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Rapid detection of Burkholderia pseudomallei with a lateral flow recombinase polymerase amplification assay.
PLoS One. 2019; 14(7):e0213416.Plos

Abstract

Melioidosis is a severe infectious disease caused by gram-negative, facultative intracellular pathogen Burkholderia pseudomallei (B. pseudomallei). Although cases are increasing reported from other parts of the world, it is an illness of tropical and subtropical climates primarily found in southeast Asia and northern Australia. Because of a 40% mortality rate, this life-threatening disease poses a public health risk in endemic area. Early detection of B. pseudomallei infection is vital for prognosis of a melioidosis patient. In this study, a novel isothermal recombinase polymerase amplification combined with lateral flow dipstick (LF-RPA) assay was established for rapid detection of B. pseudomallei. A set of primer-probe targeting orf2 gene within the putative type III secretion system (T3SS) cluster genes was generated and parameters for the LF-RPA assay were optimized. Result can be easy visualized in 30 minutes with the limit of detection (LOD) as low as 20 femtogram (fg) (ca. 25.6 copies) of B. pseudomallei genomic DNA without a specific equipment. The assay is highly specific as no cross amplification was observed with Burkholderia mallei, members of the Burkholderia cepacia-complex and 35 non-B. pseudomallei bacteria species. Moreover, isolates from patients in Hainan (N = 19), Guangdong (N = 1), Guangxi (N = 3) province of China as well as in Australia (N = 3) and Thailand (N = 1) were retrospectively confirmed by the newly developed method. LODs for B. pseudomallei-spiked soil and blood samples were 2.1×103 CFU/g and 4.2×103 CFU/ml respectively. The sensitivity of the LF-RPA assay was comparable to TaqMan Real-Time PCR (TaqMan PCR). In addition, the LF-RPA assay exhibited a better tolerance to inhibitors in blood than TaqMan PCR. Our results showed that the LF-RPA assay is an alternative to existing PCR-based methods for detection of B. pseudomallei with a potentiality of early accurate diagnosis of melioidosis at point of care or in-field use.

Authors+Show Affiliations

Department of Pestis, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Beijing, China.Department of Pestis, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Beijing, China.Department of Diarrheal Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Beijing, China.Department of Pestis, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Beijing, China.Department of Bioinformatics, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Beijing, China.Department of Preservation Center for Standard Strain, China Institute of Veterinary Drug Control, Beijing, China.Department of Hospital Antibiotics Resistance, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Beijing, China.State Key Laboratory of Infectious Diseases Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Beijing, China.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Validation Study

Language

eng

PubMed ID

31283772

Citation

Peng, Yao, et al. "Rapid Detection of Burkholderia Pseudomallei With a Lateral Flow Recombinase Polymerase Amplification Assay." PloS One, vol. 14, no. 7, 2019, pp. e0213416.
Peng Y, Zheng X, Kan B, et al. Rapid detection of Burkholderia pseudomallei with a lateral flow recombinase polymerase amplification assay. PLoS ONE. 2019;14(7):e0213416.
Peng, Y., Zheng, X., Kan, B., Li, W., Zhang, W., Jiang, T., Lu, J., & Qin, A. (2019). Rapid detection of Burkholderia pseudomallei with a lateral flow recombinase polymerase amplification assay. PloS One, 14(7), e0213416. https://doi.org/10.1371/journal.pone.0213416
Peng Y, et al. Rapid Detection of Burkholderia Pseudomallei With a Lateral Flow Recombinase Polymerase Amplification Assay. PLoS ONE. 2019;14(7):e0213416. PubMed PMID: 31283772.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Rapid detection of Burkholderia pseudomallei with a lateral flow recombinase polymerase amplification assay. AU - Peng,Yao, AU - Zheng,Xiao, AU - Kan,Biao, AU - Li,Wei, AU - Zhang,Wen, AU - Jiang,Taozhen, AU - Lu,Jinxing, AU - Qin,Aiping, Y1 - 2019/07/08/ PY - 2019/02/19/received PY - 2019/06/17/accepted PY - 2019/7/9/entrez PY - 2019/7/10/pubmed PY - 2020/2/19/medline SP - e0213416 EP - e0213416 JF - PloS one JO - PLoS ONE VL - 14 IS - 7 N2 - Melioidosis is a severe infectious disease caused by gram-negative, facultative intracellular pathogen Burkholderia pseudomallei (B. pseudomallei). Although cases are increasing reported from other parts of the world, it is an illness of tropical and subtropical climates primarily found in southeast Asia and northern Australia. Because of a 40% mortality rate, this life-threatening disease poses a public health risk in endemic area. Early detection of B. pseudomallei infection is vital for prognosis of a melioidosis patient. In this study, a novel isothermal recombinase polymerase amplification combined with lateral flow dipstick (LF-RPA) assay was established for rapid detection of B. pseudomallei. A set of primer-probe targeting orf2 gene within the putative type III secretion system (T3SS) cluster genes was generated and parameters for the LF-RPA assay were optimized. Result can be easy visualized in 30 minutes with the limit of detection (LOD) as low as 20 femtogram (fg) (ca. 25.6 copies) of B. pseudomallei genomic DNA without a specific equipment. The assay is highly specific as no cross amplification was observed with Burkholderia mallei, members of the Burkholderia cepacia-complex and 35 non-B. pseudomallei bacteria species. Moreover, isolates from patients in Hainan (N = 19), Guangdong (N = 1), Guangxi (N = 3) province of China as well as in Australia (N = 3) and Thailand (N = 1) were retrospectively confirmed by the newly developed method. LODs for B. pseudomallei-spiked soil and blood samples were 2.1×103 CFU/g and 4.2×103 CFU/ml respectively. The sensitivity of the LF-RPA assay was comparable to TaqMan Real-Time PCR (TaqMan PCR). In addition, the LF-RPA assay exhibited a better tolerance to inhibitors in blood than TaqMan PCR. Our results showed that the LF-RPA assay is an alternative to existing PCR-based methods for detection of B. pseudomallei with a potentiality of early accurate diagnosis of melioidosis at point of care or in-field use. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/31283772/Rapid_detection_of_Burkholderia_pseudomallei_with_a_lateral_flow_recombinase_polymerase_amplification_assay_ L2 - http://dx.plos.org/10.1371/journal.pone.0213416 DB - PRIME DP - Unbound Medicine ER -