Date Published: July 8, 2019
Publisher: Public Library of Science
Author(s): Yao Peng, Xiao Zheng, Biao Kan, Wei Li, Wen Zhang, Taozhen Jiang, Jinxing Lu, Aiping Qin, Ruslan Kalendar.
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.
Melioidosis, also called Whitmores disease, is an emerging infectious disease caused by the environmental bacterium B. pseudomallei. Although reported in many regions of the world, it is primarily distributed in tropical and subtropical regions. Melioidosis is estimated to account for 89,000 deaths worldwide every year [1, 2]. B. pseudomallei. has been classified as a category B bioterrorism agent by the Centers for Disease Control and Prevention of USA . In China, the epidemic areas of melioidosis are mainly in Hainan, Guangdong and Guangxi province [4, 5]. Isolates from Hainan alone exhibited highly genetic diversity when tested by multilocus sequence typing (MLST) . The main routes of B. pseudomallei infection are through inoculation of compromised skin, inhalation of contaminated soil during extreme weather event  and ingestion of contaminated water . Manifestations of melioidosis are various and hard to differentiate from common pneumonia, flu or tuberculosis. In addition, B. pseudomallei is intrinsically resistance to a wide range of antibiotics, such as penicillin, ampicillin, first and second-generation of cephalosporins, gentamicin, tobramycin, streptomycin, and polymyxin .
First introduced in 2006, RPA represents an innovative DNA Isothermal detecting technology that has been used to detect a range of pathogens. It is an alternative to existing PCR-based method. One of the advantages of RPA method is its minimum equipment requirement. The assay was conducted successfully under human body heat . The LF-RPA assay reported here to detect B. pseudomallei is as sensitive as TaqMan PCR and is able to amplify DNA to detectable levels in a temperature range of 25°C to 50°C, indicating it is feasible to carry the assay under human body temperature. This is of a great advantage for field application in low resource settings. The assay possesses a comparative LOD with LAMP assay which detects BPSS1406, a gene also located within the cluster genes encoding T3SS of B. pseudomallei . The orf2 gene was successfully used to detect B. pseudomallei of 27 clinical isolates but was negative with 35 non-B. pseudomallei bacteria species and B. mallei, a relative of B. pseudomallei and also a category A bioterrorism relevant organism. In addition, the assay can distinguish B. pseudomallei from members of B. cepacian-complex and non-human pathogen of B. thailandensis , suggesting the high specificity of this gene for detecting of B. pseudomallei. The orf2 gene, within the type III secretion system gene cluster of the B. pseudomallei (GenBank accession no. AF074878), was no significant similarity to other Burkholderia subspecies, and previous targeted to distinguish B. pseudomallei from other microbial species by TaqMan PCR [18, 22]. The LF-RPA method is a very rapid technique, providing instructive information in less than 30 minutes (20 minutes reaction and 5 minutes detection) with a high sensitivity (LOD of 20 fg (ca.25.6 copies) on pure gDNA of B. pseudomallei, or 5±0.45 CFU/ml on B. pseudomallei cells. LOD of LF-RPA for detection of Salmonella was 10.5 CFU/ml . It seems that template prepared by boiling sample at 95°C for 5 minutes is better than by isolating gDNA with a purification kit. As demonstrated in this study, the LOD of boiling method is 5 CFU/ml verse 420 CFU/ml of kit purified gDNA. This may due to the loss of gDNA during the purification procedures.