A rapid nucleic acid concentration measurement system with large-field-of-view for droplet digital PCR microfluidic chip

Droplet digital polymerase chain reaction(ddPCR) is an effective technique for the absolute quantification of target mucleic acid unparalleled sensitivity. However, current commerical ddPCR device for the detection of the gene...

Application and research of loop-mediated isothermal amplification and lateral flow dipstick in detection of Porcine circovirus type 3

Background：Porcine circovirus type 3 (PCV3) is a newly detected pathogen from pigs in the past few years. As the infection rate of PCV3 in the herd is getting higher and wider, the infection is more and more serious. The virus with unclear clinical symptoms and pathogenesis is also paid more and more attention. And detection of pathogenic antibodies has become an important issue. Methods:In this experiment, Loop-mediated isothermal amplification (LAMP) was combined with Lateral flow dipstick (LFD) to establish a rapid and convenient detection method (LAMP-LFD). Results:The results showed that the band was darkened at a nucleic acid concentration of 0.2 pg/ μL using a conventional PCR method, and a clear detection line was observed at a nucleic acid concentration of 0.2 fg/μL using the LAMP-LFD method. The primer and hybridization probe are only compatible with PCV3.Compared to the PCR method, the LAMP-LFD method has a shorter time (about 1 h) and requires less instrumentation. Conclusions: The test results show that the LAMP-LFD method has extremely high sensitivity and specificity. The operation is simple and quick. The visual effect of the detection results is better than that of PCR and single LAMP test, which can bring convenience in practical production applications.

Optimization of the Separation Method for Bioleaching Bacteria DNA and RNA Extracted Simultaneously

DNA and RNA based analysis was a useful way to characterize microbial communities during biohydrometallurgical processes. However, feasible, affordable and efficient DNA and RNA separation methods are rarely reported although several simultaneous DNA and RNA extraction methods have been developed recently. In this study, various salts including NaCl, CaCl2 and LiCl were tested for separation of DNA and RNA. Salt concentration, nucleic acid concentration, precipitation temperature and time were optimized. The results showed that LiCl was more efficient to separate DNA and RNA than the other two salts. The optimized conditions were as follows: 1/4 volume saturated LiCl was used for precipitating RNA first at -20°C for 30 min for total nucleic acid concentration of approximately 200-400 ng/μL, and then centrifuged at 12,000×g at room temperature for 20 min to collect RNA. DNA in the supernatant was precipitated using 0.6 volume isopropanol and then collected by centrifugation at 12,000×g at room temperature for 20 min. The results indicated that DNA and RNA could be extracted from not only pure culture of Acidithiobacillus ferrooxidans(A.ferrooxidans), Acidithiobacillus caldus(A. caldus), Acidithiobacillus albertensis(A. albertensis), Leptospirillum ferrooxidans(L. ferrooxidans), Ferroplasma thermophilium(F. thermophilium), but also the acid mine drainage(AMD) water samples from Daye copper mine, Xiangxi gold mine, Axi gold mine. The resulted DNA and RNA could be amplified and A260/280 was 1.8-2.0, A260/230 was 1.8-2.2, which indicated high quality of DNA and RNA. This method could be widely used for separation of bioleaching bacteria DNA and RNA extracted simultaneously.