More sensitive and specific Salmonella detection methods are urgently needed. Especially, many diarrheal pathogenic bacteria, including Salmonella, can cause severe infectious diseases at low infectious doses. These methods are time-consuming, or poor in specificity or of low sensitivity, and sometimes require expensive instruments for laboratory setup. To date, the detection methods for Salmonella, including traditional biochemical culture, immune testing, and molecular biological approaches, are represented by polymerase chain reaction (PCR)/real-time PCR. Timely screening of Salmonella is the key to prevent and control diarrheal disease outbreaks. In China, the previous study has estimated the incidence of non-typhoid salmonellosis at 626.6 cases per 100,000 people ( Yue et al., 2020). In the European Union, European Centre for Disease Prevention and Control reported 91,857 Salmonella infection cases in humans, and Salmonella caused 30.7% of all food-borne outbreaks during 2018 ( European Food Safety Authority, & European Centre for Disease Prevention and Control, 2020). In the United States, the Centers for Disease Control and Prevention (CDC) estimated food was one of the main sources of Salmonella infection Salmonella caused approximately 1.35 million infections, 26,500 hospitalizations, and 420 deaths every year ( CDC, 2020). According to statistics by the World Health Organization, the number of outbreaks of different food-borne intestinal diseases since 2010 has reached 582 million, with nearly 350,000 deaths, including 52,000 Salmonella cases ( World Health Organization, 2016)]. Salmonella, a gram-negative bacillus, is the most common diarrheal pathogenic bacteria and infects millions of people across the world every year, with the most common clinical manifestations being acute gastroenteritis, encephalitis, pericarditis, sepsis, and even death ( Hugas and Beloeil, 2014 Ferrari et al., 2019). While two-step assay was more sensitive and suitable for samples at low abundance. One-tube RPA-Cas13a developed in this study provides a simple, rapid, and specific detection method for Salmonella spp. The detection results of one-tube or two-step RPA-Cas13a and real-time PCR were highly consistent in clinical samples. The one-tube RPA-Cas13a could detect the Salmonella genome with the limit of 10 2 copies, which was the same as real-time polymerase chain reaction (PCR), but less sensitive than two-step RPA-Cas13a (10 0 copies). and had no cross-reaction with the other nine diarrheal bacteria. The designed primers were highly specific to Salmonella spp. The established one-tube RPA-Cas13a method can complete the detection within 20 min and the two-step RPA-Cas13a method detection time within 45 min. detection, by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats associated protein 13a) cleavage. In this study, a conserved short fragment of the Salmonella invA gene was selected and used to design primers and specific crRNA (CRISPR RNA) for establishing a one-tube and two-step reaction system for Salmonella spp. is advantageous to the control of its spread. Rapid and sensitive detecting Salmonella spp. is one of the most common foodborne disease-causing pathogens that can cause severe diseases in very low infectious doses.
0 kommentar(er)
