African Swine Fever (ASF) is a highly infectious viral disease with high mortality. The most recent ASF outbreak in Vietnam occurred in 2019, posing a threat to spread to the neighboring Asian countries. Without a commercial vaccine or efficient chemotherapeutics successfully developed, rapid diagnosis and necessary biosecurity procedures are required to control the disease. While the diagnosis method of ASF recommended by the World Organization of Animal Health is real-time PCR, it is not . CC-BY-NC-ND 4.

The COVID-19, caused by the novel coronavirus SARS-CoV-2, has broken out of control all over the globe and put the majority of the world under lockdown. There have been no specific antiviral medications for SARS-CoV-2 while vaccines are still under development. Thus, rapid diagnosis and necessary public health measures are currently key parts to contain the pandemic. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection. However, this method is not suitable for point-of-care (POC) diagnosis because of the timeconsuming procedure, the requirements of biosafety conditions and expensive equipment.In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were developed and compared. The three methods exhibited similar performance with the limit of detection (LOD) as low as just 1 copy per reaction when evaluated on the synthetic DNA fragments. The results can be read with naked eyes within 30 minutes without crossreactivity to closely related coronaviruses. When tested with SARS-CoV-2 extracted genomic-RNA, LAMP outperformed both CPA and PSR assays. Moreover, the direct detection of SARS-CoV-2 in simulated patient samples (oropharyngeal and nasopharyngeal swabs) by colorimetric iNAATs was also successful. Further preparation of the lyophilized reagents for LAMP reactions revealed that the freeze-dried, ready-touse kit maintained the sensitivity and LOD value of the liquid assays. These results strongly indicate that the colorimetric lyophilized LAMP test kit developed herein is highly suitable for detecting SARS-CoV-2 at POC.

Foodborne illness undermines human health by causing fever, stomachache and even lethality. Among foodborne bacterial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa are of extraordinary significance which drive reasons of food and beverage poisoning in numerous cases. Today, PCR has been widely used to examine the presence of different foodborne pathogens. However, PCR requires specialized equipment and skillful personnel which limit its application in the field. Recently, there is an emerging of isothermal PCR methods in which the reactions occur at low and constant temperatures, allowing their application in restricted-resource settings. In this work, multiplex Recombinase Polymerase Amplification (RPA) was used to simultaneously detect S. aureus and P. aeruginosa with high sensitivity and specificity. The limit detection of multiplex RPA was 10 and 30 fg/reaction of genomic DNAs of S. aureus and P. aeruginosa, respectively. Besides, the reaction time was reduced to only 25 minutes with a low incubation temperature of 39 °C. Markedly, multiplex RPA reactions succeeded to directly detect as low as 1 and 5 CFU/reaction of S. aureus and P. aeruginosa cells, respectively without the requirement of extracting DNA genome. Moreover, the multiplex RPA reliably detected the two foodborne bacteria in milk, fruit juice and bottled water samples. In general, the direct multiplex RPA described in this study is a rapid, simple, sensitive and efficient alternative tool that could be used to detect the presence of S. aureus and P. aeruginosa without the necessity of costly devices and high-trained staff.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is one of the most complicated and dangerous diseases in pigs with high mortality since it modulates the immune system of the lungs and has been closely associated with secondary infection of other lethal bacteria and viruses. The gold standard of molecular diagnosis for PRRSV, reverse transcription (RT)-PCR, is time-consuming, expensive and requires transportation of samples to a specialized laboratory. In this study, a direct colorimetric RT-loop-mediated isothermal amplification (RT-LAMP) method was developed to specifically and rapidly detect PRRSV. The RT-LAMP outcomes can be visualized by the naked eye after 45 min of incubation at 65˚C without any cross-reactivity recorded with the bacteria and other viruses tested. In particular, the mobile, non-instrumented, commercial pocket hand warmers were demonstrated to successfully provide constant temperature for consistent nucleic acid amplification throughout the RT-LAMP reactions. The limit of detection of the assay was defined as the genomic RNA concentration extracted from a known viral titer of 10 -2.5 TCID 50 /ml. The direct use of clinical serum samples required a simple dilution to maintain the performance of the colorimetric RT-LAMP assay. Therefore, the direct colorimetric RT-LAMP assay developed is well-qualified for producing a ready-to-use kit for PRRSV diagnosis in the field.