Multiplex SYBR green real time PCR for the simultaneous detection and differentiation of four important reproductive infectious pathogens

2019 ◽  
Vol 1 (2) ◽  
pp. 69-77
Author(s):  
Omnia El-Sayed ◽  
Alaa Hussein
2011 ◽  
Vol 60 (4) ◽  
pp. 459-466 ◽  
Author(s):  
Spencer D. Polley ◽  
Samuel Boadi ◽  
Julie Watson ◽  
Alan Curry ◽  
Peter L. Chiodini

Diagnosis of microsporidial infections is routinely performed by light microscopy, with unequivocal non-molecular species identification achievable only through electron microscopy. This study describes a single SYBR Green real-time PCR assay for the simultaneous detection and species identification of such infections. This assay was highly sensitive, routinely detecting infections containing 400 parasites (g stool sample)−1, whilst species identification was achieved by differential melt curves on a Corbett Life Science Rotor-Gene 3000. A modification of the QIAamp DNA tissue extraction protocol allowed the semi-automated extraction of DNA from stools for the routine diagnosis of microsporidial infection by real-time PCR. Of 168 stool samples routinely analysed for microsporidian spores, only five were positive by microscopy. By comparison, 17 were positive for microsporidial DNA by real-time analysis, comprising 14 Enterocytozoon bieneusi, one Encephalitozoon cuniculi and two separate Pleistophora species infections.


2014 ◽  
Vol 40 (01) ◽  
pp. 1-9 ◽  
Author(s):  
Chao-Nan Lin ◽  
Chi-Hsien Chien ◽  
Ming-Tang Chiou ◽  
Jou-Wei Wang ◽  
Ya-Ling Lin ◽  
...  

Parvovirus is now considered to be one of the most important infectious agents affecting canine, feline and porcine domestic animals. Canine parvovirus 2 (CPV 2) and feline parvovirus (FPV) are major infectious causes of mortality in puppies and kittens. Both CPV 2 and FPV have recently been shown to infect both dogs and cats. The characteristic symptoms of canine, feline and porcine parvovirus disease are intestinal hemorrhage with severe bloody diarrhea, leukopenia and reproductive failure, respectively. In this work, we describe the development of a novel real-time PCR system that is based on the use of SYBR Green and that allows the simultaneous detection of VP2 gene of CPV, FPV and porcine parvovirus (PPV). This system yielded low coefficients of variation for intra-assay and inter-assay variabilities. This novel SYBR Green-based real-time PCR assay was sensitive, specific and reliable for the amplification of CPV 2, FPV and PPV DNA, with a reproducible limit of detection of as few as 10 copies/μL of target DNA per reaction. The methods described in this study have been used successfully in our veterinary diagnostic laboratory and have been shown to be helpful tools for the diagnosis and quantification of parvovirus infection in canines, felines and swine.


2013 ◽  
Vol 30 (3) ◽  
pp. 160-164
Author(s):  
Natsuko IIDA ◽  
Hiroshi FUKUSHIMA ◽  
Midori HIROI ◽  
Miya YAGI ◽  
Takashi KANDA ◽  
...  

2017 ◽  
Vol 39 (2) ◽  
pp. 175-184
Author(s):  
Z. Zheng ◽  
P. Zhang ◽  
G. He ◽  
K. Liao ◽  
Z. Wang ◽  
...  

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