https://doi.org/10.1016/j.jpha.2023.03.008

DNA barcoding in herbal medicine: Retrospective and prospective

Rapid DNA template preparation directly from a single rice (Oryza sativa) grain or rice flour of its equivalent weight was developed for loop-mediated isothermal amplification (LAMP). LAMP efficiency using DNA extract obtained from consecutive addition of alkaline lysis reagent (25 mM NaOH, 0.2 mM EDTA) and neutralizing reagent (40 mM Tris-HCl [pH 5]) was comparable to that using an equivalent amount of purified DNA as template. The stability of the prepared DNA extract was confirmed for up to six-day storage at room temperature. Without using any special laboratory devices, the developed method enabled a rapid, simple, and low-cost DNA template preparation method for reliable LAMP testing to detect rice genes.

Rapid DNA template preparation directly from a rice sample without purification for loop-mediated isothermal amplification (LAMP) of rice genes.

(1) Background: Rapid and accurate determination of the content of the chemical dye Auramine O(AO) in traditional Chinese medicines (TCMs) is critical for controlling the quality of TCMs. (2) Methods: Firstly, various models were developed to detect AO content in Dendrobium officinale (D. officinale). Then, the detection of AO content in Saffron and Curcuma using the D. officinale training set as a calibration model. Finally, Saffron and Curcuma samples were added to the training set of D. officinale to predict the AO content in Saffron and Curcuma using secondary wavelength screening. (3) Results: The results show that the sparrow search algorithm (SSA)-backpropagation (BP) neural network (SSA-BP) model can accurately predict AO content in D. officinale, with Rp2 = 0.962, and RMSEP = 0.080 mg/mL. Some Curcuma samples and Saffron samples were added to the training set and after the secondary feature wavelength screening: The Support Vector Machines (SVM) quantitative model predicted Rp2 fluctuated in the range of 0.780 ± 0.035 for the content of AO in Saffron when 579, 781, 1195, 1363, 1440, 1553 and 1657 cm−1 were selected as characteristic wavelengths; the Partial Least Squares Regression (PLSR) model predicted Rp2 fluctuated in the range of 0.500 ± 0.035 for the content of AO in Curcuma when 579, 811, 1195, 1353, 1440, 1553 and 1635 cm−1 were selected as the characteristic wavelengths. The robustness and generalization performance of the model were improved. (4) Conclusion: In this study, it has been discovered that the combination of surface-enhanced Raman spectroscopy (SERS) and machine learning algorithms can effectively and promptly detect the content of AO in various types of TCMs.

Rapid Indentification of Auramine O Dyeing Adulteration in Dendrobium officinale, Saffron and Curcuma by SERS Raman Spectroscopy Combined with SSA-BP Neural Networks Model

Tick-borne hemoprotozoan and rickettsial diseases affect the health and productivity of small ruminants in tropical and subtropical regions. Despite the large population of goats in the southern part of Thailand, there is limited information on the prevalence of tick-borne pathogens. In this study, polymerase chain reaction was used to detect the presence of Theileria spp., T. ovis, T. orientalis, Babesia ovis, Anaplasma ovis, and A. marginale in 262 goats from three provinces in the southern part of Thailand. In this investigation, Theileria spp. and A. ovis were detected while T. ovis, B. ovis, and A. marginale were not detected. Overall infection rates of Theileria spp. and A. ovis were 10.3% and 1.5%, respectively. The co-infections of two parasites was observed in 1.5% of goats. Sequence analysis showed the presence of T. luwenshuni and T. orientalis in the goat samples. This study is the first to use the molecular detection of T. orientalis in Thai goats, and presents genetic characterization using the major piroplasm surface protein (MPSP) gene. In the phylogenetic analysis, the T. orientalis MPSP sequence was classified as type 7. The A. ovis major surface protein 4 (MSP4) gene sequences shared high identities and similarity with each other and clustered with isolates from other regions. This study provides information about the prevalence and genetic diversity of tick-borne pathogens in goats in the study area, and is expected to be valuable for the development of effective control measures to prevent disease in animals in Thailand.

/pdf/molecular-detection-and-genetic-diversity-of-tick-borne-dmnru1u5.pdf

Molecular Detection and Genetic Diversity of Tick-Borne Pathogens in Goats from the Southern Part of Thailand

Ticks are small and adaptable arachnid ectoparasites and global carriers of various pathogens that threaten both human and animal health. They are present in many parts of China. A total of 858 ticks were collected from various regions and hosts, then subjected to species identification based on morphological and molecular characteristics, as described in the authors' previous study. Eighty-three individual tick samples were selected for screening pathogens based on metagenomic next-generation sequencing (mNGS) and polymerase chain reaction (PCR) assays. The genomic DNA of tick species was extracted, and amplification of the bacterial 16S rRNA gene was carried out from DNA of individual ticks using V3-V4 hypervariable regions, before subjecting to metagenomic analysis. Each tick underwent specific PCR tests for identifying the bacterial species present, including Anaplasma, Ehrlichia, Coxiella, and Rickettsia, and also protozoans such as Babesia, Theileria, and Hepatozoon. Illumina NovaSeq sequencing results revealed that the dominant phylum and family in Rhipicephalus spp. were Bacteroidota and Muribaculaceae, respectively. Alpha diversity patterns varied depending on tick sex (R. linnaei only), species and location, but not on host. Furthermore, bacterial pathogens, including A. marginale (58 %, 29/50), A. platys (6 %, 3/50), E. minasensis (2 %, 1/50), Ehrlichia sp. (10 %, 5/50), T. sinensis (24 %, 12/50), T. orientalis (54 %, 27/50) and Coxiella-like bacteria (CLB) (80 %, 40/50) were detected in R. microplus, while E. canis (33.33 %, 10/30), H. canis (20 %, 6/30) and CLB (100 %, 30/30) were detected in R. linnaei. Also, Anaplasma sp. (33.33 %, 1/3), A. marginale (33.33 %, 1/3), R. felis (33.33 %, 1/3) and CLB (100 %, 3/3) were detected in R. haemaphysaloides. Dual and triple co-infections involving pathogens or CLB were detected in 84.00 % of R. microplus, 66.66 % of R. haemaphysaloides, and 33.00 % of R. linnaei. The report on microbial communities and pathogens, which found from Rhipicephalus spp. in Hainan Island, is an important step towards a better understanding of tick-borne disease transmission. This is the first report in the area on the presence of Anaplasma sp., A. marginale, R. felis and Coxiella, in R. haemaphysaloides. 

Molecular and Next-Generation Sequencing Analysis of Tick-Borne Pathogens of Rhipicephalus Ticks (Acari: Ixodidae) in cattle and dogs.

Theileria spp. are a group of parasites primarily transmitted by ticks and can pose a significant threat to domestic and wild animals globally. The main objective of this study was to understand the epidemiology of Theileria spp. in goats of Hainan Island/province, which is the only tropical region of China, and to study their hematological profiles in naturally infected goats. A total of 464 blood samples were collected from randomly selected local adult goats (Capra hircus, local domestic breed with black hair), from six cities and eight counties of Hainan, from November 2017 to October 2020. Blood smear microscopy of the sample and a nested polymerase chain reaction (nPCR) targeting the 18S rRNA gene combined with DNA sequencing were used to detect piroplasm infections in goats. Data analysis of the obtained sequences revealed that all the sequences were highly similar to the Theileria luwenshuni 18S rRNA gene sequence from the database. This result is consistent with the microscopic examination. In the hematological test, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin of the goats naturally infected with T. luwenshuni significantly increased, while mean corpuscular hemoglobin concentration and red blood cell distribution width (RDW) were significantly decreased. Results showed that T. luwenshuni could cause macrocytic, hypochromic anemia in goats. This study provides reliable and comprehensive information about the epidemiology of the parasite infections and hematological profile of the infected goats in Hainan, which encourages further investigations to develop practical control strategies for Theileria spp. infections in tropical areas.

/pdf/identification-of-theileria-spp-and-investigation-of-2csm719g.pdf

Identification of Theileria spp. and investigation of hematological profiles of their infections in goats in Hainan Island, China

Background: L-lysine is widely used in the feed, food, and pharmaceutical industries, and screening for high L-lysine-producing strains has become a key goal for the industry. Methods: We constructed the rare L-lysine codon AAA by corresponding tRNA promoter replacement in C. glutamicum. Additionally, a screening marker related to the intracellular L-lysine content was constructed by converting all L-lysine codons of enhanced green fluorescent protein (EGFP) into the artificial rare codon AAA. The artificial EGFP was then ligated into pEC-XK99E and transformed into competent Corynebacterium glutamicum 23604 cells with the rare L-lysine codon. After atmospheric and room-temperature plasma mutation and induction culture, 55 mutants (0.01% of total cells) with stronger fluorescence were sorted using flow cytometry, and further screened by fermentation in a 96-deep-well plate and 500 mL shaker. Results: The fermentation results showed that the L-lysine production was increased by up to 9.7% in the mutant strains with higher fluorescence intensities, and that the highest screening positive rate was 69%, compared with that in the wild-type strain. Conclusion: The application of artificially constructed rare codons in this study represents an efficient, accurate, and simple method for screening other amino acid-producing microorganisms.

/pdf/design-and-application-of-artificial-rare-l-lysine-codons-in-3us4r7xr.pdf

Design and application of artificial rare L-lysine codons in Corynebacterium glutamicum

Rapid identification of Dendrobium officinale using Loop-Mediated Isothermal Amplification (LAMP) method.

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Identification of Theileria spp. and investigation of hematological profiles of their infections in goats in Hainan Island, China

Design and application of artificial rare L-lysine codons in Corynebacterium glutamicum

Rapid identification of Dendrobium officinale using Loop-Mediated Isothermal Amplification (LAMP) method.