A Rapid Digital PCR System with a Pressurized Thermal Cycler

We designed a silicon-based fast-generated static droplets array (SDA) chip and developed a rapid digital polymerase chain reaction (dPCR) detection platform that is easy to load samples for fluorescence monitoring. By using the direct scraping method for sample loading, a droplet array of 2704 microwells with each volume of about 0.785 nL can be easily realized. It was determined that the sample loading time was less than 10 s with very simple and efficient characteristics. In this platform, a pressurized thermal cycling device was first used to solve the evaporation problem usually encountered for dPCR experiments, which is critical to ensuring the successful amplification of templates at the nanoliter scale. We used a gradient dilution of the hepatitis B virus (HBV) plasmid as the target DNA for a dPCR reaction to test the feasibility of the dPCR chip. Our experimental results demonstrated that the dPCR chip could be used to quantitatively detect DNA molecules. Furthermore, the platform can measure the fluorescence intensity in real-time. To test the accuracy of the digital PCR system, we chose three-channel silicon-based chips to operate real-time fluorescent PCR experiments on this platform.

Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis

The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines.

Multiplex Site-Directed Gene Editing Using Polyethylene Glycol-Mediated Delivery of CRISPR gRNA:Cas9 Ribonucleoprotein (RNP) Complexes to Carrot Protoplasts

The aim of this work was to show an efficient, recombinant DNA-free, multiplex gene-editing method using gRNA:Cas9 ribonucleoprotein (RNP) complexes delivered directly to plant protoplasts. For this purpose, three RNPs were formed in the tube, their activity was confirmed by DNA cleavage in vitro, and then they were delivered to carrot protoplasts incubated with polyethylene glycol (PEG). After 48 h of incubation, single nucleotide deletions and insertions and small deletions at target DNA sites were identified by using fluorescent-PCR capillary electrophoresis and sequencing. When two or three RNPs were delivered simultaneously, long deletions of 33–152 nt between the gRNA target sites were generated. Such mutations occurred with an efficiency of up to 12%, while the overall editing effectiveness was very high, reaching 71%. This highly efficient multiplex gene-editing method, without the need for recombinant DNA technology, can be adapted to other plants for which protoplast culture methods have been established.

Detection of precisely edited CRISPR/Cas9 alleles through cointroduced restriction-fragment length polymorphisms

CRISPR/Cas9 is a powerful tool for producing genomic insertions and deletions (indels) to interrogate gene function. Modified CRISPR/Cas9 protocols can produce targeted genetic changes that are more precise than indels, but founder recovery is less efficient. Focusing on producing missense mutations in zebrafish using single-stranded oligo deoxynucleotide (ssODN) donor templates, we pioneered a strategy of adding synonymous changes to create novel restriction-enzyme (RE) sites, allowing detection of rare precise edits in a modified fluorescent-PCR fragment assay. We have named this process TIARS (test for incorporation of added recognition sites). Aided by TIARS, we induced two distinct amino-acid substitutions (T979I and P1387S) in the atp7a gene among somatic tissues of CRISPR-Cas9 treated F0 zebrafish. One of these F0s transmitted the allele to atp7aT979I/+ F1 progeny, and trans heterozygosity of this allele against a null atp7a allele causes hypopigmentation, consistent with more severe pigment deficits in zebrafish or humans carrying only null mutations in atp7a/ATP7A. Design of ssODNs with novel RE recognition sites is labor-intensive, so we developed an in silico tool, TIARS Designer, and performed bioinformatic validation indicating that TIARS should be generalizable to other genes and experimental systems that employ donor template DNA.

Detection of meat from horse, donkey and their hybrids (mule/hinny) by duplex real-time fluorescent PCR

Meat adulteration is currently a common practice worldwide. In China, adulteration of donkey meat products with the similar species (horse and mule/hinny) meat and mislabeling are becoming widespread concerns. In this study, a sensitive and species-specific duplex real-time PCR assay based on the simultaneous amplification of fragments of the creatine kinase muscle gene family, was developed and optimized for the identification of horse, donkey and mule /hinny species in raw and heat-processed meat products. Duplex real-time PCR results showed different fluorescence amplification curves for horse and donkey. Both kinds of fluorescence amplification curves appeared simultaneously for mule/hinny. The limit of detection (LOD) of the method was up to 0.01 ng /μL. The method and strategy developed in this study could be applied to detect the presence of adulterants from horse and mule /hinny meat in raw donkey meat and meat products.

Study on the Method of Isolating the Aptamer from the Surface of HepG2 Cells

The hepatocellular carcinoma (HCC) is a malignant tumor that occurs in the liver. It is a common malignant tumor in clinic. The main reason for its high mortality is its early latency. Therefore, how to accurately determine and test the hepatocellular carcinoma in the early stage has a very positive significance for the treatment. It is an important method for the early diagnosis of the hepatocellular carcinoma to use aptamers specifically binding to hepatocellular carcinoma cells, which has good application prospects. In order to improve the efficiency of aptamer selection of tumor cells, our group designed and developed an automated instrument for the aptamer selection. In this paper, the method to separate bound aptamers from the surface of HepG2 cells in automated selection process was studied, and the feasibility of separating binding aptamers from the HepG2 cell surface using ultrapure water and the effect of different temperature environments on its isolation were discussed. Results of the real-time fluorescent PCR and flow cytometry showed that ultrapure water could be used to isolate bound HepG2 cells and aptamers, and the concentration of the aptamers increased with the rise of the temperature between 25 and 80 degrees Celsius. This result will contribute to the improvement on the efficiency of automated selections for aptamers corresponding to HepG2 cells.

Capillary Electrophoresis of PCR fragments with 5’-labelled primers for testing the SARS-Cov-2

BackgroundDue to the huge demand for SARS-Cov-2 determination, alternatives to the standard qtPCR tests are potentially useful for increasing the number of samples screened. Our aim was to develop a direct fluorescent PCR capillary-electrophoresis detection of the viral genome. We validated this approach on several SARS-Cov-2 positive and negative samples.Study designWe isolated the naso-pharingeal RNA from 20 positive and 10 negative samples. The cDNA was synthesised and two fragments of the SARS-Cov-2 were amplified. One of the primers for each pair was 5’-end fluorochrome labelled. The amplifications were subjected to capillary electrophoresis in ABI3130 sequencers to visualize the fluorescent peaks.ResultsThe two SARS-Cov-2 fragments were successfully amplified in the positive samples, while the negative samples did not render fluorescent peaks.ConclusionWe describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5 hours. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR (lack of reactive and real-time PCR equipment) and increase the testing capacity.

The construction and application of a new 17-plex Y-STR system using universal fluorescent PCR

Y-chromosomal short tandem repeat (Y-STR) polymorphisms are useful in forensic identification, population genetics and human structures. However, the current Y-STR systems are limited in discriminating distant relatives in a family with a low discrimination power. Increasing the capacity of detecting Y chromosomal polymorphisms will drastically narrow down the matching number of genealogy populations or pedigrees. In this study, we developed a system containing 17 Y-STRs that are complementary to the current commercially available Y-STR kits. This system was constructed by multiplex PCR with expected sizes of 126-400 bp labeled by different fluorescence molecules (DYS715, DYS709, DYS716, DYS713 and DYS607 labeled by FAM; DYS718, DYS723, DYS708 and DYS714 labeled by JOE; DYS712, DYS717, DYS721 and DYS605 labeled by TAMRA; and DYS719, DYS726, DYS598 and DYS722 labeled by ROX). The system was extensively tested for sensitivity, male specificity, species specificity, mixture, population genetics and mutation rates following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The genetic data were obtained from eight populations with a total of 1260 individuals. Our results showed that all the 17 Y-STRs are human- and male-specific and include only one copy of the Y-chromosome. The 17 Y-STR system detects 143 alleles and has a high discrimination power (0.996031746). Mutation rates were different among the 17 Y-STRs, ranging from 0.30% to 3.03%. In conclusion, our study provides a robust, sensitive and cost-effective genotyping method for human identification, which will be beneficial for narrowing the search scope when applied to genealogy searching with the Y-STR DNA databank.