Patrón de neumonía organizativa en TC de tórax: prevalencia y asociación con resultados clínicos en una cohorte de pacientes con COVID-19 grave/crítico / Organizing Pneumonia Pattern on Chest CT: Prevalence and Association with Clinical Outcomes in a Cohort of Patients with Severe/Critical COVID-19

Introducción: La neumonía por COVID-19 puede presentarse con dos patrones radio-lógicos: daño alveolar difuso o neumonía organizativa. Estos patrones tienen diferente evolución y pronóstico en pacientes sin infección por COVID-19. Nuestro objetivo fue evaluar la prevalencia del patrón radiológico de neumonía organizativa y su asociación con los desenlaces clínicos.Métodos: Se realizó un estudio de cohorte retrospectivo que incluyó a pacientes adultos hospitalizados por COVID-19 grave/crítica a los que se les realizó una tomografía computarizada de tórax en los 21 días posteriores al diagnóstico. Los patrones radiológicos fueron revisados y clasificados por dos radiólogos expertos. Resultados: De los 80 pacientes incluidos, el 89% (n=71) presentaron un patrón compatible con neumonía organizativa. Los principales hallazgos radiológicos fueron la distribución multilobar (98,7%) y bilateral (97,6%) con opacidades en vidrio esmerilado (97,6%). El 44% (n=33) de los sujetos requirió ingreso en cuidados intensivos, de los cuales el 24% (n=19) recibió ventilación mecánica. La presencia de neumonía organizativa se asoció de forma independiente con una disminución de las probabilidades de ventilación mecánica o muerte (Odds ratio 0,14; intervalo de confianza del 95%: 0,02 - 0,96; valor de p 0,045) en un modelo multivariado que incluía la edad, el sexo, el IMC y la afectación pulmonar en la TC.Conclusiones: Un patrón radiológico de neumonía organizativa es altamente prevalen-te en pacientes con COVID-19 grave/crítico y se asocia con mejores resultados clínico

Introduction: COVID-19 pneumonia can present with two distinct radiologic patterns: diffuse alveolar damage or organizing pneumonia. These patterns have been linked to different outcomes in non-COVID-19 settings. We sought to assess the prevalence of organizing pneumonia radiologic pattern and its association with clinical outcomes. Methods: We performed a retrospective cohort study including adult patients hospita- lized for severe/critical COVID-19 who underwent chest computed tomography within 21 days of diagnosis. Radiologic patterns were reviewed and classified by two expert radiologists. Results: Among 80 patients included, 89% (n=71) presented a pattern consistent with organizing pneumonia. The main radiologic findings were multilobar (98.7%) and bilateral (97.6%) distribution with ground glass opacities (97.6%). Intensive care admission was required for 44% (n=33) of subjects, of which 24% (n=19) received mechanical ventilation. The presence of organizing pneumonia was independently associated with a decreased odds of mechanical ventilation or death (Odds ratio 0.14; 95% confidence interval 0.02 - 0.96; p value 0.045) in a multivariate model including age, gender, BMI and lung involvement on CT. Conclusion: A radiologic pattern of organizing pneumonia is highly prevalent in patients with severe/critical COVID-19 and is associated with improved clinical outcomes.

[Site-specific PCR identification of animal species and formula particles of Scorpio].

Scorpio, a commonly used animal medicine in China, is derived from Buthus martensii as recorded in the Chinese Pharmacopoeia. China harbors rich species of Scorpionida and adulterants exist in the raw medicinal material and deep-processed products of Scorpio. The microscopic characteristics of the deep-processed products may be incomplete or lost during processing, which makes the identification difficult. In this study, the maximum likelihood(ML) tree was constructed based on the morphology and cytochrome C oxidase subunit I(COⅠ) to identify the species of Scorpio products. The results showed that the main adulterant of Scorpio was Lychas mucronatus. According to the specific SNP sites in the COⅠ sequence of B. martensii, the stable primers were designed for the identification of the medicinal material and formula granules of Scorpio. The polymerase chain reaction(PCR) at the annealing temperature of 61 ℃ and 30 cycles produced bright specific bands at about 150 bp for both B. martensii and its formula particles and no band for adulterants. The adaptability of the method was investigated, which showed that the bands at about 150 bp were produced for Scorpio medicinal material, lyophilized powder, and formula granules, and commercially available formula granules. The results showed that the established method could be used to identify the adulterants of Scorpio and its formula granules, which could help to improve the quality control system and ensure the safe clinical application of Scorpio formula granules.

[Identification of Cervi Cornu (Cervus elaphus) and its formula granules based on PCR-RFLP].

Cervi Cornu is the ossified antler, or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C. nippon, as a precious traditional Chinese medicine, has been recognized for its medicinal value and widely used in clinical practice. However, the origins of Cervi Cornu are miscellaneous, and Cervi Cornu is even mixed with adulterants in the market. Currently, there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cornu. So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants. In this study, the differences in the mitochondrial barcode cytochrome b(Cytb) gene sequences of C. elaphus, C. nippon and their related species were compared and the specific single nucleotide polymorphism(SNP) sites on the Cytb sequences of Cervi Cornu were screened out. According to the screened SNPs, Cervi Cornu-specific primers dishmy-F and dishmy-R were designed. The PCR system was established and optimized, and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated. The amplification products of C. elaphus and C. nippon were digested using the restriction enzyme MseⅠ. The results showed that after electrophoresis of the product from PCR with the annealing temperature of 56 ℃ and 35 cycles, a single specific band at about 100 bp was observed for C. elaphus samples, and the product of C. elaphus samples was 60 bp shorter than that of C. nippon samples. There was no band for adulterants from other similar species such as Alces alces, Rangifer tarandus, Odocoileus virginianus, O. hemionus, Cap-reolus pygargus, Przewalskium albirostis and negative controls. The polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method established in this study can quickly and accurately identify Cervi Cornu originated from C. elaphus in crude drugs, standard decoctions, and formula granules, and distinguish the origins of Cervi Cornu products, i.e., C. nippon and similar species. This study can be a reference for other studies on the quality standard of other formula granules of traditional Chinese medicines.

Development of an allele-specific PCR (AS-PCR) method for identifying high-methyl eugenol-containing purple Tulsi (Ocimum tenuiflorum L.) in market samples.

BACKGROUND: Ocimum tenuiflorum L. is a highly traded medicinal with several therapeutic values. Green Tulsi and purple Tulsi are two subtypes in O. tenuiflorum and both have the same medicinal properties. Recent reports have revealed that purple Tulsi contains higher quantities of methyl eugenol (ME), which is moderately toxic and potentially carcinogenic. Therefore, we developed an allele-specific PCR (AS-PCR) method to distinguish the green and purple Tulsi. METHODS AND RESULT: Using the green Tulsi as a reference, 12 single nucleotide polymorphisms (SNPs) and 10 insertions/deletions (InDels) were identified in the chloroplast genome of the purple Tulsi. The C > T SNP at the 1,26,029 position in the ycf1 gene was selected for the development of the AS-PCR method. The primers were designed to amplify 521 bp and 291 bp fragments specific to green and purple Tulsi, respectively. This AS-PCR method was validated in 10 accessions from each subtype and subsequently verified using Sanger sequencing. Subsequently, 30 Tulsi powder samples collected from the market were subjected to molecular identification by AS-PCR. The results showed that 80% of the samples were purple Tulsi, and only 3.5% were green Tulsi. About 10% of the samples were a mixture of both green and purple Tulsi. Two samples (6.5%) did not contain O. tenuiflorum and were identified as O. gratissimum. CONCLUSION: The market samples of Tulsi were predominantly derived from purple Tulsi. The AS-PCR method will be helpful for quality control and market surveillance of Tulsi herbal powders.

Culture-independent detection of low-abundant Clostridioides difficile in environmental DNA via PCR.

Clostridioides difficile represents a major burden to public health. As a well-known nosocomial pathogen whose occurrence is highly associated with antibiotic treatment, most examined C. difficile strains originated from clinical specimen and were isolated under selective conditions employing antibiotics. This suggests a significant bias among analyzed C. difficile strains, which impedes a holistic view on this pathogen. In order to support extensive isolation of C. difficile strains from environmental samples, we designed a detection PCR that targets the hpdBCA-operon and thereby identifies low abundances of C. difficile in environmental samples. This operon encodes the 4-hydroxyphenylacetate decarboxylase, which catalyzes the production of the antimicrobial compound para-cresol. Amplicon-based analyses of diverse environmental samples demonstrated that the designed PCR is highly specific for C. difficile and successfully detected C. difficile despite its absence in general 16S rRNA gene-based detection strategies. Further analyses revealed the potential of the hpdBCA detection PCR sequence for initial phylogenetic classification, which allows assessment of C. difficile diversity in environmental samples via amplicon sequencing. Our findings furthermore showed that C. difficile strains isolated under antibiotic treatment from environmental samples were originally dominated by other strains according to PCR amplicon results. This provided evidence for selective cultivation of under-represented but antibiotic-resistant isolates. Thereby, we revealed a substantial bias in C. difficile isolation and research.IMPORTANCEClostridioides difficile is a main cause of diarrheic infections after antibiotic treatment with serious morbidity and mortality worldwide. Research on this pathogen and its virulence has focused on bacterial isolation from clinical specimens under antibiotic treatment, which implies a substantial bias in isolated strains. Comprehensive studies, however, require an unbiased strain collection, which is accomplished by isolation of C. difficile from diverse environmental samples and avoidance of antibiotic-based enrichment strategies. Thus, isolation can significantly benefit from our C. difficile-specific detection PCR, which rapidly verifies C. difficile presence in environmental samples and further allows estimation of the C. difficile diversity by using next-generation sequencing.

Isolation of hitherto-uncultivated microorganisms- Application of radical scavengers.

The culture filtrates of the predominant bacterial strains isolated from soil samples have been shown to increase the microbial colony counts on agar plates used for the isolation of uncultured bacteria. One of the factors in the culture filtrates responsible for this increase was identified to be superoxide dismutase (SOD). The generation of reactive oxygen species (O2-, H2O2, and ・OH) was detected from conventional laboratory agar media. The use of agar media supplemented with radical scavengers (SOD, catalase, ascorbic acid, or rutin) effectively increased the colony counts and kinds of microbial strains that grew from soil samples. Taxonomical studies on these isolates revealed new taxa for phylum Actinomycetota; one family, three genera, and nine species were newly described. One of the strains, Patulibacter minatonensis KV-614T belonging to the new family Patulibacteraceae, was isolated on agar medium supplemented with SOD. P. minatonensis KV-614T represents a novel lineage within the phylum Actinomycetota. A polymerase chain reaction (PCR) study using specific primers for the detection of strains related to the genus Patulibacter, order Solirubrobacterales, showed a high distribution frequency, with detection in over 70% of the soil samples tested. These data suggest that the use of radical scavengers may facilitate the isolation of some hitherto-uncultivated microorganisms widely distributed in soil.

Bilateral Scleritis with Heterogeneous Etiologies: A Diagnostic Dilemma.

PURPOSE: To report a patient with bilateral scleritis who was initially treated for infectious scleritis, and subsequently diagnosed as granulomatosis with polyangiitis (GPA). METHOD: Retrospective chart review. RESULT: A 48-year-old female with a known history of diabetes presented with pain, redness, and blurring of vision in the right eye. She was diagnosed as scleritis with retinal detachment and underwent vitrectomy, with silicone oil tamponade and intravitreal injections of antibiotics, and antifungal agents. She presented with active scleritis with additional multiple pus points. Several scleral biopsies failed to yield any microorganism and finally, the painful blind eye was enucleated. The enucleated specimen grew gram-positive bacteria which were identified as Staphylococcus arlettae by polymerase chain reaction (PCR)-based sequencing of the 16S rRNA gene. Nine months after the onset of symptoms in the right eye, the patient developed necrotizing scleritis in the left eye. Laboratory investigation revealed a positive cytoplasmic- anti-neutrophil cytoplasmic autoantibody, which was previously negative during the right eye involvement. She was diagnosed with GPA by a rheumatologist and treated accordingly. CONCLUSION: Retinal detachment may be associated with scleritis, and ANCA testing may not detect GPA in its early stages. Once infection has been excluded, clinicians should not be afraid to use high-dose immunosuppression instead of surgery to treat retinal detachment associated with scleritis.

Comparison of the Performance of Bioresonance, Electrophoresis and RT-PCR in the Diagnosis of Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) continues to be one of the most researched infectious diseases of cats. The diagnosis of FIP is challenging, and diverse techniques have been developed for its accurate diagnosis. However, they have some limitations. The present study was conducted to investigate the efficacy of specific modulation frequency (SMF), compared to other routine diagnostic methods for detecting feline coronavirus. Blood samples were collected from 30 diseased cats suspected of having FIP based on clinical signs. Electrophoresis, polymerase chain reaction (PCR), and SMF tests were performed for each sample. The sensitivity and specificity of each test, as well as the agreement between the tests and the gold standard (the combination of PCR, electrophoresis, and bioresonance results), were calculated using the Kappa coefficient method. The sensitivity and specificity of electrophoresis, PCR, and SMF for the diagnosis of FIP were 70.6%, 70.6%, 100%, and 100%, 72.7%, 81.8%, respectively. According to the findings of the present study, SMF is effective and safe in FIP diagnosis, which is a challenge in veterinary medicine diagnosis.

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla.

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology.

Biosensing with Polymerase Chain Reaction-Stable DNA-Functionalized Magnetically Susceptible Carbon-Iron Microparticles.

We demonstrate a rapid and sensitive method for DNA detection without the need for fluorescence. This is based on carbon-coated magnetic iron (Fe) microparticles with a covalent surface attachment of DNA. We show that these magnetic microparticles can capture complementary DNA. Significantly, the DNA covalent surface bonds are robust to high temperatures and can be included in a sample during polymerase chain reaction (PCR). This method is employed for the detection of targeted DNA sequences (40-50 bp). Hybridization probes on the surface of the magnetically susceptible Fe microparticle recognize the target DNA sequence-specifically. The double-stranded DNA (dsDNA) microparticles are then quickly captured with a magnet from the sample matrix. This foregoes postpurification processes, such as electrophoresis, which make our technique time- and cost-effective. Captured dsDNA can be detected with intercalating dyes such as ethidium bromide through a loss in the UV absorption signal with a limit of detection (LOD) of 24 nM within 15 min. Likewise, surface-bound DNA can act as a primer in PCR to decrease the LOD to 5 pM within 2 h. This is the first instance of a nucleotide-modified magnetically susceptible carbon substrate that is PCR-compatible. Besides DNA capture, this strategy can eventually be applied to sequence-specific nucleic acid purification and enrichment, PCR cleanup, and single-strand generation. The DNA-coated particles are stable under PCR conditions (unlike commonly used polystyrene or gold particles).

Identification and Genome Characterization of a Dahlia Common Mosaic Virus Isolate from China.

Dahlia (Dahlia variabilis) is a widely cultivated ornamental and medicinal plant in China. Recently, dahlia plants with symptoms of leaf mottling and distortion were collected in Hohhot, Inner Mongolia, China. The presence of dahlia common mosaic virus (DCMV), an unassigned species in the genus Caulimovirus, was confirmed by high-throughput sequencing. Three fragments of DCMV Inner Mongolia isolate (DCMV-IN) were PCR-amplified with specific primers, sequenced and assembled into the complete genome sequence with a GenBank accession number of OR494328. The double-stranded circular DNA genome of DCMV-IN consists of 7949 bp and contains six open reading frames (ORFs). Sequence analysis showed that DCMV-IN shared high sequence identities with other DCMV isolates available in the GenBank database. Phylogenetic analysis of DCMV isolates and other representative caulimoviruses based on genome sequence clustered four DCMV isolates to a single branch which was closest to dahlia mosaic virus (DMV). No recombination event was detected among the four DCMV isolates.

[Update: Diagnostics and treatment of pulmonary tuberculosis]. / Update: Diagnostik und Therapie der pulmonalen Tuberkulose.

Molecular diagnostic tools have changed the approach to the detection of Mycobacterium tuberculosis and associated drug-resistance substantially. PCR-based technologies allow a more rapid detection with higher diagnostic sensitivity in pulmonary and extrapulmonary specimens. However, a real point of care test, which needs minimal technical resources remains missing. Genome sequencing technologies are currently changing tuberculosis drug resistance testing, and for some questions are replacing phenotypic drug resistance testing, based on culture.New evidence on treatment for drug-sensitive tuberculosis allows shortening of treatment to 4 months, or in selected cases even to 2 months based on the use of fluoroquinolones, high dose rifamycins and newly developed TB medicines.Such developments will very likely simplify the management of tuberculosis, although prevention remains the most important pillar of any tuberculosis related public health strategy.

Application of a modified tetra-primer ARMS-PCR assay for rapid Panax species identity authentication in ginseng products.

Panax ginseng products can be adulterated with materials from other Panax species. The purpose of this study is to provide a rapid P. ginseng authentication method for simultaneous identification of P. ginseng and detection of adulteration in ginseng products at different processing stages. First, a tetra-primer ARMS-PCR assay was designed based on a single-nucleotide polymorphism (SNP) within the trnL-trnF region and was tested at 28 PCR cycles with DNA extracted from Botanical Reference Materials (BRMs). Next, 5' end random nucleotide and 3' terminus phosphorothioates linkage modifications were incorporated into the inner primers to improve sensitivity and specificity at 40 PCR cycles. Finally, the modified assay was validated using characterized market ginseng materials and the detection limit was determined. The modified tetra-primer ARMS-PCR assay can achieve the desired sensitivity and specificity using one set of reaction conditions in ginseng materials at different stages. In validation, it was able to correctly identify target species P. ginseng and differentiate it from closely related species. This study suggests that the modified tetra-primer ARMS-PCR assay can be used for the rapid, species identity authentication of P. ginseng material in ginseng products. This assay can be used to complement chemical analytical methods in quality control, so both species identity and processing attributes of ginseng products can be efficiently addressed.

Development of a tetra-primer ARMS-PCR for identification of sika and red deer and their hybrids.

Accurate identification of deer-derived components is significant in food and drug authenticity. Over the years, several methods have been developed to authenticate these products; however, identifying whether female deer products are hybrids is challenging. In this study, the zinc finger protein X-linked (ZFX) gene sequences of sika deer (Cervus nippon), red deer (Cervus elaphus) and their hybrid offspring were amplified and sequenced, the X221 and X428 species-specific single nucleotide polymorphisms (SNP) loci were verified, and a tetra-primer amplification refractory mutation system (T-ARMS-PCR) assay was developed to identify the parent-of-origin of female sika deer, red deer, and their hybrid deer. The T-ARMS-PCR developed based on the X221 locus could identify sika deer, red deer, and their hybrid offspring according to the presence or absence of PCR product sizes of 486 bp, 352 bp, and 179 bp, respectively, just as X428 locus could identify sika deer, red deer, and their hybrid offspring according to the presence or absence of PCR product sizes of 549 bp, 213 bp, and 383 bp, respectively. Forty products labeled deer-derived ingredients randomly purchased were tested using this assay, and the results showed that the identification results based on the two SNP loci were utterly consistent with the actual sources. In addition, this method was found to be accurate, simple, convenient, and with high specificity, thus providing an essential technical reference for deer product species identification. It is also an important supplement to the identification methods of the original ingredients of existing deer products.

Comprehensive analysis using DNA metabarcoding, SCAR marker based PCR assay, and HPLC unveils the adulteration in Brahmi herbal products.

BACKGROUND: Brahmi is one of the important nootropic botanicals, widely sold in the market, with the name "Brahmi'' being used to describe both Bacopa monnieri and Centella asiatica species. The Brahmi herbal products market is expanding; hence, economically motivated adulteration is highly prevalent. METHODS AND RESULTS: This study aimed to develop DNA-based methods, including SCAR marker-based PCR and metabarcoding, to authenticate Brahmi herbal products and compare these methods with HPLC. These methods have been validated using mock controls (in-house blended formulations). All targeted plant species in mock controls were detected successfully with all three methods, whereas, in market samples, only 22.2%, 55.6%, and 50.0% were found positive for Brahmi by PCR assay, DNA metabarcoding, and HPLC, respectively. Metabarcoding can detect the presence of non-labeled plants together with targeted species, which is an advantage over PCR assay or HPLC. CONCLUSION: SCAR marker-based PCR is a rapid and cost-effective method for detecting the presence of B. monnieri and C. asiatica. However, in this study, the success rate of PCR amplification was relatively low because the primers targeted either RAPD or ITS-based SCAR markers. HPLC assay, although an alternative, was unable to detect the presence of other botanicals, just like the SCAR marker-based PCR assay. On the other hand, metabarcoding can be utilized to identify the target plants, even in very small quantities, while also providing simulated identification of other botanicals. This study successfully addressed the need for quality control of Brahmi herbal products and provided the first-time report of DNA metabarcoding for such products.

[Distinguishing between Artemisia stolonifera and A. argyi by specific PCR of leaves and non-glandular trichomes].

Artemisia stolonifera is a relative of A. argyi. The two species are difficult to be distinguished due to the similarity in leaf shape and have even less distinctive features after processing. This study aims to establish a method to quickly distinguish between them. At the same time, we examined the reasonability and applicability of the specific polymerase chain reaction(PCR) method. The C/T single nucleotide polymorphism was detected at the position 202 of the sequence, based on which specific primers were designed to identify these two species. The PCR with the specific primer JNC-F and the universal primer ITS3R produced a specific band at 218 bp for A. argyi and no band for A. stolonifera, which can be used to detect at least 3% of A. argyi samples mixed in A. stolonifera samples. The PCR with the specific primer KY-F and the universal primer ITS3R produced a specific band at 218 bp for A. stolonifera and no band for A. argyi, which can be used to detect at least 5% of A. stolonifera samples mixed with A. argyi. The limit of detection of the established method was 5 ng DNA. The established PCR method can accurately distinguish between A. stolonifera and A. argyi, which provides an experimental basis for the quality control of A. stolonifera and determines whether the herbs are adulterated.

Genetic Authentication of the Medicinal Plant Portulaca oleracea Using a Quick, Precise, and Sensitive Isothermal DNA Amplification Assay.

Portulaca oleracea (PO) is a commonly known medicinal crop that is an important ingredient for traditional Chinese medicine (TCM) due to its use as a vegetable in the diet. PO has been recorded to be frequently adulterated by other related species in the market of herbal plants, distorting the PO plant identity. Thus, identification of the botanical origin of PO is a crucial step before pharmaceutical or functional food application. In this research, a quick assay named "loop-mediated isothermal amplification (LAMP)" was built for the specific and sensitive authentication of PO DNA. On the basis of the divergences in the internal transcribed spacer 2 (ITS2) sequence between PO and its adulterant species, the LAMP primers were designed and verified their specificity, sensitivity, and application for the PO DNA authentication. The detection limit of the LAMP assay for PO DNA identification specifically was 100 fg under isothermal conditions at 63 °C for 30 min. In addition, different heat-processed PO samples can be applied for use in PO authentication in the LAMP assay. These samples of PO were more susceptible to the effect of steaming in authentication by PCR than boiling and drying treatment. Furthermore, commercial PO samples pursued from herbal markets were used to display their applicability of the developed LAMP analysis for PO postharvest authentication, and the investigation found that approximately 68.4% of PO specimens in the marketplace of herbal remedies were adulterated. In summary, the specific, sensitive, and rapid LAMP assay for PO authentication was first successfully developed herein, and its practical application for the inspection of adulteration in PO samples from the herbal market was shown. This LAMP assay created in this study will be useful to authenticate the botanical origin of PO and its commercial products.

Development of PCR based SSR markers for Wilsonomyces carpophilus and a PCR based diagnosis protocol for the early detection of shot hole disease in stone fruit crops.

BACKGROUND: The conidial Ascomycota fungus Wilsonomyces carpophilus causing shot hole in stone fruits is a major constraint in the production of stone fruits worldwide. Shothole disease symptoms appear on leaves, fruits, and twigs. Successful isolation of the pathogen from different hosts on synthetic culture medium is a time consuming and tedious procedure for identification of the pathogen based on morpho-cultural characterization. METHODS AND RESULTS: The present research was carried out to develop a successful PCR based early detection protocol for the shot hole disease of stone fruits, viz., peach, plum, apricot, cherry, and almond using the pathogen specific SSR markers developed from the Wilsonomyces carpophilus genome using Genome-wide Microsatellite Analysing Tool package (GMATA) software. Diseased leaf samples of different stone fruits were collected from the SKUAST-K orchard and the pathogen was isolated on potato dextrose agar (PDA) medium and maintained on Asthana and Hawkers' medium with a total of 50 pathogen isolates comprised of 10 isolates each from peach, plum, apricot, cherry and almond. The DNA was extracted from both healthy and infected leaf samples of different stone fruits. The DNA was also extracted from the isolated pathogen cultures (50 isolates). Out of 2851 SSR markers developed, 30 SSRs were used for the successful amplification of DNA extracted from all the 50 pathogen isolates. These SSRs were used for the amplification DNA from shot hole infected leaf samples of different stone fruits, but the amplification was not observed in the control samples (DNA from healthy leaves), thus confirming the detection of this disease directly from the shot hole infected samples using PCR based SSR markers. To our knowledge, this forms the first report of SSR development for the Wilsonomyces carpophilus and their validation for the detection of shot hole disease directly from infected leaves. CONCLUSION: PCR based SSR makers were successfully developed and used for the detection of Wilsonomyces carpophilus causing shot hole disease in stone fruits including almond in nuts for the first time. These SSR markers could successfully detect the pathogen directly from the infected leaves of stone fruits namely peach, plum, apricot and cherry including almond from the nuts.

A Cas12a-based platform combined with gold nanoparticles for sensitive and visual detection of Alternaria solani.

Alternaria solani (A. solani), the causal agent of early blight in potatoes, poses a serious and persistent threat to potato production worldwide. Therefore, developing a method that can accurately detect A. solani in the early stage to avoid further spread is urgent. However, the conventional PCR-based method is not appropriate for application in the fields. Recently, the CRISPR-Cas system has been developed for nucleic acids analysis at point-of-care. Here, we propose a gold nanoparticles-based visual assay combining loop-mediated isothermal amplification with CRISPR-Cas12a to detect A. solani. After optimization, the method could detect 10-3 ng/µL genomic gene of A. solani. The specificity of the method was confirmed by discriminating A. solani from other three highly homologous pathogens. We also developed a portable device that could be used in the fields. By integrating with the smartphone readout, this platform holds significant potential in high-throughput detection of multiple pathogens in the fields.

Genome skimming as an efficient tool for authenticating commercial products of the pharmaceutically important Paris yunnanensis (Melanthiaceae).

BACKGROUND: Paris yunnanensis (Melanthiaceae) is a traditional Chinese medicinal plant of significant pharmaceutical importance. Due to previous taxonomic confusion, a congeneric species, Paris liiana, has been mistaken for P. yunnanensis and cultivated on a large scale, leading to the mixing of commercial products (i.e., seedlings and processed rhizomes) of P. yunnanensis with those of P. liiana. This may have adverse effects on quality control in the standardization of P. yunnanensis productions. As the lack of PCR amplifiable genomic DNA within processed rhizomes is an intractable obstacle to the authentication of P. yunnanensis products using PCR-based diagnostic tools, this study aimed to develop a PCR-free method to authenticate commercial P. yunnanensis products, by applying genome skimming to generate complete plastomes and nrDNA arrays for use as the molecular tags. RESULTS: Based on a dense intraspecies sampling of P. liiana and P. yunnanensis, the robustness of the proposed authentication systems was evaluated by phylogenetic inferences and experimental authentication of commercial seedling and processed rhizome samples. The results indicate that the genetic criteria of both complete plastomes and nrDNA arrays were consistent with the species boundaries to achieve accurate discrimination of P. yunnanensis and P. liinna. Owing to its desirable accuracy and sensitivity, genome skimming can serve as an effective and sensitive tool for monitoring and controlling the trade of P. yunnanensis products. CONCLUSION: This study provides a new way to solve the long-standing problem of the molecular authentication of processed plant products due to the lack of PCR amplifiable genomic DNA. The proposed authentication system will support quality control in the standardization of P. yunnanensis products in cultivation and drug production. This study also provides molecular evidence to clarify the long-standing taxonomic confusion regarding the species delimitation of P. yunnanensis, which will contribute to the rational exploration and conservation of the species.