Development of a single tube nested polymerase chain reaction assay for the detection of Neospora caninum DNA

Ellis, John; McMillan, D.; Ryce, Cheryl; Payne, Suzanne; Atkinson, R; Harper, P. A. W.

doi:10.1016/s0020-7519(99)00144-7

Cited by 48 publications

(34 citation statements)

References 28 publications

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“…In addition, such analysis should compensate for the presence of potential PCR-inhibiting compounds and exclude false-negative results due to poor-quality DNA. Another criterion for the design of both 28S rRNA and Neospora Nc5 primers was that the resultant amplicon should be as short as practical since DNA in some clinical samples may be degraded due to autolysis, aging, or chemical fixation (14).…”

Section: Discussionmentioning

confidence: 99%

“…Immunohistochemistry (IHC) is a relatively insensitive technique for detecting the parasite in host tissues due to low parasite numbers and, sometimes, due to the low quality of the fetal tissue that could be autolyzed, mummified, or macerated (6,12). As an alternative, several PCR-based methods have been developed in the last few years targeting the parasite ITS1 region (27) and the repeated Neospora-specific Nc5 sequence (18) with different modifications, such as nested or seminested PCR test, trying to increase the sensitivity and specificity of the technique (6,13,14). PCR techniques have been useful as diagnostic tools for detection of the parasite in bovine aborted fetuses (6,14,16,28) and to study the parasite distribution in experimentally infected mice (20,31), as a first step to characterize an experimental model of the infection to be used in the near future in drug and vaccine testing.…”

mentioning

confidence: 99%

“…As an alternative, several PCR-based methods have been developed in the last few years targeting the parasite ITS1 region (27) and the repeated Neospora-specific Nc5 sequence (18) with different modifications, such as nested or seminested PCR test, trying to increase the sensitivity and specificity of the technique (6,13,14). PCR techniques have been useful as diagnostic tools for detection of the parasite in bovine aborted fetuses (6,14,16,28) and to study the parasite distribution in experimentally infected mice (20,31), as a first step to characterize an experimental model of the infection to be used in the near future in drug and vaccine testing. At present, only a quantitative-competitive PCR (QC-PCR) technique for the detection of Neospora in host tissues has been reported (19), based on the inclusion of a known competitor to the target Neospora-specific Nc5 genomic sequences.…”

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Quantitative Detection ofNeospora caninumin Bovine Aborted Fetuses and Experimentally Infected Mice by Real-Time PCR

et al. 2002

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We report the development of a real-time PCR assay for the quantitative detection of Neospora caninum in infected host tissues. The assay uses the double-stranded DNA-binding dye SYBR Green I to continuously monitor product formation. Oligonucleotide primers were designed to amplify a 76-bp DNA fragment corresponding to the Nc5 sequence of N. caninum. A similar method was developed to quantify the 28S rRNA host gene in order to compare the parasite load of different samples and to correct for the presence of potential PCR-inhibiting compounds in the DNA samples. A linear quantitative detection range of 6 logs with a calculated detection limit of 10 ؊1 tachyzoite per assay was observed with excellent linearity (R 2 ‫؍‬ 0.998). Assay specificity was confirmed by using DNA from the closely related parasite Toxoplasma gondii. The applicability of the technique was successfully tested in a variety of host brain tissues: (i) aborted bovine fetuses classified into negative or positive Neospora-infected animals according to the observation of compatible lesions by histopathological study and (ii) experimentally infected BALB/c mice, divided into three groups, inoculated animals with or without compatible lesions and negative controls. All samples were also tested by ITS1 Neospora nested PCR and a high degree of agreement was shown between both PCR techniques ( ‫؍‬ 0.86). This technique represents a useful quantitative diagnostic tool to be used in the study of the pathogenicity, immunoprophylaxis, and treatment of Neospora infection.

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Quantitative Detection ofNeospora caninumin Bovine Aborted Fetuses and Experimentally Infected Mice by Real-Time PCR

et al. 2002

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“…2,5,6,9 In the present study, PCR assay was applied in routine detection of C. burnetii, C. abortus, Salmonella enterica Serovar abortusovis, T. gondii, and Neospora caninum in fetal tissues from naturally aborted ewes and goats in 2003-2005. A total of 315 fetuses (292 ovine and 23 caprine) and 84 placentae (including 76 ovine and 8 caprine samples) were analyzed during [2003][2004][2005] (Table 1). Following macroscopic examination, the brain, skeletal muscle, liver, spleen, and abomasum were removed from each fetus.…”

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Detection of Pathogens in Ovine and Caprine Abortion Samples from Sardinia, Italy, by PCR

et al. 2007

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Abstract. During 2003During -2005 abortion samples (315 fetuses and 84 placentae) were collected from 107 ovine and caprine farms in northern Sardinia. Tissues from aborted fetuses and placentae were examined by PCR assay to detect DNA from Coxiella burnetii, Chlamydophila abortus, Salmonella enterica Serovar abortusovis, Toxoplasma gondii, and Neospora caninum. The DNA from at least 1 of these 5 infectious agents was amplified in 41% of ovine fetuses, while only 17% of the caprine fetuses yielded a positive amplification result for at least 1 of the 5 agents. Out of a total of 366 ovine aborted samples, T. gondii DNA was detected most frequently (18.1% of fetuses and 13.1% of placentae), followed by S. abortusovis (13% of fetuses and 14.4% of placentae), C. burnetii (10.9% of fetuses, of 9.2% placentae), C. abortus (2.4% of fetuses, 6.5% of placentae), and N. caninum (2% of placentae). In 33 fetuses and 9 placentae, the simultaneous presence of pathogens with different associations was detected. Out of a total of 31 caprine aborted samples, T. gondii was detected most frequently (13% of fetuses and 25% of placentae), followed by C. abortus (12.5% of placentae), C. burnetii (12.5% of placentae), and N. caninum (8.6%).

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“…Brain tissue is commonly used in PCR because cyst formation is common in this structure. In epidemiological surveys the detection of antibodies to the parasite is achieved by ELISA technique or indirect immunofluorescence (IFAT) (THURMOND; HIETALA, 1999;ELLIS et al, 1999). Even though these procedures are well known, a great variation in the infection rate of neosporosis is found among cattle herds (THILSTED; DUBEY, 1989;RAGOSO, 2003).…”

Section: Introductionmentioning

confidence: 99%

Incidência e transmissão transplacentária de Neospora caninum em fêmeas primíparas da raça Bos indicus abatidos em Presidente Prudente, São Paulo, Brasil

et al. 2008

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Development of a single tube nested polymerase chain reaction assay for the detection of Neospora caninum DNA

Cited by 48 publications

References 28 publications

Quantitative Detection ofNeospora caninumin Bovine Aborted Fetuses and Experimentally Infected Mice by Real-Time PCR

Quantitative Detection ofNeospora caninumin Bovine Aborted Fetuses and Experimentally Infected Mice by Real-Time PCR

Detection of Pathogens in Ovine and Caprine Abortion Samples from Sardinia, Italy, by PCR

Incidência e transmissão transplacentária de Neospora caninum em fêmeas primíparas da raça Bos indicus abatidos em Presidente Prudente, São Paulo, Brasil

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