Singleplex real-time PCR and duplex PCR platform for the rapid detection of hypervirulent Klebsiella pneumoniae

Jiaqi Wang¹Yishuai Wu¹Liying Zhao^1,2Mengran Xing^1,2Yangyang Huang¹Shengjie Peng¹Xu Chunyan¹Hong Yao¹CHENGLONG LI^1*Xiang-Dang Du¹

• ¹Henan Agricultural University, Zhengzhou, China
• ²Huazhong Agricultural University, Wuhan, Hubei Province, China

Hypervirulent Klebsiella pneumoniae (HvKP) is a notorious zoonotic pathogen that poses a significant threat to public health, as it can cause severe infections with high morbidity and mortality among young and healthy individuals. Commonly, a positive string test is primarily used to identify HvKP strains, but which is laborious and timeconsuming. A rapid assay to identify HvKP is needed for public health personnel to track the spread of these strains and provide timely efforts for their control. Given this context, rapid SYBR Green I-based singleplex real-time PCR assays were developed for defining HvKP on the basis of the biomarkers iroB, iucA, peg-344 and plasmidborne rmpA, respectively. These four singleplex PCR assays all displayed a high degree of linearity (R 2 >0.99) in the range of 10 4 to 10 9 cfu/mL, and the limit of detection (LOD) was 10 3 cfu/mL, which was equivalent to 10 cfu/reaction. To improve the efficiency and reduce the cost of diagnostic testing, SYBR Green I-based duplex PCR melting curve assays were developed with average melting temperatures of 85.0, 87.5, 76.0 and 78.5°C for iroB, iucA, peg-344 and plasmid-borne rmpA, respectively. The LODs for the developed duplex PCRs for iroB/peg-344, iucA/peg-344 and iucA/rmpA combination were 10 4 cfu/mL (that was 10 2 cfu/reaction), 10 4 cfu/mL (that was 10 2 cfu/reaction) and 10 5 cfu/mL (that was 10 3 cfu/reaction), respectively. High specificity was shown when other bacterial pathogens were detected in this study. These assays could be used as rapid, sensitive and specific diagnostic tools for the practical identification of HvKP strains.