scholarly journals Diagnóstico de ectima contagioso em pequenos ruminantes através da Reação em Cadeia da Polimerase em Tempo Real

2021 ◽  
Vol 4 (5) ◽  
pp. 4-11
Author(s):  
Rosana Leo de Santana ◽  
Keyword(s):  
Foot And Mouth Disease ◽  
Contagious Ecthyma ◽  
Dissociation Curve ◽  
Affected Animal ◽  
Amplification Product ◽  
Chain Reaction ◽  
Field Samples ◽  
Restriction Sites ◽  
Specific Amplification ◽  
Polymerase Chain

The virus of contagious ecthyma (CEV), also known as orf virus (ORFV) is the etiological agent of contagious ecthyma (CE) in sheep and goat and belongs to the Parapoxvirus genus, family Poxviridae. In some cases, CE can be confused with vesicular diseases so there is need for differentiation especially because, according to the standards of the National Program for the Eradication of FMD (PNEFA), goats and sheep are not vaccinated against Foot and Mouth Disease (FMD), acting as sentinel animals. Although initial studies have demonstrated the usefulness of the polymerase chain reaction (PCR) as a diagnostic test, there are no studies involving its use on Brazilian field samples, which may be genetically distinct from previously studied samples, as described in a study of restriction sites analysis of Brazilian CE samples. This work was conducted with the goal of standardizing a PCR (qPCR) test using SYBR Green I dye for molecular diagnosis of EC in DNA extracted from lesions of affected animal or cell culture inoculated in field samples. The products were detected with qPCR dissociation curve analysis which showed a peak at 88 ºC indicating that positive samples have only one specific amplification product. All DNA samples tested (29 animals crusts and their cell cultures) were positive in the qPCR. The qPCR was able to detect the DNA of at least 10,000 times dilution corresponding to 0.056 ng of DNA. It is believed that with the additional qPCR validations reported in this study, it can be used for differential diagnosis in the health surveillance of PNEFA.

scholarly journals Identification and phylogenetic analysis of contagious ecthyma virus from camels (Camelus dromedarius) in Iran

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Ahmad Oryan ◽  
Mahboobe Mosadeghhesari ◽  
Saeed Zibaee ◽  
Ali Mohammadi
Keyword(s):  
Polymerase Chain Reaction ◽  
Phylogenetic Analysis ◽  
Camelus Dromedarius ◽  
Contagious Ecthyma ◽  
Dromedary Camels ◽  
Chain Reaction ◽  
B2l Gene ◽  
Bovine Papular Stomatitis Virus ◽  
Specific Amplification ◽  
Polymerase Chain

Contagious ecthyma is a highly contagious disease affecting domestic and wild ruminants such as sheep, goats and camels. The identification and characterisation of a parapoxvirus (PPV) infecting camels is described here. The virus was detected in dromedary camels (Camelus dromedarius) from Kerman and Shiraz in Iran. PPV-specific amplification by polymerase chain reaction (PCR) further confirmed that the disease was associated with PPV infection. Phylogenetic analysis of ORF011 (B2L) gene sequences showed 99.79% and 82.13% similarity of the PPV identified in this study with the Jodhpur isolate and the bovine papular stomatitis virus (BPSV) isolates (CE41), respectively. Moreover, phylogenetic analysis of the ORF045 gene indicated that the Shiraz sample was in all probability closely related to VR634 and to F00.120R and PCPV776. In conclusion, the results suggest that camel PPV (CPPV) is a likely cause of contagious ecthyma in dromedary camels in Iran.

scholarly journals Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽  
2021 ◽  
pp. 1-6
Author(s):  
Salman Khan ◽  
Syed Asad Ali Shah ◽  
Syed Muhammad Jamal
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Kappa Value ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Level Of Agreement

<b><i>Background:</i></b> Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. <b><i>Methods:</i></b> A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. <b><i>Results:</i></b> S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (<i>p</i> = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. <b><i>Conclusions:</i></b> The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

PCR BY THERMAL CONVECTION

2004 ◽  
Vol 18 (16) ◽  
pp. 775-784 ◽  
Author(s):  
DIETER BRAUN
Keyword(s):  
Polymerase Chain Reaction ◽  
Thermal Convection ◽  
Dna Amplification ◽  
Reaction Vessel ◽  
Cold Region ◽  
Chain Reaction ◽  
Heating And Cooling ◽  
Highly Sensitive ◽  
Specific Amplification ◽  
Polymerase Chain

The Polymerase Chain Reaction (PCR) allows for highly sensitive and specific amplification of DNA. It is the backbone of many genetic experiments and tests. Recently, three labs independently uncovered a novel and simple way to perform a PCR reaction. Instead of repetitive heating and cooling, a temperature gradient across the reaction vessel drives thermal convection. By convection, the reaction liquid circulates between hot and cold regions of the chamber. The convection triggers DNA amplification as the DNA melts into two single strands in the hot region and replicates into twice the amount in the cold region. The amplification progresses exponentially as the convection moves on. We review the characteristics of the different approaches and show the benefits and prospects of the method.

scholarly journals A PML/retinoic acid receptor alpha fusion transcript is constantly detected by RNA-based polymerase chain reaction in acute promyelocytic leukemia

Blood ◽  
1992 ◽  
Vol 79 (12) ◽  
pp. 3110-3115 ◽  
Author(s):  
S Castaigne ◽  
N Balitrand ◽  
H de The ◽  
A Dejean ◽  
L Degos ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Retinoic Acid ◽  
Retinoic Acid Receptor ◽  
Promyelocytic Leukemia ◽  
Retinoic Acid Receptor Alpha ◽  
Rt Pcr ◽  
Amplification Product ◽  
Chain Reaction ◽  
Acid Receptor ◽  
Polymerase Chain

Abstract The t(15;17) translocation is specifically observed in patients with promyelocytic leukemia (AML3). The chromosomal rearrangement juxtaposes the retinoic acid receptor alpha (RAR alpha) and PML genes, resulting in PML/RAR alpha fusion transcripts. Our previous studies have shown that a polymerase chain reaction (PCR) amplification product could be obtained from the cDNA of the NB4 promyelocytic cell line from which the chimaeric PML/RAR alpha was cloned. We report here that in all 14 AML3 patients tested, reverse transcriptase-PCR (RT-PCR) allows the detection of three specific fusion products. In eight patients, one amplification product was detected corresponding to the previously described abnormal fusion. Five patients displayed a different amplified fragment corresponding to a different fusion point. One other patient always showed a third different-sized product. The different types of fusion transcripts amplified were correlated to the size of the abnormal RAR alpha transcripts detected in these patients by Northern analysis, but did not prove determinant for either the phenotypic features or the retinoic acid responsiveness in AML3 cells in this group of patients. The consistent identification by RT-PCR of the fusion of the PML and RAR alpha genes in AML3 patients suggest that this method will provide a useful tool for the diagnosis and detection of minimal residual disease in these patients.

scholarly journals Field evaluation of a quantitative polymerase chain reaction assay for Mycoplasma hyorhinis

2014 ◽  
Vol 26 (6) ◽  
pp. 755-760 ◽  
Author(s):  
Maria J. Clavijo ◽  
Simone Oliveira ◽  
Jeffrey Zimmerman ◽  
Aaron Rendahl ◽  
Albert Rovira
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Assay ◽  
Quantitative Polymerase Chain Reaction ◽  
Systemic Disease ◽  
Analytical Sensitivity ◽  
Upper Respiratory Tract ◽  
Chain Reaction ◽  
Mycoplasma Hyorhinis ◽  
Field Samples ◽  
Polymerase Chain

Mycoplasma hyorhinis has emerged as an important cause of systemic disease in nursery pigs. However, this bacterium can also be found in the upper respiratory tract of healthy swine. The current study describes the development of a quantitative polymerase chain reaction assay for the detection of M. hyorhinis and the evaluation of the assay in both disease diagnosis and disease surveillance using a large number of field samples. The analytical sensitivity was estimated to be 12 genome equivalents/μl. The assay was highly specific, detecting all 25 M. hyorhinis isolates tested and none of the 19 nontarget species tested. Assay repeatability was evaluated by testing different matrices spiked with known amounts of M. hyorhinis. Overall, assessment of the repeatability of the assay showed suitable precision within and between runs for all matrices. The coefficient of variation ranged from 10% to 24%. Mycoplasma hyorhinis DNA was detected in 48% of samples (pericardium, pleura, joints, nasal cavity, and lungs) from pigs with systemic disease. Mycoplasma hyorhinis was detected in nasal (92%) and oropharyngeal swabs (66%), as well as in oral fluids (100%). Potential uses of this tool involve the characterization of the prevalence of this pathogen in swine herds as well as bacterial quantification to evaluate intervention efficacy.

Development of polymerase chain reaction and fluorescent in situ hybridisation techniques for the detection of a bacterial strain that degrades the cyanobacterial toxin microcystin LR

2005 ◽  
Vol 56 (8) ◽  
pp. 1127 ◽  
Author(s):  
D. G. Bourne ◽  
R. L. Blakeley ◽  
P. Riddles ◽  
G. J. Jones
Keyword(s):  
Polymerase Chain Reaction ◽  
Environmental Samples ◽  
In Situ Hybridisation ◽  
Cyanobacterial Blooms ◽  
Chain Reaction ◽  
Fluorescent In Situ Hybridisation ◽  
Cyanobacterial Toxin ◽  
Specific Amplification ◽  
Polymerase Chain

Polymerase chain reaction (PCR) and fluorescent in situ hybridisation (FISH) techniques were developed for the detection of a Sphingomonas bacterium (strain MJ-PV), previously demonstrated to degrade the cyanobacterial toxin microcystin LR. A PCR amplification protocol using the primer set Sph-f1008/Sph-r1243 demonstrated specific amplification of the target 16S ribosomal DNA (rDNA) of strain MJ-PV. A 16S ribosomal RNA (rRNA) targeted probe, Sph-r1264, labelled with a rhodamine fluorescent dye was successfully used in whole-cell FISH for the detection of MJ-PV in seeded controls. DNA primers and a PCR protocol were developed for the specific amplification of a gene, mlrA, which codes for the enzyme MlrA, responsible for hydrolysis of the cyanobacterial toxin microcystin LR. A survey using 16S rDNA and mlrA primers on extracted DNA from environmental samples of a lake that suffers regular toxic cyanobacterial blooms demonstrated no amplified products indicative of the presence of MJ-PV or mlrA. Although not detecting the MJ-PV strain in the tested environmental samples, these developed methods are useful to study the distribution of strain MJ-PV demonstrated to degrade mycrocystin LR in seeded bioremediation trails, as well as the distribution and the regulation of mlrA shown to be involved in mycrocystin LR degradation.

scholarly journals Evaluation of Polymerase Chain Reaction (PCR)-Based Methods for Rapid, Accurate Detection and Monitoring of Verticillium dahliae in Woody Hosts by Real-Time PCR

Plant Disease ◽  
2015 ◽  
Vol 99 (6) ◽  
pp. 866-873 ◽  
Author(s):  
Baker Aljawasim ◽  
Paul Vincelli
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Verticillium Dahliae ◽  
Sugar Maple ◽  
Broad Host Range ◽  
Chain Reaction ◽  
Accurate Detection ◽  
Field Samples ◽  
Vascular Discoloration ◽  
Polymerase Chain

Verticillium wilt, caused by Verticillium dahliae, is one of the most economically important diseases of woody hosts such as ash (Fraxinus spp.), sugar maple (Acer saccharum), and redbud (Cercis canadensis). The causal agent has a broad host range, including not only woody hosts but also important vegetable and field crops, and it is distributed worldwide. Diagnosis of V. dahliae in infected woody hosts is often based on the occurrence of vascular discoloration and time-consuming isolation. However, not all woody hosts exhibit vascular discoloration, and not all vascular discoloration symptoms are due to infection by V. dahliae. In this study, real-time polymerase chain reaction (PCR)-based assays were evaluated and employed for rapid and accurate detection of V. dahliae in different woody hosts. High-quality DNA was extracted in large quantities from presumptively infected woody hosts by collecting drill-press shavings from sample tissue, bead beating, and extracting using a cetyltrimethylammonium bromide method. Six published primer sets were evaluated against genomic DNA of V. dahliae as well as selected negative controls, and two sets (VertBt-F/VertBt-R and VDS1/VDS2) showed promise for further evaluation using DNA extracts from field samples. The VertBt primers amplified a species-specific 115-bp fragment of the expected size, while the VDS primers amplified the expected specific 540-bp fragment. However, the VertBt primer set exhibited higher sensitivity in detection of V. dahliae even in asymptomatic trees. The PCR-based methods developed here could be used as rapid tools for pathogen detection and monitoring, thus informing plant pathogen management decisions.

scholarly journals Detection of minimal residual disease in acute myelomonocytic leukemia with abnormal marrow eosinophils by nested polymerase chain reaction with allele specific amplification

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2291-2296 ◽  
Author(s):  
J Hebert ◽  
JM Cayuela ◽  
MT Daniel ◽  
R Berger ◽  
F Sigaux
Keyword(s):  
Residual Disease ◽  
Fusion Transcript ◽  
Type A ◽  
Chain Reaction ◽  
Allele Specific ◽  
Acute Myelomonocytic Leukemia ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Myelomonocytic Leukemia ◽  
Minimal Residual

Abstract Acute myelomonocytic leukemia with bone marrow eosinophilia (AML-M4Eo in the French-American-British FAB] classification) is frequently associated with pericentric inversion of chromosome 16, inv(16)(p13q22). Recently, the molecular cloning of teh breakpoints has led to the identification of the two fused genes, CBFB on 16q and MYH11 on 16p. We have analyzed 24 patients with AML-M4Eo at diagnosis and 47 patients with AML of other FAB subtypes, by a reverse-transcriptase polymerase chain reaction (RT-PCR) assay for the CBFB/MYH11 fusion mRNAs. Three types of fusion mRNAs were detected in 22 samples of AML- M4Eo (type A, n = 20; type C, n = 1; and type D, n = 1). Among these 22 positive samples, inv(16) was found in the 20 cytogenetically studied cases. No fusion transcript was detected in two patients with AML-M4Eo and in patients with other types of AML. These results confirm that CBFB/MYH11 transcripts (with a predominant type A form) are present in most cases of inv(16) AML. Moreover, detection of the hybrid transcript is closely associated with the finding of abnormal bone marrow (BM) eosinophils in AML-M4Eo as it is not found in other, FAB subtypes of AML, including AML-M4. To assess the presence of type A CBFB/MYH11 fusion transcripts in five AML-M4Eo patients in remission, we designed a sensitive assay combining nested PCR and allele-specific amplification (NPASA). Residual leukemia cells were detected in four patients who were in remission from 4 to 22 months, but not in one patient in long-term remission (5 years). The clinical relevance of persistent CBFB/MYH11 fusion transcripts in remission remains to be established by studying a large prospective series of patients. NPASA provides a useful and sensitive tool for the detection of minimal residual disease in inv(16) AML and, potentially, in other leukemias associated with translocations that result in a predominant fusion transcript.

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