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Rapid detection of Pseudomonas aeruginosa by cross priming amplification
XIANG Yong, YAN Ling, ZHENG Xiao-cui, LI Li-zhen, LIU Peng, CAO Wei-sheng
2020, 19 (10): 2523-2529.   DOI: 10.1016/S2095-3119(20)63187-2
Abstract109)      PDF in ScienceDirect      
Pseudomonas aeruginosa (PA) is an opportunistic pathogen of humans and animals and a common source of nosocomial infections especially of the respiratory tract.  Pseudomonas aeruginosa is also a major bacterial disease of poultry and in particular, eggs and newly hatched chicks.  In this study, we developed a simple, accurate and rapid molecular detection method using cross priming amplification (CPA) with a nucleic acid test strip to detect P. aeruginosa.  The assay efficiently amplified the target gene within 45 min at 62°C only using a simple water bath.  The detection limit of the method was 1.18×102 copies μL–1 for plasmid DNA and 4.4 CFU mL–1 for bacteria in pure culture, and was 100 times more sensitive than conventional PCR.  We screened 83 clinical samples from yellow-feather broiler breeder chickens and hospitalized/treated dogs and cats using CPA, PCR and traditional culture methods.  The positive-sample ratios were 15.3% (13/83) by CPA, 13.3% (11/83) by PCR and 12.1% (10/83) by the culture method.  The established CPA method has significant advantages for detecting P. aeruginosa.  The method is easy to use and possesses high specificity and sensitivity without the requirements of complicated experimental equipment.  The PA-CPA assay is especially fit for outdoor and primary medical units and is an ideal system for the rapid detection and monitoring of P. aeruginosa.
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Establishment of an avian leukosis virus subgroup A-resistant cell line
FENG Min, DAI Man-man, LIAO Ming, CAO Wei-sheng
2017, 16 (04): 930-936.   DOI: 10.1016/S2095-3119(16)61453-3
Abstract907)      PDF in ScienceDirect      
Rapid diagnostic methods for classifying avian leukosis subgroups in the field were needed for routine, large-scale screening.  As a first step in method development, we inserted the avian leukosis virus subgroup A (ALV-A) env gene into plasmid pcDNA3.1/Zeo (+) and used this construct to transfect DF-1 cells.  Zeocin-resistant cells were obtained after 2 weeks of zeocin selection.  Then, the cells were analyzed using PCR, immunofluorescence, and Western blot for expression of the envA-encoded envelope protein after 30 serial passages.  The DF-1/A cell line was completely resistant to 104 TCID50/0.1 mL (50% tissue culture infective dose) ALV-A and was partially resistant to 105 TCID50/0.1 mL ALV-A viral particles.  By comparing the DF-1/A and DF-1 cell lines, an ALV-A isolate was identified using a gag-specific ELISA for capsid protein p27.  Thus, we established a DF-1/A cell line that was resistant to ALV-A infection.  This cell line will be useful as a diagnostic tool.
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