Enterobacter cloacae belongs to the Enterobacter genus and is a Gram-negative bacterium [1]. It has been widely present in soil, plants, and various aquatic environments [2]. E. cloacae has strong adhesion and invasion properties that contribute to its ability to infect the host [3]. It is also an important opportunistic pathogen and the leading cause of nosocomial infections worldwide [4, 5]. It can cause septicemia, meningitis, endocarditis, septic arthritis and bone marrow. Inflammation can also result in infections of the lower respiratory tract, skin, soft tissue, urinary tract, abdominal cavity, and eyes [4, 6]. Recently, there have been many reports of E. cloacae infections in veterinary clinics. E. cloacae can also cause diseases in aquatic animals and cause a large number of deaths [7, 8]. In remote areas, it can result in the deaths of many aquatic animals on farms, due to a failure to detect and treat in time.
Now, with the overuse of antibiotics, E. cloacae has a high prevalence of multi-drug resistance [9]. This brings several challenges to clinical treatment and infection control [10, 11]. To deal with the global epidemic of E. cloacae, monitoring and early identification is important for human life, farm animals, and aquaculture.
For patients and hospitals, pathogen diagnosis is important for disease prevention and treatment [12]. Traditional culture methods can identify pathogen accurately, but are a lengthy process. Recently, many functional enzymes have been used to establish conventional methods of biochemical identification of clinical pathogens, such as polymerase chain reaction (PCR)-based, quantitative PCR (qPCR)-based, biochemical analysis, conventional culture procedure, and immunology-based diagnosis tests[13–16]. Although many useful detection methods have been developed, they all suffer from a variety of drawbacks. Most of these methods can be performed in well-equipped laboratories, but it is often necessary to detect pathogens in peoples’ houses, poorly equipped hospitals, and farms; in these cases, it can be difficult to detect pathogens in a short period of time [17]. It is particularly important to establish a relatively simple and straightforward method as there is often a lack of trained personnel. For this reason, isothermal amplification methods have been developed, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) [18, 19].
Recombinase polymerase amplification (RPA) was reported in 2006 [20], it can rely on recombinase (UvsX and UvsY), single stranded binding protein (gp32), and strand displacing DNA polymerase (Bsu) to accomplish nucleic acid amplification, and it does not need a stringent incubation temperature; exponential amplification can be performed at 37°C rather than thermocycling between 55°C and 95°C. It can complete reactions in 30 minutes or less. The amplification products of RPA can be detected using gel electrophoresis, real-time fluorescence, lateral flow strip (LFS), and other methods [19, 21–22]. Of these detection methods, LFS is a simple tool, detection results of LFS can be analyzed with the naked eye and do not require complex instruments and trained personnel.
In this study, a reliable RPA-LFS assay for E. cloacae was established after screening several primers and probes. Reactions could be completed within 30 min at 37°C, and the results could be observed on a LFS in 5 min. This method had the same limit of detection (101 CFU/reaction) as the qPCR method when the results of 217 clinical samples were compared. Thus, the method established in this study may be useful in remote areas or farms with poor resources.