A multiplex TaqMan PCR assay for the detection of measles and rubella virus

Hübschen, Judith M.; Kremer, Jacques R.; Landtsheer, Sébastien De

doi:10.1016/j.jviromet.2008.01.032

Cited by 81 publications

(53 citation statements)

References 34 publications

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“…A total of 200 l of eluted nucleic acids were divided into 3 aliquots and stored at Ϫ80°C. Reverse transcription of total RNA with random primers (Invitrogen, Merelbeke, Belgium) and the measles virus PCR (for quality control) were performed as described previously (27). Specific detection PCRs of all pathogens were carried out with previously published primers using 5 l of raw DNA or cDNA…”

Section: Methodsmentioning

confidence: 99%

Prevalence and Seasonality of Tick-Borne Pathogens in Questing Ixodes ricinus Ticks from Luxembourg

Reye

Hübschen

Sausy

et al. 2010

Appl Environ Microbiol

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In Europe, ixodid ticks are important arthropod vectors of human and animal pathogens, but comprehensive studies of the prevalence of all relevant pathogens in Central Europe are scarce. As a result of ecological changes, the incidences of tick-borne infections are expected to increase. In this study, 1,394 nymphal and adult Ixodes ricinus ticks sampled monthly during the active season from 33 ecologically distinct collection sites throughout Luxembourg were screened for all human tick-borne pathogens relevant in Central Europe. Species were identified by sequence analysis of detection PCR amplicons. Mean infection rates of ticks were 11.3% for Borrelia burgdorferi sensu lato, 5.1% for Rickettsia sp., 2.7% for Babesia sp., and 1.9% for Anaplasma phagocytophilum. No tick was found to be infected with Coxiella sp., Francisella tularensis subsp., or Tick-borne encephalitis virus (TBEV). A total of 3.2% of ticks were infected with more than one pathogen species, including mixed Borrelia infections (1.5%). Seasonal variations of tick infection rates were observed for Borrelia, Babesia, and Anaplasma, possibly reflecting a behavioral adaptation strategy of questing ticks. A positive correlation between the grade of urbanization and Borrelia infection rate of ticks was observed, suggesting an established urban zoonotic cycle. We also found Hepatozoon canis (0.1%) and Bartonella henselae (0.3%), which so far have not been found in questing Ixodes ricinus ticks in Central Europe.In Western Europe, the hard tick Ixodes ricinus is the main arthropod vector of various human and animal pathogens, causing several tens of thousands of severe infections in humans every year (25, 37). The most common tick-borne infection is Lyme borreliosis. This multisystemic disorder is caused by spirochetes of the Borrelia burgdorferi sensu lato complex, which is comprised of at least 12 species worldwide (45). Among the 6 European species, only Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto are known as human pathogens, whereas the significance of Borrelia valaisiana, Borrelia spielmanii, and Borrelia lusitaniae for human health is not clear (24). In a metaanalysis of 154 European studies, a mean of 13.7% of ticks were found to be infected with Borrelia spp., predominantly with B. afzelii and B. garinii. However, the prevalence of Borrelia species varies from 2 to 49% between different regions (43).Other tick-borne bacteria which cause disease in humans are Rickettsia sp., Anaplasma phagocytophilum, Bartonella henselae and Bartonella quintana, Coxiella burnetii, and Francisella tularensis subsp., all of which show only relatively low prevalence rates of 0.1 to 4.8% for European ticks (16,23,26,42,48,52). In addition, three species of the parasitic protozoan Babesia are known to infect humans, namely, B. divergens, B. microti, and the newly described Babesia sp. EU1 (5). Also, in Western Europe, Tick-borne encephalitis virus (TBEV) has a relatively low prevalence; however, this pathogen deserves special attention b...

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Section: Methodsmentioning

confidence: 99%

Prevalence and Seasonality of Tick-Borne Pathogens in Questing Ixodes ricinus Ticks from Luxembourg

Reye

Hübschen

Sausy

et al. 2010

Appl Environ Microbiol

Self Cite

145

105

View full text Add to dashboard Cite

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“…The throat swabs were used for PCR analysis as described previously [1] and for virus isolation [2]. Phylogenetic analysis based on the rubella virus E1 glycoprotein gene was done with MEGA [3] and sequences were compared to published sequences by BLAST.…”

Section: Number Of Casesmentioning

confidence: 99%

Ongoing rubella outbreak in Bosnia and Herzegovina, March-July 2009 - preliminary report

Novo¹,

Huebschen

Tesanovic

et al. 2009

Eurosurveillance

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“…Current procedures for the diagnosis of acute gastroenteritis in individual patients are based on viral RNA or DNA detection by conventional PCR assays and specific real-time RT-PCR assays. To optimize the use of sometimes limited quantities of patient materials, random primers can be used in the RT step required for the detection of RNA viruses (9,19,21). The cDNA formed in such a reaction can be used in PCR assays specific for different viruses (9,19,21).…”

mentioning

confidence: 99%

“…To optimize the use of sometimes limited quantities of patient materials, random primers can be used in the RT step required for the detection of RNA viruses (9,19,21). The cDNA formed in such a reaction can be used in PCR assays specific for different viruses (9,19,21). The detection of several different viruses can be accelerated and simplified by use of multiplex real-time PCR assays with multiple primer pairs in a single tube, making it possible to amplify multiple target sequences (9,15,18,19,21).…”

mentioning

confidence: 99%

“…The cDNA formed in such a reaction can be used in PCR assays specific for different viruses (9,19,21). The detection of several different viruses can be accelerated and simplified by use of multiplex real-time PCR assays with multiple primer pairs in a single tube, making it possible to amplify multiple target sequences (9,15,18,19,21). The use of random primers and multiplex PCR assays in combination reduces reagent and labor costs, as well as the turnaround time.…”

mentioning

confidence: 99%

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Novel Approach for Detection of Enteric Viruses To Enable Syndrome Surveillance of Acute Viral Gastroenteritis

et al. 2009

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Acute gastroenteritis is one of the most common diseases worldwide, with viruses, particularly noroviruses, being the leading cause in developed countries. In The Netherlands, systematic surveillance of gastroenteritis outbreaks of suspected viral etiology was established by the National Institute for Public Health and the Environment in 1994. Since 2002, the total number of outbreaks reported has been increasing, and with that comes the need for sensitive assays that can be performed quickly. In addition, the diagnostic demand changed so that now the proportion of samples from hospitals is higher and there is a need for patient-based test results. In order to target the diagnosis of acute gastroenteritis, we reviewed our data on outbreaks of gastroenteritis and the prevalence of individual viruses to provide a priority list of viruses for which samples should be evaluated. Random primers were used to replace the separate specific primers for each virus used in the reverse transcription steps. The individual PCR assays were replaced by multiplex PCR assays. We employed a two-step method in which in the first step we screened for the most common causes of viral gastroenteritis, noroviruses of genogroup II and rotaviruses of group A, with equine arteritis virus used as the internal control. Subsequently, in the second step, two parallel PCR assays were developed for the detection of noroviruses of genogroup I and equine arteritis virus in one run and adenoviruses, sapoviruses, and astroviruses in the other run. The specificities of the assays were calculated to be 92.5% for the assay for noroviruses of genogroup I and 100% for the assays for all other viruses, the detection limits were equal for all viruses, and the turnaround time was reduced to 1 day compared to the at least 3 days required for the methods used previously. This approach allows the targeted, rapid, and cost-effective elucidation of the causes of acute gastroenteritis outbreaks.

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A multiplex TaqMan PCR assay for the detection of measles and rubella virus

Cited by 81 publications

References 34 publications

Prevalence and Seasonality of Tick-Borne Pathogens in Questing Ixodes ricinus Ticks from Luxembourg

Prevalence and Seasonality of Tick-Borne Pathogens in Questing Ixodes ricinus Ticks from Luxembourg

Ongoing rubella outbreak in Bosnia and Herzegovina, March-July 2009 - preliminary report

Novel Approach for Detection of Enteric Viruses To Enable Syndrome Surveillance of Acute Viral Gastroenteritis

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