scholarly journals Discrimination between sheep-associated and wildebeest-associated malignant catarrhal fever virus by means of a single-tube duplex nested PCR

2005 ◽  
Vol 72 (4) ◽  
Author(s):  
C.W. Bremer ◽  
H. Swart ◽  
F.A. Doboro ◽  
B. Dungu ◽  
M. Romito ◽  
...  
Keyword(s):  
Dna Amplification ◽  
Malignant Catarrhal Fever ◽  
Nested Polymerase Chain Reaction ◽  
Variable Regions ◽  
Single Tube ◽  
Annealing Temperatures ◽  
High Annealing ◽  
Herpesvirus 1 ◽  
Polymerase Chain ◽  
Primer Sets

A single-tube duplex nested polymerase chain reaction (sdn-PCR) was developed for the detection of and discrimination between ovine herpesvirus-2 (OvHV-2) and alcelaphine herpesvirus-1 (AlHV-1). These viruses respectively cause sheep- and wildebeest-associated malignant catarrhal fever (SAMCF and WA-MCF). In the first step of the sdn-PCR, two primers with high annealing temperatures based on conserved regions of the tegument genes were used for DNA amplification. In the second step, two primer sets based on variable regions of the respective OvHV-2 and AlHV-1 genes and with annealing temperatures > 11 °C below the primers used in the first step, were used. Internal regions of different sizes from amplicons produced in the first step were amplified. This single-tube test obviates the need for two separate assays to detect both viral types, thereby reducing time, labour and cost.

scholarly journals Discrimination between sheep-associated and wildebeest-associated malignant catarrhal fever virus by means of a single-tube duplex nested PCR

2005 ◽  
Vol 72 (4) ◽  
pp. 285-291
Author(s):  
C.W. Bremer ◽  
H. Swart ◽  
F.A. Doboro ◽  
B. Dungu ◽  
M. Romito ◽  
...  
Keyword(s):  
Dna Amplification ◽  
Malignant Catarrhal Fever ◽  
Nested Polymerase Chain Reaction ◽  
Variable Regions ◽  
Single Tube ◽  
Annealing Temperatures ◽  
High Annealing ◽  
Herpesvirus 1 ◽  
Polymerase Chain ◽  
Primer Sets

A single-tube duplex nested polymerase chain reaction (sdn-PCR) was developed for the detection of and discrimination between ovine herpesvirus-2 (OvHV-2) and alcelaphine herpesvirus-1 (AlHV-1). These viruses respectively cause sheep- and wildebeest-associated malignant catarrhal fever (SAMCF and WA-MCF). In the first step of the sdn-PCR, two primers with high annealing temperatures based on conserved regions of the tegument genes were used for DNA amplification. In the second step, two primer sets based on variable regions of the respective OvHV-2 and AlHV-1 genes and with annealing temperatures > 11 °C below the primers used in the first step, were used. Internal regions of different sizes from amplicons produced in the first step were amplified. This single-tube test obviates the need for two separate assays to detect both viral types, thereby reducing time, labour and cost.

Rapid cycle allele-specific amplification: studies with the cystic fibrosis delta F508 locus

1993 ◽  
Vol 39 (5) ◽  
pp. 804-809 ◽  
Author(s):  
C T Wittwer ◽  
B C Marshall ◽  
G H Reed ◽  
J L Cherry
Keyword(s):  
Cystic Fibrosis ◽  
Dna Amplification ◽  
Temperature Cycling ◽  
Rapid Cycling ◽  
Cycle Times ◽  
Product Specificity ◽  
Annealing Temperatures ◽  
Allele Specific ◽  
Specific Amplification ◽  
Polymerase Chain

Abstract Rapid cycle DNA amplification is a polymerase chain reaction technique with improved product specificity and cycle times of 20-60 s, allowing complete 30-cycle reactions in 10-30 min. The presence or absence of the delta F508 deletion and wild-type allele was determined in 104 cystic fibrosis patients by rapid cycle DNA amplification. In separate allele-specific assays, sequences on both sides of the delta F508 locus were amplified with the 3' end of a discriminating primer at the delta F508 locus, with either a 3-bp or a 1-bp mismatch. With rapid cycling (35-s cycles), single-base discrimination was achieved over a broad range of annealing temperatures (50 degrees C or lower); with conventional cycling and "hot starts" (160-s cycles), only annealing temperatures of 61-62 degrees C sufficiently discriminated between alleles. With rapid cycling, genotype could still be assessed with annealing temperatures as low as 25 degrees C. We conclude that faster temperature cycling can improve the results of allele-specific amplification.

scholarly journals Detection of ovine herpesvirus-2 in clinical cases of sheep-associated malignant catarrhal fever in balinese cattle and apparently healthy sheep in East Nusa Tenggara

2021 ◽  
Vol 33 ◽  
pp. 06006
Author(s):  
Agus Wiyono ◽  
Harimurti Nuradji ◽  
Maxs UE Sanam ◽  
Yohanes TRMR Simarmata ◽  
Rini Damayanti
Keyword(s):  
Fatal Outcome ◽  
Clinical Signs ◽  
Economic Losses ◽  
Malignant Catarrhal Fever ◽  
Test Results ◽  
Nested Polymerase Chain Reaction ◽  
Healthy Cattle ◽  
Healthy Sheep ◽  
Vaginal Swabs ◽  
Polymerase Chain

Malignant catarrhal fever (MCF) is a disease causing a fatal outcome in cattle and generates economic losses worldwide. This study aims to detect the cause of the disease in Balinese cattle showing clinical signs such as high fever, serous ocular mucopurulent nasal discharges, and enlargement of pre-scapularis and pre-femoralis lymphnodes. These cattle were previously housed 50 meters away from a flock of sheep which were brought from Sabu Island 3 months earlier. Samples including blood, ocular, nasal, and vaginal swabs were collected from 22 sheep, 30 goats, 33 clinically healthy cattle (22 Balinese and 11 Ongole cattle), and 3 infected Balinese cattle. Samples were processed and tested using A nested polymerase chain reaction (PCR) test. Results showed t hat 12 sheep out of 22 and 3 out of 3 infected Balinese cattle were positive MCF, suggesting a potential spread of the disease from sheep to Balinese cattle. No goats and Ongole cattle that were positive indicate that these animals are less susceptible to Ovine Herpesvirus-2 (OvHV-2) infection compared to Balinese cattle. The finding of 5 positive samples from 22 healthy Balinese cattle shows the potential of sub-clinical infection of OvHV-2.

scholarly journals The prevalence of ovine herpesvirus-2 in 4 sheep breeds from different regions in South Africa

2010 ◽  
Vol 81 (2) ◽  
Author(s):  
C.W. Bremer
Keyword(s):  
South Africa ◽  
Nested Pcr ◽  
Lower Incidence ◽  
The Other ◽  
Malignant Catarrhal Fever ◽  
Single Tube ◽  
Sheep Breeds ◽  
Significant Difference ◽  
Herpesvirus 1 ◽  
Low Incidence

About 90% of bovine malignant catarrhal fever (BMCF) PCR-positive cases in South Africa are caused by alcelaphine herpesvirus-1 (AlHV-1) and the other 10 % by ovine herpesvirus-2 (OvHV-2). The prevalence of OvHV-2 in different sheep breeds in South Africa was determined in order to investigate whether the lower incidence of BMCF caused by OvHV-2 in comparison with AlHV-1 can be ascribed to a low incidence of the virus in sheep. A single-tube hemi-nested PCR was developed, evaluated and applied to detect OvHV-2 DNA. The prevalence of the virus in 4 sheep breeds from various regions in South Africa was shown to be 77 %. No statistically significant difference was found amongst the sheep breeds tested.

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