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The mechanically transmitted haemoflagellate,Trypanosoma evansicauses 'surra', a wasting disease of domestic animals and is highly endemic in distribution in Southeast Asia. The detection ofT. evansiis important for improving the epizootiological and animal health status of the region. The specificity and sensitivity of polymerase chain reaction (PCR) using oligonucleotide primers constructed fromT. evansirepetitive DNA sequences were studied in the present investigation. Using the assay, it was possible to amplify template DNA ofT. evansiderived from buffaloes, camels and horses to a threshold sensitivity level of 0.5 pg and to detect DNA from as few as five organisms in 10 (l crude blood samples. Following experimental infection of calves with 5 × 105T. evansi, positive signals could be observed as early as 12 h post-infection. DNAs from two common haemoflagellates of cattle,Babesia bigeminaandTheileria annulatawere not amplified with the primers.
Glare EM, Patton JC, Premier RR, Lawrence AJ, Nisbett IT: Analysis of a repetitive DNA sequence from Bordetella pertussis and its application to the diagnosis of pertussis using the polymerase chain reaction. J Clin Microbiol 28, 1982–1987 (1990
Carvalho, R., Oliveira, A. M., Souza, A. M., Passos, L. M. F. and Martins, A. S. (2000): Prevalence of equine herpesvirus type 1 latency detected by polymerase chain reaction. Arch. Virol. 145 , 1773-1787. Prevalence of equine
northern Mexican states, prevalence between 12.4% and 16.6% were found in female sex workers [ 16 – 17 ]. In the present study, by using a different study design (case–control) and laboratory method (polymerase chain reaction [PCR]) compared to those used
A polymerase chain reaction (PCR) assay was used to detect the Indian isolate of banana bunchy top virus at early stages of infection in banana suckers before symptom expression. The viral DNA was detected from a single viruliferous banana aphid (Pentalonia nigronervosa) at 1.0 kb. The six plant species viz., Zingiber officinale, Colocasia esculenta, Catheranthus roseus, Canna indica, Hedychium coronarium and Alphinium sp. upon inoculation with BBTV showed negative results in PCR assay. Using PCR assay the BBTV could be detected in meristem tip cultured banana plants before symptom expression. In field condition the meristem tip cultured banana plants expressed BBTV symptom 45 days after planting.
Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in pharyngeal and rectal swab samples of a cat seropositive for adenovirus and suffering from transient hepatic failure. The samples were taken at a one-year interval, and both faecal samples as well as the second pharyngeal sample were positive in PCR performed with general adenovirus primers. The size of the amplified products corresponded to that of the positive control. The identity of the amplicons was also confirmed by DNA sequencing. The 301 bp long hexon gene fragment was very similar to but distinguishable from the corresponding hexon sequence of human adenovirus type 2. This result suggests the possibility of persistent carrier status and shedding of adenovirus in cats.
In this study, bovine viral diarrhoea virus (BVDV) was detected and biotypically characterised in clinical samples using reverse transcriptase-polymerase chain reaction (RT-PCR). The RT-PCR technique produced two different amplicons (402 and approx. 680 bp in size) in case of the presence of both biotypes (cp and ncp) in the sample. The mixture of the biotypes as detected by RT-PCR was verified by the immunoplaque assay (IPA). Purification of biotypes was carried out by native plaque isolation and subsequent RT-PCR revealed single products (402 or approx. 680 bp in size) in each clone. The results showed that RT-PCR can be used for accurate molecular differentiation between the BVDV biotypes.
Polymerase chain reaction (PCR) was used to amplify the spacer regions between the 16S and 23S genes of rRNA genetic loci of Salmonella serovars for their rapid identification. These genetic loci revealed a significant level of polymorphism in length across the species/serovar lines. When the 16S-23S spacer region amplification products were subjected to agarose electrophoresis, the patterns observed could be used to distinguish all the serovars of Salmonella tested. Unique elements obtained in amplification products were mostly clustered at serovar level, although certain genus-specific patterns were also observed. On the basis of the results obtained, the amplification of 16S-23S ribosomal spacer region could suitably be used in a PCR-based identification method for Salmonella serovars.
The typing of non-tuberculous mycobacteria (NTM) is important from a clinical and epidemiological perspective. The polymerase chain reaction-restriction enzyme analysis (PRA) method and DNA sequence analysis method were utilized to target a gene region that codes the 65-kDa heat-shock protein for typing 150 suspected NTM samples isolated from the respiratory tract. Mycobacterium abscessus, Mycobacterium xenopi, Mycobacterium fortuitum, and Mycobacterium peregrinum were most frequently found by both methods. Six isolates that could not be defined by the PRA method were defined as Nocardia cyriacigeorgica, Nocardia abscessus, and Mycobacterium intracellulare by DNA sequence analysis. Discordance between the results of the two methods was observed for only one isolate. The isolate that was defined as Mycobacterium gordonae type 6 by the PRA method was defined as Mycobacterium senegalense by sequence analysis. The PRA method is simple and gives rapid results. Compared with DNA sequence analysis, it gives consistent and reliable results up to a ratio of 90%. DNA sequence analysis is the gold standard method in which all strains can be defined. However, given our laboratory conditions, its disadvantage is that it takes longer to reach a diagnosis than through the PRA method.
Primary human cytomegalovirus infection and the viral reactivation from latency are major complications in organ transplant recipients. In the peripheral blood the replicating virus can be detected either by nucleic acid based tests or by demonstrating the HCMV structural proteins in antigenemia test. We developed a quantitative competitive PCR method to assess the HCMV load in the peripheral blood. The viral load in nine healthy blood donors and in four renal transplant recipients with negative antigenemia test was in the same range: 5-124 (median: 18) HCMV copies/106 b-globin copies for healthy blood donors and 16-48 (median: 37) HCMV copies/106 b-globin copies for the transplant recipients. Three antigenemia positive renal transplant recipients had a HCMV load of 2,2´105/106 b-globin, 1,5´104/106 b-globin and 6,5´103/106 b-globin, respectively. In conclusion, the quantitative measurement of HCMV load in the peripheral blood correlated well with the routine HCMV antigenemia test. The DNA-based test, however can detect earlier the reactivation of the HCMV infection.
