DNA Logical Computing on a Transistor for Cancer Molecular Diagnosis.

Given the high degree of variability and complexity of cancer, precise monitoring and logical analysis of different nucleic acid markers are crucial for improving diagnostic precision and patient survival rates. However, existing molecular diagnostic methods normally suffer from high cost, cumbersome procedures, dependence on specialized equipment and the requirement of in-depth expertise in data analysis, failing to analyze multiple cancer-associated nucleic acid markers and provide immediate results in a point-of-care manner. Herein, we demonstrate a transistor-based DNA molecular computing (TDMC) platform that enables simultaneous detection and logical analysis of multiple microRNA (miRNA) markers on a single transistor. TDMC can perform not only basic logical operations such as "AND" and "OR", but also complex cascading computing, opening up new dimensions for multi-index logical analysis. Owing to the high efficiency, sensing and computations of multi-analytes can be operated on a transistor at a concentration as low as 2×10-16 M, reaching the lowest concentration for DNA molecular computing. Thus, TDMC achieves an accuracy of 98.4% in the diagnosis of hepatocellular carcinoma from 62 serum samples. As a convenient and accurate platform, TDMC holds promise for applications in "one-stop" personalized medicine.

Research progress of severe acute respiratory syndrome coronavirus 2 on aerosol collection and detection.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019 has negatively affected people's lives and productivity. Because the mode of transmission of SARS-CoV-2 is of great concern, this review discusses the sources of virus aerosols and possible transmission routes. First, we discuss virus aerosol collection methods, including natural sedimentation, solid impact, liquid impact, centrifugal, cyclone and electrostatic adsorption methods. Then, we review common virus aerosol detection methods, including virus culture, metabolic detection, nucleic acid-based detection and immunology-based detection methods. Finally, possible solutions for the detection of SARS-CoV-2 aerosols are introduced. Point-of-care testing has long been a focus of attention. In the near future, the development of an instrument that integrates sampling and output results will enable the real-time, automatic monitoring of patients.

Novel aerosol detection platform for SARS­CoV­2: Based on specific magnetic nanoparticles adsorption sampling and digital droplet PCR detection.

The SARS­CoV­2 virus is released from an infectious source (such as a sick person) and adsorbed on aerosols, which can form pathogenic microorganism aerosols, which can affect human health through airborne transmission. Efficient sampling and accurate detection of microorganisms in aerosols are the premise and basis for studying their properties and evaluating their hazard. In this study, we built a set of sub-micron aerosol detection platform, and carried out a simulation experiment on the SARS­CoV­2 aerosol in the air by wet-wall cyclone combined with immunomagnetic nanoparticle adsorption sampling and ddPCR. The feasibility of the system in aerosol detection was verified, and the influencing factors in the detection process were experimentally tested. As a result, the sampling efficiency was 29.77%, and extraction efficiency was 98.57%. The minimum detection limit per unit volume of aerosols was 250 copies (102 copies/mL, concentration factor 2.5).

SSR marker development and intraspecific genetic divergence exploration of Chrysanthemum indicum based on transcriptome analysis.

BACKGROUND: Chrysanthemum indicum L., an important ancestral species of the flowering plant chrysanthemum, can be used as medicine and for functional food development. Due to the lack of hereditary information for this species and the difficulty of germplasm identification, we herein provide new genetic insight from the perspective of intraspecific transcriptome comparison and present single sequence repeat (SSR) molecular marker recognition technology. RESULTS: Through the study of a diploid germplasm (DIWNT) and a tetraploid germplasm (DIWT), the following outcome were obtained. (1) A significant difference in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations for specific homologous genes was observed using the OrthoMCL method for the identification of homologous gene families between the two cytotypes. Ka/Ks analysis of common, single-copy homologous family members also revealed a greater difference among genes that experienced positive selection than among those experiencing positive selection. (2) Of more practical value, 2575 SSR markers were predicted and partly verified. We used TaxonGap as a visual tool to inspect genotype uniqueness and screen for high-performance molecular loci; we recommend four primers of 65 randomly selected primers with a combined identification success rate of 88.6% as priorities for further development of DNA fingerprinting of C. indicum germplasm. CONCLUSIONS: The SSR technology based on next-generation sequencing was proved to be successful in the identification of C. indicum germplasms. And the information on the intraspecfic genetic divergence generated by transcriptome comparison deepened the understanding of this complex species' nature.

Identification of reference genes for tissue-specific gene expression in Panax notoginseng using quantitative real-time PCR.

Validated internal controls are prerequisites to accurately normalize gene expression levels. Here, 14 candidate reference genes in Panax notoginseng were characterized. Primer specificity and amplification efficiency were evaluated for each gene. Candidates were subjected to transcript quantification in the root, fibrous root, rhizome, leaf, receptacle, pedicel, and fruit tissues. Expression stability (M value) and normalization factor variation (Vn/Vn+1) were determined by geNorm. 26S-2 and ACT-2 exhibited the highest expression stability among the tissues. Gene expression of dammarenediol synthase was accurately detected after normalization to 26S-2 and ACT-2 was performed. Results were consistent when each or both of 26S-2 and ACT-2 were applied as internal control. Hence, this study provides useful information to normalize gene expression accurately in the tissue-specific transcripts of P. notoginseng.

Triterpenoid saponin biosynthetic pathway profiling and candidate gene mining of the Ilex asprella root using RNA-Seq.

Ilex asprella, which contains abundant α-amyrin type triterpenoid saponins, is an anti-influenza herbal drug widely used in south China. In this work, we first analysed the transcriptome of the I. asprella root using RNA-Seq, which provided a dataset for functional gene mining. mRNA was isolated from the total RNA of the I. asprella root and reverse-transcribed into cDNA. Then, the cDNA library was sequenced using an Illumina HiSeq™ 2000, which generated 55,028,452 clean reads. De novo assembly of these reads generated 51,865 unigenes, in which 39,269 unigenes were annotated (75.71% yield). According to the structures of the triterpenoid saponins of I. asprella, a putative biosynthetic pathway downstream of 2,3-oxidosqualene was proposed and candidate unigenes in the transcriptome data that were potentially involved in the pathway were screened using homology-based BLAST and phylogenetic analysis. Further amplification and functional analysis of these putative unigenes will provide insight into the biosynthesis of Ilex triterpenoid saponins.

Use of the psbA-trnH region to authenticate medicinal species of Fabaceae.

Fabaceae is a huge family that contains a large number of medicinal plants, many of which are commonly used in Chinese traditional medicine. However, traditional taxonomy has not been able to meet the complicated demands of species discrimination within Fabaceae. Thus, we employed a famous DNA barcode, the psbA-trnH region, to discriminate commonly used medicinal species of the family Fabaceae. Here, the psbA-trnH regions derived from 152 samples were amplified. These samples represented 104 Fabaceae medicinal species from 60 genera, including 25 authentic Fabaceae species listed in the Chinese pharmacopoeia and common adulterant species. The results indicate that the psbA-trnH region performed well in terms of its universality and high variability in length and composition. Species discriminative power analysis of the psbA-trnH region showed that 91.3% of species could be identified successfully by the BLAST1 method in conjunction with the nearest distance method. And, the species resolution rate of the TaxonGap method exceeded 93%. The results provide support for the use of the psbA-trnH plastid region as a sensitive marker to the authentication of Fabaceae medicinal plants.

Identification of Fabaceae plants using the DNA barcode matK.

In this study, we tested the applicability of the core DNA barcode MATK for identifying species within the Fabaceae family. Based on an evaluation of genetic variation, DNA barcoding gaps, and species discrimination power, MATK is a useful barcode for Fabaceae species. Of 1355 plant samples collected from 1079 species belonging to 409 diverse genera, MATK precisely identified approximately 80 % and 96 % of them at the species and genus levels, respectively. Therefore, our research indicates that the MATK region is a valuable marker for plant species within Fabaceae.

Species identification of medicinal pteridophytes by a DNA barcode marker, the chloroplast psbA-trnH intergenic region.

Medicinal pteridophytes are an important group used in traditional Chinese medicine; however, there is no simple and universal way to differentiate various species of this group by morphological traits. A novel technology termed "DNA barcoding" could discriminate species by a standard DNA sequence with universal primers and sufficient variation. To determine whether DNA barcoding would be effective for differentiating pteridophyte species, we first analyzed five DNA sequence markers (psbA-trnH intergenic region, rbcL, rpoB, rpoC1, and matK) using six chloroplast genomic sequences from GeneBank and found psbA-trnH intergenic region the best candidate for availability of universal primers. Next, we amplified the psbA-trnH region from 79 samples of medicinal pteridophyte plants. These samples represented 51 species from 24 families, including all the authentic pteridophyte species listed in the Chinese pharmacopoeia (2005 version) and some commonly used adulterants. We found that the sequence of the psbA-trnH intergenic region can be determined with both high polymerase chain reaction (PCR) amplification efficiency (94.1%) and high direct sequencing success rate (81.3%). Combined with GeneBank data (54 species cross 12 pteridophyte families), species discriminative power analysis showed that 90.2% of species could be separated/identified successfully by the TaxonGap method in conjunction with the Basic Local Alignment Search Tool 1 (BLAST1) method. The TaxonGap method results further showed that, for 37 out of 39 separable species with at least two samples each, between-species variation was higher than the relevant within-species variation. Thus, the psbA-trnH intergenic region is a suitable DNA marker for species identification in medicinal pteridophytes.

Construction of EVA/chitosan based PEG-PCL micelles nanocomposite films with controlled release of iprodione and its application in pre-harvest treatment of grapes.

A novel nanocomposite poly(ethylene-co-vinyl acetate) (EVA) film with controlled in vitro release of iprodione (ID) was prepared. Chitosan (CS) was used as the reinforcement which enhances the water and oxygen permeability of films. ID loaded poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) (IPP) micelles were used as the drug carrier which endows the films with antifungal and controlled release ability. IPP micelles with spherical shape and uniform size were obtained, and the maximum encapsulation efficacy (EE) was 91.17 ± 5.03% by well controlling the feeding amount of ID. Incorporation CS could improve the oxygen and moisture permeability of films, and the maximum oxygen permeability (OP) and water vapor transmission rate (WVTR) were 477.84 ± 13.03 cc/(m2·d·0.1 MPa) and 8.60 ± 0.25 g m-2 d-1, respectively. After loading IPP micelles, the films showed an improved antifungal ability and temperature-sensitive drug release behavior, and were found to enhance the quality of grapes by pre-harvest spraying.

Identification of Dendrobium species by a candidate DNA barcode sequence: the chloroplast psbA-trnH intergenic region.

DNA barcoding is a novel technology that uses a standard DNA sequence to facilitate species identification. Although a consensus has not been reached regarding which DNA sequences can be used as the best plant barcodes, the psbA-trnH spacer region has been tested extensively in recent years. In this study, we hypothesize that the psbA-trnH spacer regions are also effective barcodes for Dendrobium species. We have sequenced the chloroplast psbA-trnH intergenic spacers of 17 Dendrobium species to test this hypothesis. The sequences were found to be significantly different from those of other species, with percentages of variation ranging from 0.3 % to 2.3 % and an average of 1.2 %. In contrast, the intraspecific variation among the Dendrobium species studied ranged from 0 % to 0.1 %. The sequence difference between the psbA-trnH sequences of 17 Dendrobium species and one Bulbophyllum odoratissimum ranged from 2.0 % to 3.1 %, with an average of 2.5 %. Our results support the notion that the psbA-trnH intergenic spacer region could be used as a barcode to distinguish various Dendrobium species and to differentiate Dendrobium species from other adulterating species.

SNP typing for germplasm identification of Amomum villosum Lour. Based on DNA barcoding markers.

Amomum villosum Lour., produced from Yangchun, Guangdong Province, China, is a Daodi medicinal material of Amomi Fructus in traditional Chinese medicine. This herb germplasm should be accurately identified and collected to ensure its quality and safety in medication. In the present study, single nucleotide polymorphism typing method was evaluated on the basis of DNA barcoding markers to identify the germplasm of Amomi Fructus. Genomic DNA was extracted from the leaves of 29 landraces representing three Amomum species (A. villosum Lour., A. xanthioides Wall. ex Baker and A. longiligulare T. L. Wu) by using the CTAB method. Six barcoding markers (ITS, ITS2, LSU D1-D3, matK, rbcL and trnH-psbA) were PCR amplified and sequenced; SNP typing and phylogenetic analysis were performed to differentiate the landraces. Results showed that high-quality bidirectional sequences were acquired for five candidate regions (ITS, ITS2, LSU D1-D3, matK, and rbcL) except trnH-psbA. Three ribosomal regions, namely, ITS, ITS2, and LSU D1-D3, contained more SNP genotypes (STs) than the plastid genes rbcL and matK. In the 29 specimens, 19 STs were detected from the combination of four regions (ITS, LSU D1-D3, rbcL, and matK). Phylogenetic analysis results further revealed two clades. Minimum-spanning tree demonstrated the existence of two main groups: group I was consisting of 9 STs (ST1-8 and ST11) of A. villosum Lour., and group II was composed of 3 STs (ST16-18) of A. longiligulare T.L. Wu. Our results suggested that ITS and LSU D1-D3 should be incorporated with the core barcodes rbcL and matK. The four combined regions could be used as a multiregional DNA barcode to precisely differentiate the Amomi Fructus landraces in different producing areas.

Identification of medicinal plants in the family Fabaceae using a potential DNA barcode ITS2.

AIM OF THE STUDY: To test whether the ITS2 region is an effective marker for use in authenticating of the family Fabaceae which contains many important medicinal plants. MATERIALS AND METHODS: The ITS2 regions of 114 samples in Fabaceae were amplified. Sequence assembly was assembled by CodonCode Aligner V3.0. In combination with sequences from public database, the sequences were aligned by Clustal W, and genetic distances were computed using MEGA V4.0. The intra- vs. inter-specific variations were assessed by six metrics, wilcoxon two-sample tests and "barcoding gaps". Species identification was accomplished using TaxonGAP V2.4, BLAST1 and the nearest distance method. RESULTS: ITS2 sequences had considerable variation at the genus and species level. The intra-specific divergence ranged from 0% to 14.4%, with an average of 1.7%, and the inter-specific divergence ranged from 0% to 63.0%, with an average of 8.6%. Twenty-four species found in the Chinese Pharmacopoeia, along with another 66 species including their adulterants, were successfully identified based on ITS2 sequences. In addition, ITS2 worked well, with over 80.0% of species and 100% of genera being correctly differentiated for the 1507 sequences derived from 1126 species belonging to 196 genera. CONCLUSIONS: Our findings support the notion that ITS2 can be used as an efficient and powerful marker and a potential barcode to distinguish various species in Fabaceae.

Assessment of candidate plant DNA barcodes using the Rutaceae family.

DNA barcoding is a rapidly developing frontier technology that is gaining worldwide attention. Here, seven regions (psbA-trnH, matK, ycf5, rpoC1, rbcL, ITS2, and ITS) with potential for use as DNA barcodes were tested for their ability to identify 300 samples of 192 species from 72 genera of the family Rutaceae. To evaluate each barcode's utility for species authentication, PCR amplification efficiency, genetic divergence, and barcoding gaps were assessed. We found that the ITS2 region exhibited the highest inter-specific divergence, and that this was significantly higher than the intra-specific variation in the "DNA barcoding gap" assessment and Wilcoxon two-sample tests. The ITS2 locus had the highest identification efficiency among all tested regions. In a previous study, we found that ITS2 was able to discriminate a wide range of plant taxa, and here we confirmed that ITS2 was also able to discriminate a number of closely related species. Therefore, we propose that ITS2 is a promising candidate barcode for plant species identification.

Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species.

BACKGROUND: The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL+matK as the plant barcode, yet the combination was shown to successfully discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over. METHODOLOGY/PRINCIPAL FINDINGS: Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level. CONCLUSIONS: The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.