Sanguineus ◽

Ehrlichia Canis ◽

Clinical Parameters ◽

Blood Samples ◽

Persistently Infected ◽

Doxycycline Treatment ◽

Canine Monocytic Ehrlichiosis ◽

Three Phases

ABSTRACT Doxycycline is the treatment of choice for canine monocytic ehrlichiosis (CME), a well-characterized disease and valuable model for tick-borne zoonoses. Conflicting reports of clearance of Ehrlichia canis after treatment with doxycycline suggested that the disease phase during which treatment is initiated influences outcomes of these treatments. The purpose of this study was to evaluate the efficacy of a 28-day doxycycline regimen for clearance of experimental E. canis infections from dogs treated during three phases of the disease. Ten dogs were inoculated with blood from E. canis carriers and treated with doxycycline during acute, subclinical, or chronic phases of CME. Daily rectal temperatures and semiweekly blood samples were monitored from each dog, and Rhipicephalus sanguineus ticks were acquisition fed on each dog for xenodiagnosis. Blood collected from dogs treated during acute or subclinical CME became PCR negative for E. canis as clinical parameters improved, but blood samples collected from dogs treated during chronic CME remained intermittently PCR positive. R. sanguineus ticks fed on dogs after doxycycline treatments became PCR positive for E. canis, regardless of when treatment was initiated. However, fewer ticks became PCR positive after feeding on two persistently infected dogs treated with doxycycline followed by rifampin, suggesting that antibiotic therapy can reduce tick acquisition of E. canis.

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Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of novel-goose parvovirus in vivo

10.1101/2021.07.09.451858 ◽

2021 ◽

Author(s):

Haibin Ma ◽

Yahui Li ◽

Junzheng Yang

Keyword(s):

Viral Load ◽

Real Time ◽

Real Time Pcr ◽

Bursa Of Fabricius ◽

Pcr Assay ◽

Standard Curve ◽

Goose Parvovirus ◽

Detection And Quantification

Objectives: To develop a sensitive, highly specific fluorescent quantitative real-time PCR assay for accurate detection and quantification of novel-goose parvovirus (N-GPV) in vitro and in vivo. Methods: Specific primers was designed based on N-GPV inverted terminal repeats region; virus RNA (DFV, NDV, AIV, DHV-1, DHV-3) and virus DNA (MDPV, GPV, N-GPV) were extracted, cDNA (DFV, NDV, AIV, DHV-1, DHV-3) were prepared from viral RNAs using M-MLV Reverse Transcriptase, and prepared cDNA (DFV, NDV, AIV, DHV-1, DHV-3) and DNA (MDPV, GPV, N-GPV) amplified by real-time PCR; the sensitivity, specificity and reproducibility of established real-time PCR methods were evaluated, and finally we validated the reliability of real-time PCR methods in ducklings models in vivo. Results: The standard curve of established real-time PCR had a good linearity (slope was -0.3098, Y-intercept was 37.865, efficiency of standard curve was 0.995); the detection limit of established real-time PCR for N-GPV was 10 copies/reaction. The sensitivity of real-time PCR was 10 copies/uL, which was 1000 times higher than conventional gel-based PCR assay. The results of intra-assay CVs (0.04-0.74%) and inter-assay CVs (0.16-0.53%) showed that the real-time PCR assay had an excellent repeatability. This method also could efficiently detect viral load in heart, liver, spleen, lung, kidney, pancreas, bursa of Fabricius, brain, blood and excrement from ducklings models after N-GPV infection from 6h to 28 days, which could provided us a dynamic distribution observation of N-GPV viral load using this real-time PCR assay in vivo. Conclusion: In the study, we developed a high sensitive, specific and reproducible real-time PCR assay for N-GPV detection. The established real-time PCR assay was suitable for parvovirus detection and quantification simultaneously, no matter sample obtained from blood, internal organs or ileac contents; the present work may provide insight into the pathogenesis of N-GPV and will contributes to better understanding of this newly emerged novel GPV related virus in cherry valley ducks.

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Development of a Broad-Range 23S rDNA Real-Time PCR Assay for the Detection and Quantification of Pathogenic Bacteria in Human Whole Blood and Plasma Specimens

BioMed Research International ◽

10.1155/2013/264651 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 15

Author(s):

Paolo Gaibani ◽

Mara Mariconti ◽

Gloria Bua ◽

Sonia Bonora ◽

Davide Sassera ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Whole Blood ◽

Extraction Method ◽

Sample Type ◽

Blood Samples ◽

Human Whole Blood ◽

Rdna Gene ◽

Whole Blood Samples ◽

Pcr Targeting

Molecular methods are important tools in the diagnosis of bloodstream bacterial infections, in particular in patients treated with antimicrobial therapy, due to their quick turn-around time. Here we describe a new broad-range real-time PCR targeting the 23S rDNA gene and capable to detect as low as 10 plasmid copies per reaction of targeted bacterial 23S rDNA gene. Two commercially available DNA extraction kits were evaluated to assess their efficiency for the extraction of plasma and whole blood samples spiked with different amount of eitherStaphylococcus aureusorEscherichia coli, in order to find the optimal extraction method to be used. Manual QIAmp extraction method with enzyme pre-treatment resulted the most sensitive for detection of bacterial load. Sensitivity of this novel assay ranged between 10 and 103 CFU per PCR reaction forE. coliandS. aureusin human whole blood samples depending on the extraction methods used. Analysis of plasma samples showed a 10- to 100-fold reduction of bacterial 23S rDNA in comparison to the corresponding whole blood specimens, thus indicating that whole blood is the preferential sample type to be used in this real-time PCR protocol. Our results thus show that the 23S rDNA gene represents an optimal target for bacteria quantification in human whole blood.

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ASSESSMENT OF THE POSSIBILITY OF APPLICATION IN LABORATORY PRACTICE OF REAGENT KIT FOR DIAGNOSIS OF GLANDERS AND MELIOIDOSIS BY REAL-TIME POLYMERASE CHAIN REACTION

Russian Clinical Laboratory Diagnostics ◽

10.18821/0869-2084-2019-64-11-700-704 ◽

2019 ◽

Vol 64 (11) ◽

pp. 700-704

Author(s):

L. V. Lemasova ◽

G. A. Tkachenko ◽

E. V. Prokhvatilova ◽

L. I. Belitskaya ◽

D. V. Viktorov ◽

...

Keyword(s):

Real Time ◽

Diagnostic Value ◽

Medical Laboratory ◽

Analytical Sensitivity ◽

Rt Pcr ◽

Laboratory Practice ◽

Environmental Objects ◽

Polymerase Chain ◽

State Examination

The reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR is designed for detecting in vitro diagnostics and differentiate the DNA of glanders and melioidosis pathogens by real-time multiplex PCR in biological (clinical) material and cultures of microorganisms, as well as environmental objects and solid food products (rice). During clinical testing diagnostic value of reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR has been studied. Based on the results obtained, a high analytical sensitivity (1×103 microbe cells/ml) and specificity (100%) of PCR-RT with the developed reagent kit were established, regardless of the type of material being studied. The diagnostic sensitivity of PCR-RT using a set of reagents was at least 98.0% and specificity at least 99%. The stages of state examination have been completed, a registration certificate has been obtained at Roszdravnadzor, production, sale and use of reagent kit in medical laboratory practice have been permitted.

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Performance assessment of the Allplex™ STI Essential real-time PCR assay for the diagnosis of Neisseria gonorrhoeae and Chlamydia trachomatis infections in genital and extra-genital sites

Journal of Laboratory Medicine ◽

10.1515/labmed-2019-0030 ◽

2019 ◽

Vol 43 (4) ◽

pp. 191-200

Author(s):

Sylvain Robinet ◽

François Parisot

Keyword(s):

Detection Limit ◽

Chlamydia Trachomatis ◽

Neisseria Gonorrhoeae ◽

Real Time ◽

Evaluation Agency ◽

Nucleic Acid Amplification ◽

Molecular Diagnostic ◽

Standard Curve ◽

Commensal Flora

Abstract Background Commercial kits performing Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) nucleic acid amplification tests (NAATs) for genital samples are recommended in association with culture, but the majority of real-time polymerase chain reaction (PCR) methods have not received regulatory approval for diagnostics in extra-genital sites. Since 2017, only the Hologic® Aptima Combo2 assay has an in vitro diagnostic (IVD) certification from the European Medicine Evaluation Agency. Methods We assessed the Allplex™ STI-Essential Assay (EA) for the diagnosis of NG and CT in both genital and extra-genital sites. The performance of the extraction step was studied by means of a standard curve between the concentration of expected cultivable gonococci and the cycle threshold (Ct). Three later-generation NAATs were used as comparators, particularly to assess the specificity (Sp). Results A relation between the gonococcal concentration, expressed as colony-forming unit (CFU) per milliliter logarithm, and the Ct was shown to be linear irrespective of the matrices (95% confidence interval [CI]). The detection limit was 10 CFU/mL, contrasting with the relatively poor sensitivity of culture due to inhibitory effects such as pH and the overgrowth of the commensal flora. NG molecular diagnostic is complex and the method comparisons showed some discrepancies when Ct was above 34. We decided to include interpretative comments on our reports on the basis of the Ct result. For CT, comparisons displayed a satisfactory agreement, and the detection limit was 50 copies/mL. Conclusions The Seegene Allplex™ STI-EA showed acceptable performance characteristics for the detection of genital and extra-genital NG and CT.

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Seroprevalence and Genotypic Analysis of Ehrlichia canis Infection in Dogs and Humans in Cauca, Colombia

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.20-0965 ◽

2021 ◽

Author(s):

Elkin Forero-Becerra ◽

Jignesh Patel ◽

Heidy-C Martínez-Diaz ◽

Paola Betancourt-Ruiz ◽

Efraín Benavides ◽

...

Keyword(s):

16S Rrna ◽

Amplicon Sequencing ◽

Immunofluorescence Assay ◽

Positive Sample ◽

Human Infection ◽

Ehrlichia Canis ◽

Rrna Gene ◽

Blood Samples ◽

Genotypic Analysis ◽

Pcr Targeting

Ehrlichia canis infections have been reported in humans in Venezuela and Costa Rica. In this study, 506 healthy residents and 114 dogs from four municipalities (Cauca, Colombia) were surveyed and blood samples collected. Antibodies to E. canis in human and canine sera were evaluated using the Tandem repeat protein 19 (TRP19) peptide ELISA and indirect immunofluorescence assay (IFA). Ehrlichia canis TRP19 antibodies were detected in only 1/506 human sera, but the single positive sample was negative by IFA. The majority (75/114; 66%) of dogs surveyed had antibodies to the E. canis TRP19 peptide by ELISA, and eight randomly selected sera were further confirmed by E. canis IFA. Genomic DNA samples obtained from 73 E. canis TRP19 ELISA–positive dog blood samples were examined by PCR targeting the 16S rRNA gene. Ehrlichia canis 16S rRNA was amplified in 30 (41%) of the dogs, and 16 amplicons were selected for DNA sequencing, which confirmed that all were E. canis. A second PCR was performed on the 16 confirmed E. canis 16S rRNA PCR–positive samples to determine the TRP36 genotype by amplifying the trp36 gene. TRP36 PCR amplicon sequencing identified nine dogs infected with the U.S. E. canis TRP36 genotype (56%), one dog with the Brazilian genotype (6%), and six dogs with the Costa Rican genotype (38%). Moreover, these molecular genotype signatures were consistent with serologic analysis using TRP36 genotype–specific peptides. Notably, there was no serologic evidence of E. canis infection in humans, suggesting that E. canis infection in dogs in Cauca is not associated with zoonotic human infection.

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Molecular Prevalence and Identification of Ehrlichia canis and Anaplasma platys from Dogs in Nay Pyi Taw Area, Myanmar

Veterinary Medicine International ◽

10.1155/2021/8827206 ◽

2021 ◽

Vol 2021 ◽

pp. 1-7

Author(s):

Myint Myint Hmoon ◽

Lat Lat Htun ◽

May June Thu ◽

Hla Myet Chel ◽

Yu Nandi Thaw ◽

...

Keyword(s):

Ehrlichia Canis ◽

Rrna Gene ◽

Limited Information ◽

Anaplasma Platys ◽

Blood Samples ◽

Glta Gene ◽

Canine Monocytic Ehrlichiosis ◽

Veterinary Importance ◽

Healthy Dogs

Ticks are vectors of different types of viruses, protozoans, and other microorganisms, which include Gram-negative prokaryotes of the genera Rickettsiales, Ehrlichia, Anaplasma, and Borrelia. Canine monocytic ehrlichiosis caused by Ehrlichia canis and canine cyclic thrombocytopenia caused by Anaplasma platys are of veterinary importance worldwide. In Myanmar, there is limited information concerning tick-borne pathogens, Ehrlichia and Anaplasma spp., as well as genetic characterization of these species. We performed nested PCR for the gltA gene of the genus Ehrlichia spp. and the 16S rRNA gene of the genus Anaplasma spp. with blood samples from 400 apparently healthy dogs in Nay Pyi Taw area. These amplicon sequences were compared with other sequences from GenBank. Among the 400 blood samples from dogs, 3 (0.75%) were positive for E. canis and 1 (0.25%) was positive for A. platys. The partial sequences of the E. canis gltA and A. platys 16SrRNA genes obtained were highly similar to E. canis and A. platys isolated from different other countries.

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