One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae.
Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%-4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples, PPRV in 45, and PM in six samples. In addition, three samples showed a co-infection of PPRV and PM. Overall, the one-step multiplex RT-qPCR assay developed will be a valuable tool for rapid detection of individual and co-infections of the targeted pathogens with high specificity and sensitivity.
Animal Production and Health Laboratory (APHL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency (IAEA), Vienna, Austria.
Tirumala Bharani Kumar
0000-0002-9090-8471
TB
Settypalli
Animal Production and Health Laboratory (APHL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency (IAEA), Vienna, Austria.
Charles Euloge
CE
Lamien
Institute of Microbiology, University of Veterinary Medicine, Vienna, Austria.
Joachim
J
Spergser
Animal Production and Health Laboratory (APHL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency (IAEA), Vienna, Austria.
Mamadou
M
Lelenta
Laboratoire National Vétérinaire (LANAVET), Annex Yaoundé, Cameroon.
Abel
A
Wade
Animal Production and Health Laboratory (APHL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency (IAEA), Vienna, Austria.|Research and Diagnostic Laboratories, National Veterinary Institute, Debre Zeit, Ethiopia.
Esayas
E
Gelaye
Institute for Veterinary Disease Control, Austrian Agency for Health and Food Safety, Mödling, Austria.
Angelika
A
Loitsch
Laboratoire National d'Elevage, Ouagadougou, Burkina Faso.
Germaine
G
Minoungou
Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), UMR CMAEE, Montpellier, France.
Francois
F
Thiaucourt
Animal Production and Health Laboratory (APHL), Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency (IAEA), Vienna, Austria.|Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), UMR CMAEE, Montpellier, France.
Adama
A
Diallo
D000818:Animals; D018151:Capripoxvirus / Q000235:genetics*; D060085:Coinfection / Q000821:virology*; D015511:Goat Diseases / Q000821:virology; D006041:Goats / Q000821:virology; D045623:Mycoplasma capricolum / Q000235:genetics*; D010326:Pasteurella Infections / Q000821:virology; D016979:Pasteurella multocida / Q000254:growth & development*; D029021:Peste-des-Petits-Ruminants / Q000821:virology; D018107:Peste-des-petits-ruminants virus / Q000235:genetics*; D011002:Pleuropneumonia, Contagious / Q000821:virology; D011213:Poxviridae Infections / Q000821:virology; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep / Q000821:virology; D012757:Sheep Diseases / Q000821:virology
D012367:RNA, Viral