Establishment of Duplex PCR Diagnostic Method for Acinetobacter baumannii and Morganella morganii from Turtle
FANG Zhenhua1, MENG Meigu2, DANG Zhaohui2, HONG Meiling2, DING Li2
1.School of Tropical Agricultural Technology, Hainan College of Vocation and Technique, Haikou 570216, China; 2.Key Laboratory for Ecology of Tropical Islands, Ministry of Education, College of Life Sciences, Hainan Normal University, Haikou 571158, China
Abstract：To establish duplex PCR diagnostic methods for pathogenic Acinetobacter baumannii and Morganella morganii from turtles, the specific primers of rpoB gene of A. baumannii and gyrB gene of M.morganii were used to optimize the reaction system and reaction conditions of single and duplex PCR reaction by annealing temperature, primer concentration, MgCl2 concentration and dNTP concentration. The results showed that the specific bands of the primers of rpoB gene of A. baumannii and gyrB gene of M.morganii were obtained by PCR amplification, which were consistent with the expected results. The PCR amplification effect was the best when the forward and reverse primers of rpoB gene and gyrB gene were 0.3 μmol/L, annealing temperature of 59.3 ℃, dNTP concentration of 0.5 mmol/L and MgCl2 concentration of 5 mmol/L, respectively. It was found that the method was only used for the amplification of A. baumannii and M. morganii, not for other pathogens such as Klebsiella pneumoniae, Aeromonas veronii and Pseudomonas putida. The sensitivity results showed that the minimum detection concentrations was 4.12×10-3 ng/μL in A. baumannii and 1.257×10-3 ng/μL in M. morganii. The detection of clinical samples showed that the results were consistent with those of single PCR. The findings indicated that the duplex PCR method for the detection of the two pathogens was highly specific and sensitive, which provides references for the differential diagnosis and clinical investigation of A. baumannii and M. morganii.
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2021, Vol. 40 
