Duplex Real-Time Polymerase Chain Reaction for Simultaneous Detection and Quantification of <i>Anaplasma Marginale</i> and <i>Anaplasma Centrale</i>

Decaro, Nicola; Carelli, Grazia; Lorusso, Eleonora; Lucente, Maria Stella; Greco, Grazia; Lorusso, Alessio; Radogna, Arianna; Ceci, Luigi; Buonavoglia, Canio

doi:10.1177/104063870802000511

Cited by 49 publications

(44 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Duplex methods have been described for the simultaneous detection of A. marginale-A. centrale and A. phagocytophilum-Borrelia burgdorferi (6,8). To our knowledge, this is the first study to describe a duplex method for the detection of A. marginale and A. phagocytophilum infections in the same sample.…”

Section: Discussionmentioning

confidence: 92%

Detection of Anaplasma marginale and A. phagocytophilum in Bovine Peripheral Blood Samples by Duplex Real-Time Reverse Transcriptase PCR Assay

Reinbold

Coetzee

Sirigireddy³

et al. 2010

J Clin Microbiol

View full text Add to dashboard Cite

Insufficient diagnostic sensitivity and specificity coupled with the potential for cross-reactivity among closely related Anaplasma species has made the accurate determination of infection status problematic. A method for the development of simplex and duplex real-time quantitative reverse transcriptase PCR (qRT-PCR) assays for the detection of A. marginale and A. phagocytophilum 16S rRNA in plasma-free bovine peripheral blood samples is described. The duplex assay was able to detect as few as 100 copies of 16S rRNA of both A. marginale and A. phagocytophilum in the same reaction. The ratio of 16S rRNA to 16S DNA copies for A. marginale was determined to be 117.9:1 (95% confidence interval [95% CI], 100.7:1, 135.2:1). Therefore, the detection limit is the minimum infective unit of one A. marginale bacterium. The duplex assay detected nonequivalent molar ratios as high as 100-fold. Additionally, the duplex assay and a competitive enzyme-linked immunosorbent assay (cELISA) were used to screen 237 samples collected from herds in which anaplasmosis was endemic. When the cELISA was evaluated by the results of the qRT-PCR, its sensitivity and specificity for the detection of A. marginale infection were found to be 65.2% (95% CI, 55.3%, 75.1%) and 97.3% (95% CI, 94.7%, 99.9%), respectively. A. phagocytophilum infection was not detected in the samples analyzed. One-and two-way receiver operator characteristic curves were constructed in order to recommend the optimum negative cutoff value for the cELISA. Percentages of inhibition of 20 and 15.3% were recommended for the one-and two-way curves, respectively. In conclusion, the duplex real-time qRT-PCR assay is a highly sensitive and specific diagnostic tool for the accurate and precise detection of A. marginale and A. phagocytophilum infections in cattle.

show abstract

Section: Discussionmentioning

confidence: 92%

Detection of Anaplasma marginale and A. phagocytophilum in Bovine Peripheral Blood Samples by Duplex Real-Time Reverse Transcriptase PCR Assay

Reinbold

Coetzee

Sirigireddy³

et al. 2010

J Clin Microbiol

View full text Add to dashboard Cite

show abstract

“…However, when the two re-actions were subjected to a comparative field trial using samples from animals from an enzootic stability region, a significant difference was not observed. Previously described real-time PCR assays for the detection of Anaplasma marginale have also shown high levels of sensitivity, as well as those reported by Carelli et al (2007) and Decaro et al (2008) (detection limit = 5 copies of DNA). However, in both cases a hydrolysis probe based system was used (TaqMan system), whereas in the present stud the SYBR Green system was used.…”

Section: Discussionmentioning

confidence: 75%

Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

Bacanelli

Ramos

Araújo³

2014

Pesq. Vet. Bras.

View full text Add to dashboard Cite

Pesq. Vet. Bras. 34(1):29-33, janeiro 2014 29 RESUMO.-[Diagnóstico molecular de Anaplasma marginale em bovinos: avaliação quantitativa de uma PCR em tempo real baseada no gene msp5.] A riquétsia Anaplasma marginale é considerada o principal agente da anaplasmose bovina. Devido a não especificidade dos sinais clínicos, a confirmação da infecção nos animais depende de testes laboratoriais. Recentemente, métodos de diagnóstico molecular têm sido aplicados para detecção de A. marginale em bovinos. No entanto, a grande quantidade de testes com diferentes sensibilidade e custos tem dificultado a escolha do ensaio mais adequado. No presente estudo, uma PCR em tempo real baseada no gene msp5 foi avaliada quantitativamente e comparada a uma reação de PCR convencional. As reações foram submetidas à avaliação de sensibilidade e especificidade com DNA plasmidial e amostras provenientes de bovinos experimentalmente infectados por A. marginale. Uma avaliação comparativa a campo foi realizada entre os testes utilizando amostras provenientes de bovinos criados em uma região de estabilidade enzoótica para A. marginale. Embora os testes não tenham apresentado diferença estatisticamente significativa, a PCR em tempo real apresentou valor de sensibilidade maior do que a PCR convencional. A PCR em tempo real foi capaz de detectar uma cópia de msp5 em 100ng de DNA plasmidial, e mais de 80% de resultados positivos entre bovinos experimentalmente infectados apenas sete dias após infecção. Além disso, baseado em análise in silico, a PCR em tempo real avaliada aqui pode ser útil para detecção de Anaplasma ovis. The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR) based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR) was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

show abstract

“…Therefore, RT-PCR using Sybr Green demonstrated superior performance as a diagnostic tool. The RT-PCR described here has advantages over the recently developed RT-PCRs (DECARO et al, 2008), since they employ a Taqman system, which increases the tests costs, compared to method that uses the intercalating fluorochrome of DNA double strand. Another advantage is the use of the dissociation temperature, which confers a diagnostic specificity that is important for epizootiological studies.…”

Section: Introductionmentioning

confidence: 99%

“…Several molecular diagnostic methods with high levels of sensitivity and specificity are used, such as reverse line blotting (GUBBELS et al, 1999), conventional polymerase chain reaction (C-PCR) (FRENCH et al, 1998), semi-nested, nested PCR (MOLAD et al, 2006) and real time PCR (RT-PCR) (DECARO et al, 2008). This last technique presents some advantages, such as speed of obtaining results, quantification of the infection level, and a minor probability of contamination.…”

Section: Introductionmentioning

confidence: 99%

“…This last technique presents some advantages, such as speed of obtaining results, quantification of the infection level, and a minor probability of contamination. So far, the RT-PCR that has been developed to detect anaplasma in bovines uses the Taqman system as a reaction indicator (DECARO et al, 2008). This system requires a specific probe with a fluorescent reagent and a fluorescence emission quencher, making it more expensive than the technique that utilizes double strand DNA intercalating fluophore, the Syber Green.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Real time polymerase chain reaction to diagnose Anaplasma marginale in cattle and deer (Ozotoceros bezoarticus leucogaster) of the Brazilian Pantanal

Picoloto

Lima

Olegário

et al. 2010

Rev. Bras. Parasitol. Vet.

View full text Add to dashboard Cite

Epizootiological study of Anaplasma marginale in regions that contain various reservoir hosts, co-existence of rickettsia pathogens, and common vectors is a complicated task. To achieve diagnosis of this rickettsia in cattle and campeiro deer of Brazilian Pantanal, a comparison was made between a real time polymerase chain reaction (RT-PCR) with intercalating Sybr Green fluorochrome and primers based on msp5 gene of A. marginale; a conventional PCR (C-PCR); and parasitological examination using thin blood smear stained with Giemsa-MayGrunwald. Both PCRs showed good performance in the diagnosis of A. marginale in cattle, and were superior to the parasitological exam. The RT-PCR detected seven positive campeiro deer (16.3%). This rate was significantly higher compared to C-PCR, which identified one animal as positive (2.3%), and also compared to parasitological diagnosis, which did not find any positive animals. The dissociation temperature average of positive reactions in cattle (81.72 ºC ± 0.20) was identical to dissociation temperature found in the cervids (81.72 ºC ± 0.12), suggesting that both animal species were infected with A. marginale. We concluded that RT-PCR can be used for A. marginale diagnosis and in epizootiological studies of cattle and cervids; in spite of the small number of campeiro deer samples, the results indicated that this wildlife species has importance in the Anaplasma epizootiology in the Brazilian Pantanal.

show abstract

Duplex Real-Time Polymerase Chain Reaction for Simultaneous Detection and Quantification of Anaplasma Marginale and Anaplasma Centrale

Cited by 49 publications

References 16 publications

Detection of Anaplasma marginale and A. phagocytophilum in Bovine Peripheral Blood Samples by Duplex Real-Time Reverse Transcriptase PCR Assay

Detection of Anaplasma marginale and A. phagocytophilum in Bovine Peripheral Blood Samples by Duplex Real-Time Reverse Transcriptase PCR Assay

Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

Real time polymerase chain reaction to diagnose Anaplasma marginale in cattle and deer (Ozotoceros bezoarticus leucogaster) of the Brazilian Pantanal

Contact Info

Product

Resources

About