Recombinase polymerase amplification combined with lateral flow dipstick for equipment-free detection of Salmonella in shellfish

Abstract

Salmonella is a major pathogen that causes acute foodborne outbreaks worldwide. Seafood, particularly shellfish, is a proven source of Salmonella spp. infection because many people prefer to eat it raw or lightly cooked. However, traditional identification methods are too time-consuming and complex to detect contamination of bacteria in the food chain in a timely manner, and few studies have aimed to identify Salmonella in shellfish early in the supply chain. We herein developed a method for rapid detection of Salmonella in shellfish based on the method of recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD), which targets the invasion gene A (invA). The RPA-LFD was able to function at 30-45 °C, and at the temperature of 40 °C, it only took 8 min of amplification to reach the test threshold of amplicons. The established method had both a good specificity and a sensitivity of 100 fg DNA per reaction (20 µL). Regarding practical performance, RPA-LFD performed better than real-time PCR. Another advantage of RPA-LFD is that it was capable of being performed without expensive equipments. Thus, RPA-LFD has potential for further development as a detection kit for Salmonella in shellfish and other foods under field conditions.

Simultaneous and visual detection of infectious bronchitis virus and Newcastle disease virus by multiple LAMP and lateral flow dipstick

“…LAMP is one of the most widely used isothermal nucleic acid amplification techniques (INATs). Several studies on the diagnostic methods of other pathogens had showed that these INATs possessed equal or even higher sensitivities compared with qPCR assays (Gao et al, 2018;Khan et al, 2017;Yang et al, 2015). Our results indicated that LAMP assays possessed higher sensitivities than qPCR when detecting IBV and NDV ( Figure 5).…”

Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow Immunoassay for Rapid Detection of Salmonella in Food

“…Recombinase polymerase amplification could amplify target fragments in a shorter time (10 min). By contrast, a longer time (40 min) is required for a Loop-mediated isothermal amplification (LAMP) assay, and approximately 40 cycles (40 min) are required in the qPCR assay [1,13,28]. Clearly, the RPA assay is more efficient.…”

“…The LFD-RPA is suitable for testing in the field, and could provide low-cost and instrument-free nucleic acid detection in remote areas. In the previous reports of literatures, LFD-RPA assay was applied to detect Salmonella in shellfish with a detection limit of 5 CFU/mL in 8 min at 40 • C [1]. The testing of Salmonella typhimurium in milk could successfully reach as low as 1.95 CFU/mL in 10 min at 40-42 • C [29].…”

“…With the acceleration of economic globalization and trade liberalization, food safety has been a global public health issue prompting widespread concern [1]. Salmonella is an important food-borne pathogen causing food poisoning, which can transmit along the food production chain from farm to an retail chain, leading to severe foodborne diseases [2][3][4].…”

Multiplex Recombinase Polymerase Amplification Assay for the Simultaneous Detection of Three Foodborne Pathogens in Seafood

“…Salmonella is a major foodborne pathogen, and it can cause an illness called salmonellosis [8]. The consumption of food contaminated with Salmonella might lead to typhoid fever, septicemia, gastroenteritis, and even death [9,10]. Since these three pathogens might coexist in one sample with a relatively low concentration, it is crucial to develop a rapid, highly sensitive, and accurate detection method for the simultaneous identification of multiple targets.…”