Extraction Kit Research Articles

Evaluation of long-read sequencing for Ostreid herpesvirus type 1 genome characterization from Magallana gigas infected tissues.

Since the 1990s, the Pacific oyster Magallana gigas has faced significant mortality, which has been associated with the detection of the Ostreid Herpesvirus type 1 (OsHV-1). Due to the complex genomic architecture and the presence of multiple genomic isomers, short-read sequencing using Illumina method struggles to accurately assemble tandem and repeat regions and to identify and characterize large structural variations in the OsHV-1 genome. Third-generation sequencing technologies, as long-read real-time nanopore sequencing from Oxford Nanopore Technologies (ONT), offer new possibilities for OsHV-1 whole-genome analysis. Identification of the best method for extraction of high molecular weight (HMW) DNA and development of accurate bioinformatic pipelines for its characterization are now required. To this end, we evaluated and compared six HMW methods and one conventional DNA extraction kit for their ability to extract OsHV-1 DNA from M. gigas-infected tissues. We then evaluated the ability of ONT sequencing to produce an accurate OsHV-1 genome from both whole-genome and "adaptive sampling" (AS) sequencing approaches. Finally, we evaluated the efficiency of bioinformatics tools for de novo assembly and consensus calling to generate accurate OsHV-1 genomes. The HMW DNA extraction kit coupled with ONT sequencing and dedicated bioinformatics tools allowed us to produce accurate OsHV-1 genomes compared to those assembled using Illumina technology. The AS approach allowed up to 60% enrichment for viral data, and the long reads generated by ONT allowed the characterization of OsHV-1 isomers. Together with its portability, this sequencing shows great promise as a diagnostic tool for the characterization of unculturable aquatic viruses directly from host tissues.IMPORTANCEMany aquatic viruses threaten commercially valuable species and cause significant economic losses during outbreaks. To improve our understanding of the origin, transmission patterns and spread of these viruses, additional genomic data are essential. However, genomic characterization of unculturable large DNA viruses is a major challenge. In the present study, we have successfully evaluated the ability of ONT sequencing and adaptive sequencing (AS) to sequence and assemble the complete OsHV-1 genome. Our results show that it is now possible to sequence the whole genome of large DNA viruses directly from infected host tissue, without the need for prior in vitro propagation or prior laboratory steps for virus enrichment.

Exploring animal food microbiomes and resistomes via 16S rRNA gene amplicon sequencing and shotgun metagenomics.

As a diverse and complex food matrix, the animal food microbiota and repertoire of antimicrobial resistance (AMR) genes remain to be better understood. In this study, 16S rRNA gene amplicon sequencing and shotgun metagenomics were applied to three types of animal food samples (cattle feed, dry dog food, and poultry feed). ZymoBIOMICS mock microbial community was used for workflow optimization including DNA extraction kits and bead-beating conditions. The four DNA extraction kits (AllPrep PowerViral DNA/RNA Kit, DNeasy Blood & Tissue Kit, DNeasy PowerSoil Kit, and ZymoBIOMICS DNA Miniprep Kit) were compared in animal food as well as the use of peptide nucleic acid blockers for 16S rRNA gene amplicon sequencing. Distinct microbial community profiles were generated, which varied by animal food type and DNA extraction kit. Employing peptide nucleic acid blockers prior to 16S rRNA gene amplicon sequencing was comparable with post-sequencing in silico filtering at removing chloroplast and mitochondrial sequences. There was a good agreement between 16S rRNA gene amplicon sequencing and shotgun metagenomics on community profiles in animal feed data sets; however, they differed in taxonomic resolution, with the latter superior at resolving at the species level. Although the overall prevalence of AMR genes was low, resistome analysis of animal feed data sets by shotgun metagenomics revealed 10 AMR gene/protein families, including beta-lactamases, erythromycin/lincomycin/pristinamycin/tylosin, fosfomycin, phenicol, and quinolone. Future expansion of microbiome and resistome profiling in animal food will help better understand the bacterial and AMR gene diversity in these commodities and help guide pathogen control and AMR prevention efforts.IMPORTANCEWith the growing interest and application of metagenomics in understanding the structure/composition and function of diverse microbial communities along the One Health continuum, this study represents one of the first attempts to employ these advanced sequencing technologies to characterize the microbiota and AMR genes in animal food. We unraveled the effects of DNA extraction kits on sample analysis by 16S rRNA gene amplicon sequencing and showed similar efficacies of two strategies at removing chloroplast and mitochondrial reads. The in-depth analysis using shotgun metagenomics shed light on the community compositions and the presence of an array of AMR genes in animal food. This exploration of microbiomes and resistomes in representative animal food samples by both sequencing approaches laid important groundwork for future metagenomic investigations to gain a better understanding of the baseline/core microbiomes and associated AMR functions in these diverse commodities and help guide pathogen control and AMR prevention efforts.

A sensitivity analysis of methodological variables associated with microbiome measurements.

The experimental methods employed during metagenomic sequencing analyses of microbiome samples significantly impact the resulting data and typically vary substantially between laboratories. In this study, a full factorial experimental design was used to compare the effects of a select set of methodological choices (sample, operator, lot, extraction kit, variable region, and reference database) on the analysis of biologically diverse stool samples. For each parameter investigated, a main effect was calculated that allowed direct comparison both between methodological choices (bias effects) and between samples (real biological differences). Overall, methodological bias was found to be similar in magnitude to real biological differences while also exhibiting significant variations between individual taxa, even between closely related genera. The quantified method biases were then used to computationally improve the comparability of data sets collected under substantially different protocols. This investigation demonstrates a framework for quantitatively assessing methodological choices that could be routinely performed by individual laboratories to better understand their metagenomic sequencing workflows and to improve the scope of the datasets they produce.IMPORTANCEMethod-specific bias is a well-recognized challenge in metagenomic sequencing characterization of microbiome samples, but rigorous bias quantification is challenging. This report details a full factorial exploration of 48 experimental protocols by systematically varying microbiome sample, iterations of material production, laboratory personnel, DNA extraction kit, marker gene selection, and reference databases. Quantification of the biases associated with each parameter revealed similar magnitudes of variation arising from real biological differences and from varied analysis procedures. Furthermore, these measurement biases varied substantially with taxa, even between closely related genera. However, computational correction of method bias using a reference material was demonstrated that significantly harmonized metagenomic sequencing results collected using different analysis protocols.

First Report of Pseudomonas amygdali Causing Leaf Spot on Hibiscus rosa-sinensis in a greenhouse in New York State, U.S.A.

Hibiscus rosa-sinensis, commonly known as the "Chinese hibiscus", is a widely cultivated shrub with ornamental and medicinal applications (Jadhav et al., 2009). However, it is known to be susceptible to a range of pathogens including bacteria (Chase, 1986). In March 2019, Hibiscus rosa-sinensis plants in production at a greenhouse in New York, but originally from Florida, developed widespread, conspicuous leaf spots and associated chlorosis for a month after arrival. "Hibiscus 35-1" was isolated from a suspension of symptomatic plant tissue pieces soaked in sterile deionized water and streaked on potato dextrose agar (PDA) (González-Tobón et al., 2023), resulting in numerous white bacterial colonies with a uniform appearance. Previously we reported the complete species identification for this bacterial isolate using whole genome sequencing via Oxford Nanopore and Illumina platforms (BioProject: PRJNA765326) as a Pseudomonas amygdali with an ANI ≥ 95% ± 0.5%. (González-Tobón et al., 2023). The hypersensitive response (HR) to P. amygdali 35-1 was first evaluated in three tobacco (Nicotiana tabacum) and three tomato (Solanum lycopersicum) plants. An overnight culture was washed, resuspended, adjusted to an OD600 of 0.2-0.3, and used to inoculate two leaves per plant via syringe infiltration (Chakravarthy et al., 2017). HR was observed on all leaves at the inoculation site as early as 48hpi for tobacco and 24hpi for tomato. No symptoms were observed for mock inoculations. The pathogenicity of P. amygdali 35-1 in hibiscus was then tested using six 'Social Butterfly' plants in 4.0 in pots inoculated by hand spraying. Inoculum was made from a 72 h culture on solid King's B medium resuspended in sterile deionized water with 2 drops/L of Tween 80 and sprayed at a concentration of 3.6 x 108 bacteria/mL, as determined by dilution plating. Inoculated plants and six matching controls were sealed inside plastic bags and placed in a randomized complete block in a growth chamber at 30 ºC with a 12h photoperiod for 40 days. No changes were observed on any of the control plants but spotting and chlorosis similar to the original symptoms gradually developed on five of the inoculated plants. After 40 days, seven 2-mm tissue squares were excised from the lesion margins or from the asymptomatic tissue of controls, surface-sterilized with 0.75% NaCl, and plated on King's B. No bacteria were recovered from the control plants but white colonies resembling those of the original inoculation were recovered from several diseased tissue pieces. Genomic DNA was extracted from this bacterium using the New England Biolabs Monarch Genomic DNA Extraction Kit. Polymerase chain reaction (PCR) tests were performed to amplify five genes (16S, cts, gapA, gyrB, and rpoD) typically used for multilocus sequence typing (MLST) (Sarkar & Guttman, 2004). The amplicons were Sanger sequenced and BLAST analyses confirmed 100% match to P. amygdali 35-1 (GenBank: CP084212). To our knowledge, our lab is the first to report P. amygdali causing disease on hibiscus in New York. Similar symptoms on hibiscus plants in Florida were reported in the past as caused by other Pseudomonas species of the P. syringae G group, mainly P. syringae and P. cichorii (Chase, 1986; Gardan et al., 1999: Girard et al., 2021; Jones et al., 1986). Given the lack of consistent reporting on bacterial leaf spot pathogens of hibiscus, as well as the economic importance of this ornamental, further research on the disease is needed.

First report of hop stunt viroid infecting luffa and cantaloupe in Taiwan

Luffa (Luffa cylindrica (L.) M. Roem.) and cantaloupe (Cucumis melo L.) are economically important cucurbitaceous crops in Taiwan. In August 2018, luffa samples displaying leaf chlorosis and deformation were collected in Nantou while cantaloupe samples exhibiting the above suspected systemic viral-like symptoms were collected in Chiayi in October 2019, respectively. Reverse transcription (RT)-PCR tests were conducted to investigate potential viral infections in leaf samples from 12 luffa and nine cantaloupe plants. Total nucleic acids were extracted from symptomatic cucurbit tissues using the taco™ DNA/RNA extraction kit (GeneReach Biotechnology, Taiwan), followed by PCR or RT-PCR tests for virus detection. Seven sets of degenerate primers targeting viral genera reported in local cucurbit crops, including genera Begomovirus, Crinivirus, Cucumovirus, Polerovirus, Potyvirus, Orthotospovirus and Tobamovirus, were utilized. However, all tests yielded negative results. Consequently, degenerate primers targeting genus Pospiviroid or hop stunt viroid (HSVd)-specific primers were employed. Among these tests, only HSVd-specific primers HSVd-RTR_F/R (5’-GGAATTCTCGAGTTGCCGCA-3’/5’-CCGCGGCCCTCTCT-3’) (van Dorst and Peters 1974) successfully amplified DNA fragments of approximately 130 bp from 19 symptomatic samples (12 from luffa and 9 from cantaloupe), while normal leaf samples of luffa and cantaloupe tested negative. Based on known nucleotide sequences, primers were designed to amplify and obtain the full-length 303-nucleotide sequences of the HSVd genome derived from luffa (accession no. OP331278) and cantaloupe (accession no. OP331279). The two sequences exhibit a 98.7% nucleotide identity with each other while exhibiting 99.3% nucleotide identity with a cucumber isolate from Japan (X00524) (Sano et al. 1984) and 98.0% nucleotide identity with another isolate (X07405) from the Netherlands (Puchta et al. 1988). Comparisons with 13 HSVd citrus isolates from Taiwan revealed nucleotide similarities ranging from 96.1% to 99.0%. To confirm pathogenicity, Nicotiana benthamiana was chosen as the experimental isolation host. Mechanical inoculation of N. benthamiana with diluted sap (20 W/V) from diseased plants consistently produced leaf wrinkling and stunting symptoms in 10–60% of inoculated plants by 12 days post-inoculation (DPI). RT-PCR tests confirmed viroid infection. Additionally, luffa seedlings at the cotyledon stage back-inoculated with the purified HSVd luffa isolate developed leaf deformation symptoms 15–25 DPI, and the infection was confirmed by RT-PCR, fulfilling Koch’s postulates. HSVd infections have previously been reported in citrus and grapes in Taiwan (Lin et al. 2015). To our knowledge, this is the first report of HSVd infecting luffa and cantaloupe in Taiwan. The newly identified HSVd cucumber isolates represent a potential threat to cucurbit production and warrant further investigation.

Detection of Porcine Norovirus GII.18 Strains in Pigs Using Broadly Reactive RT-qPCR Assay for Genogroup II Noroviruses

Abstract Noroviruses, belonging to the family Caliciviridae, are classified into at least ten genogroups (G) based on their major capsid protein (VP1). The common genogroup to be identified in both humans and pigs is GII, although porcine noroviruses (PoNoVs) belong to genotypes of their own (GII.11, GII.18, and GII.19). So far, PoNoVs have not been studied much in Finland, possibly due to their rather symptomless nature in pigs. In the present study, we enrolled a total of 189 fecal samples collected from pigs from Finnish farms. Samples were taken from 12 farms in 2010, 2019 and 2020. We analyzed feces from growing pigs ranging from 2.1 to 6 months of age. RNA was extracted from fecal suspensions using a commercial viral RNA extraction kit, followed by RT (reverse transcription)-qPCR. The genotypes were determined by Sanger sequencing of the PCR fragments amplified by conventional PCR. Of the 12 farms, 6 (50%) had at least one PoNoV-infected pig. Altogether 18 (9.5%) of the 189 pigs tested positive for PoNoVs. Pigs mostly aged over 4 months were infected with PoNoVs. Eventually, 12 positive samples were determined as genotype GII.18. We could demonstrate the presence of PoNoVs in Finnish pigs. In future, more studies in which longer sequences from PoNoV genome can be obtained, are required.

Comparison of Plant Genomic DNA Extraction Kits Using the Proofman-LMTIA Amplification Assay.

The extraction of DNA is the basis of molecular biology research. The quality of the extracted DNA is one of the key factors for the success of molecular biology experiments. To select a suitable DNA extraction method for Chinese medicinal herbs and seeds. In this experiment, four commercial DNA extraction kits were used to extract the genomic DNA (gDNA) from the Pinellia ternata (Thunb.) Breit. powder; Arisaema amurense Maxim. powder as well as the seeds of Glycine max (L.) Merr. On the one hand, the concentration and purity of DNA extracted by these four kits were compared. On the other hand, nucleic acid amplification experiments were performed on three samples extracted by each of the four kits by Proofman-LMTIA methods, which is a novel nucleic acid isothermal amplification technique. The concentration and purity of DNA extracted by different kits were used to determine which methods were suitable for the dry powder of Chinese herbal medicines and seeds. The efficiency of the amplification curve to show whether the extracted DNA can be used in nucleic acid amplification experiments. The results showed that the Proofman-LMTIA methods were of high specificity and the optimal reaction temperatures were 63, 59, and 59°C for P. ternata (Thunb.) Makino; A. amurense Maxim. and G. max (L.) Merr., respectively. The concentration and purity of the gDNAs extracted with all kits were within the acceptable ranges; meanwhile, the amplification of the gDNA extracted by Kit II was of the highest efficiency. In this experiment, the principle, concentration, purity, and time taken for extracting DNA with four kits were compared. The automated extraction kit based on the magnetic method is suitable for extracting DNA from Chinese medicinal herbs and seeds. The extracted DNA is suitable for nucleic acid amplification detection.

Construction of Candida albicans pRB895-SAP2-SC5314 With SAP2 High Expression and Its Effects on Adhesion.

SAP2 is closely associated with the pathogenicity and drug resistance of Candida albicans (C. albicans). Our study aimed to construct C. albicans with SAP2 overexpression (pRB895-SAP2-SC5314) to explore the influence of SAP2 on the adhesion of C. albicans and predict the interaction between magnolol and Sap2 by molecular docking. The recombinant plasmid pRB895-SAP2 with high SAP2 expression was prepared using a plasmid extraction kit and transformed into C. albicans strain SC5314 using an improved lithium acetate conversion method to construct PRB895-SAP2-SC5314. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect the expression of adhesion-related genes in the different strains. Molecular docking and visual analysis of magnolol and Sap2 were performed using the CB-DOCK2 platform. Compared with the control SC5341 and SC5341 transfected with pRB895, SAP2 expression was significantly higher in the pRB895-SAP2-SC5314 strain (p < 0.05). Based on the sequencing and mapping results, the pRB895-SAP2-SC5314 strain was successfully prepared. SAP2 overexpression significantly downregulated ALS1 expression (p < 0.05), whereas ALS3, TEC1, HOG1, PHR1, and TUP1 expression was downregulated in C. albicans (p < 0.05). The optimal docking result for magnolol and Sap2 protein was -8.1 kcal/mol of vina score, which was considered good docking. SAP2 overexpression may strengthen the adhesion and pathogenicity of C. albicans, and magnolol may act as an Sap2 inhibitor that affects the adhesion of C. albicans.

Performance evaluation of nine reference centers and comparison of DNA extraction protocols for effective surveillance of Leishmania-infected Phlebotomine sand flies: Basis for technical recommendations.

Leishmaniasis, caused by Leishmania protozoan parasites transmitted by Phlebotomine sand flies, is a significant public health concern in the Mediterranean basin. Effective monitoring of Leishmania-infected sand flies requires standardized tools for comparing their distribution and infection prevalence. Consistent quantitative real-time PCR (qPCR) parameters and efficient DNA extraction protocols are crucial for reliable results over time and across regions. However, the absence of standardized technical recommendations for Leishmania DNA detection hinders effective surveillance. This study aimed to compare different DNA extraction protocols and conduct a qPCR-based External Quality Assessment (EQA) through a multicenter study involving nine reference laboratories, with a focus on optimizing Leishmania DNA detection in sand fly. EQA samples consisted of Leishmania infantum and L. major species, at concentrations ranging from 101 to 104 parasites/mL. All but one center detected all concentrations, demonstrating strong diagnostic proficiency. The ability to detect low concentrations highlighted the robustness of the qPCR assay used, though variations in Cq values indicated differences in sensitivity related to technical capabilities or DNA extraction kit performance. A comparative analysis of seven DNA extraction methods identified the EZ1 DSP Virus Kit and QIAamp DNA mini-kit as the most efficient, supporting their use in standardized protocols. The study also assessed the effects of lyophilization and shipment conditions, showing no significant compromise in Leishmania detection despite slight variations in Cq values. Experimentally infected sand flies were included to simulate field conditions, and all centers successfully detected positive samples with varying Cq values, probably reflecting differences in infection load. This study emphasizes the importance of standardized DNA extraction protocols and continuous quality assurance for accurate Leishmania DNA detection. The results highlight the superior performance of certain extraction kits and the need for ongoing technical training, essential for reliable leishmaniasis surveillance, particularly in field settings with low infection densities.

Development of a Collection Kit for Efficient Isolation of SARS-CoV-2 RNA from Urine and Stool Samples

Background Information: Consider adding a brief sentence about the significance of detecting SARS-CoV-2 in urine and stool samples. For example, mention how these samples can serve as alternative sources for diagnosis, potentially leading to better patient outcomes. Objective Statement: This study aimed to develop a collection kit for the effective extraction of SARS-CoV-2 RNA from urine and stool samples. The study presents a significant advancement in the field of viral diagnostics by introducing a novel collection kit specifically designed for the extraction of SARS-CoV-2 RNA from urine and stool samples. The demonstrated efficacy of the kit in isolating the virus with high success rates from alternative biological fluids, as well as the superior RNA. Methodology: The kit contains a swab of medical polyester and three tubes, two of them used to inactivate the virus, one contains 3% phenol and the other 1% phenol, and the third tube contains 2 ml of (PBS) Phosphate Buffer Saline added to it 4% of skim milk. Urine or stool samples from Covid-19 patients who reported positive by nasopharyngeal swab during RT-PCR was adde consequently in these three tubes then assessed through RT-PCR testing. Results: The kit succeeded in isolating coronavirus from the urine and stool of patients who reported positive by nasopharyngeal samples. From 20 positive nasopharyngeal samples, there was 18 (90%) of them were positive by using stool sample using this kit, and 15 (75%) of them were give positive result when urine samples were used by this kit. The concentration of RNA of coronavirus which was isolated from urine and feces was higher than in the nasopharyngeal swab during RT-PCR. Mean Ct value of the 20 samples was showed that the mean Ct of nasopharyngeal was 31.5, while urine and stool samples Ct means less concentration of virus. were 25.75 and 24.32 respectively. High The kit was able to serve RNA that was isolated from urine and feces for about 5,10, and 30 days in a percentage of 100%, 94.1%, and 94.1% respectively. Conclusion and Implications: The kit is produce to confirm that the patient is infected with by COVID-19 and to preserve the virus in stool or urine samples of the infected person for a long period until laboratory tests through RT-PCR are conducted on the samples. The kit showed great ability to test SARS-CoV-2, even if it was at low concentrations of virus compared to the examination of nasopharyngeal swabs.

Development of an infiltration-based RNA preservation method for cryogen-free storage of leaves for gene expression analyses in field-grown plants

BackgroundGene expression is a fundamental process for plants to express their phenotype, and its analysis is the basis of molecular studies. However, the instability of RNA often poses an obstacle to analyzing plants grown in fields or remote locations where the availability of liquid nitrogen or dry ice is limited. To deepen our understanding of plant phenotypes and tolerance to field-specific stresses, it is crucial to develop methodologies to maintain plant RNA intact and safely transfer it for downstream analyses such as qPCR and RNA-seq.ResultsIn this study, the author developed a novel tissue preservation method that involved the infiltration of RNA preservation solution into the leaf apoplast using a syringe and subsequent storage at 4 °C. RNA-seq using samples stored for 5 d and principal component analyses showed that rice leaves treated with the infiltration method maintained the original transcriptome pattern better than those treated with the traditional method when the leaves were simply immersed in the solution. Additionally, it was also found that extracted RNA can be transported with minimum risk of degradation when it is bound to the membrane of RNA extraction kits. The developed infiltration method was applied to rice plants grown in a local farmer's field in northern Madagascar to analyze the expression of nutrient-responsive genes, suggesting nutrient imbalances in some of the fields examined.ConclusionsThis study showed that the developed infiltration method was effective in preserving the transcriptome status of rice and sorghum leaves when liquid nitrogen or a deep freezer is not available. The developed method was useful for diagnosing plants in the field based on the expression of nutrient-responsive marker genes. Moreover, the method used to protect RNA samples from degradation during transportation offers the possibility to use them for RNA-seq. This novel technique could pave the way for revealing the molecular basis of plant phenotypes by accelerating gene expression analyses using plant samples that are unique in the field.

12 real forensic cases solved by the DNA STR-typing of skeletal remains exposed to extreme environment conditions, without the conventional bone pulverization step.

DNA identification of human skeletal remains play a valuable role in the forensic field, especially in missing persons and mass disasters investigation. Hard tissues, such as bones and teeth, represent a very common kind of samples analyzed in forensic laboratories because often they are the only biological materials remaining. However, the major limitation in using these compact samples rely on time consuming and labor-intensive treatment of grinding them into powder before proceeding with the conventional DNA purification and extraction step. In this context, a novel DNA extraction assay, called the TBone Ex kit (DNA Chip Research Inc.), was developed to digests bone chips without powdering "as reported by Kitayama (JAMA 12:84-89, 2010)." Here, we simultaneously analyzed bone and tooth samples obtained by our police laboratory that belonged to 15 different forensic cases from Sardinia (Italy). The total of 27 samples were recovered from different scenarios and were exposed to extreme environmental factors, including sunlight, seawater, soil, fauna, vegetation and high temperature and humidity. The TBone Ex kit was used prior to the EZ2 DNA extraction kit on the EZ2 Connect Fx instrument (Qiagen), and high quality autosomal and Y-chromosome STRs profiles were obtained for the 80% of the cases, in a relatively short time frame. This study provides additional support for the use of the TBone Ex kit for digesting bone fragments/whole teeth as an effective alternative to pulverization protocols. We empirically demonstrated the effectiveness of the kit in processing multiple bone samples simultaneously, largely simplifying the DNA extraction procedure, and the good yield of recovered DNA for downstream genetic typing in highly compromised forensic real specimens. In conclusion, the results of this study appear useful for forensic laboratories, to which the various actors of the criminal justice system - such as potential jury members, judges, defense attorneys and prosecutors - require immediate feedback.

DNA quality and STR success rate in different formalin-fixed tissues.

Formalin-fixed tissues possess irreplaceable value as a source of DNA for identification, especially when fresh samples are unavailable. Nonetheless, extracting and amplifying DNA from these tissues is challenging, primarily due to formaldehyde-induced cross-linking and nucleic acid fragmentation. In this study, two pre-extraction treatments, gradual dehydration using ethanol and pre-digestion heat treatments, and three DNA extraction methods, the Chelex-100 method, TIANamp FFPE DNA Kit, and ML Ultra-micro DNA extraction kit, were utilized to optimize DNA extraction from different tissues, which were fixed in 4% unbuffered formalin for different durations. The tissues include the heart, liver, spleen, lung, kidney, muscle, and brain. DNA quality was assessed, and quantification was conducted using Spectrophotometer and Quantifiler® Trio DNA Quantification Kits, while the GSTAR™ 25 kit was employed for STR detection. The results indicated that the two pre-extraction treatments exhibited no significant effect on the STR success rate. On day 9, allelic dropout was observed in the heart, liver, spleen, lung, and kidney tissues. Furthermore, allelic dropout was observed in muscle and brain at 12 days and 15 days, respectively. In conclusion, the results underscore the feasibility of effectively extracting DNA from formalin-fixed tissues within 9 days for subsequent STR analysis.

First report of potato pink rot caused by Phytophthora erythroseptica in Colorado.

Pink rot, caused by the oomycete Phytophthora erythroseptica, is a serious disease that may cause substantial losses to potato growers. In 2023, high infection rates of pink rot were noticed in the San Luis Valley (SLV), Colorado around harvest time and in storage with several growers losing complete storage pins to pink rot. In September 2023, tubers with pink rot symptoms were collected from two grower fields of Russet Norkotah (~2-3% infection) and Canela Russet (~5% infection) near the San Luis Valley Research Center at harvest time. Infected tubers showed dark discoloration on the surface at the stem end. Upon cutting, affected tissue developed salmon pink color about 20-30 min later (Fig. 1). DNA was extracted from two symptomatic tubers from each cultivar and healthy tubers using DNeasy PowerSoil Pro Kit (Qiagen, MD, USA) according to the manufacturer's instructions. The conventional PCR and qPCR assays developed by Cullen et al. (2007) were used to detect P. erythroseptica. In the conventional PCR, the primer pair Pery2F1/R1 produced expected bands of 135bp from symptomatic tubers but not from the healthy tubers. The symptomatic tubers were positive in the qPCR using the primer-probe set 99F, 177R, and 133T (Cullen et al., 2007). P. erythroseptica was isolated by culturing specimens of infected tissues on PARP selective media (Jeffers and Martin, 1986). A week later colonies of white color grew on the media. These colonies were confirmed to be of P. erythroseptica by PCR as mentioned above. The colonies of two isolates were transferred to water agar media, then three days later a mycelium tip was excised and cultured on PDA media for single isolation. On PDA media the colonies showed chrysanthemum pattern. Four days later DNA was extracted from the two isolates, RN296-1 and Canela-2, and tested positive for P. erythroseptica by the above-mentioned PCR. PDA agar plugs of two isolates were used to inoculate surface sterilized potato tubers. The isolate SLV-2023-Canela2 was inoculated on tubers of Russet Norkotah and Reveille Russet, 10 each, while10 tubers of Russet Norkotah were inoculated with the isolate SLV-2023-RN296-1. Ten tubers of Russet Norkotah were inoculated by sterilized plugs of PDA for negative controls. Tubers were left at room temperature for a week. All tubers of Russet Norkotah inoculated with SLV-2023-Canela2 and RN296-4 developed pink rot symptoms and tested positive for P. erythroseptica by PCR, while only 9 tubers of Reveille Russet inoculated with SLV-2023-Canela2 developed symptoms and were positive to P. erythroseptica by PCR. No symptoms were observed on the mock inoculated tubers and no P. erythroseptica was detected by the PCR. P. erythroseptica was isolated again from Russet Norkotah tubers inoculated with RN296-1 on PDA media and tested positive for P. erythroseptica by PCR one week later. To further confirm the identity, the ITS region was amplified using the primers ITS-1 and ITS-4 (White et al., 1990) from DNA extracted from inoculated Russet Norkotah tubers with SLV-2023-Canela2 isolate. PCR product was purified using QIAquick Gel Extraction Kit (Qiagen, MD, USA)and cloned using TOPO TA Cloning Kit for Sequencing (Invitrogen, MD, USA). Four clones were submitted for sequencing. The 846 nt sequence of the ITS was submitted to the GenBank (accession number PP587391) shared 99.76% identity with the P. erythroseptica strain BBA 62683 (KJ755119). P. erythroseptica was identified previously using morphological tools from tubers with symptoms resembling pink rot collected in the SLV (Tyler et al., 2002). However, in that report neither Koch's Postulates, nor molecular identification were conducted, hence, the current study presents the first molecular report of pink rot in the SLV, Colorado. Rapidly changing climate and increased temperature around harvest time may lead to an increase in the incidence of pink rot, hence effective management strategies need to be developed and implemented.

First Report of Leaf Blight on Paris polyphylla Caused by Ceratobasidium ramicola in China.

Paris polyphylla var. yunnanensisis a perennial herb with significant medicinal properties in anticancer, anti-inflammatory, antibacterial immunomodulatory and antispasmodic activities (Duan et al. 2018). In April 2022, leaf blight disease emerged in Xiangtan City (Hunan), affecting P. polyphylla plantings over an area of 3×104 m2 (27.904°N, 112.918°E). The disease incidence reached an average of 22% of the plants in the field, with infected plants initially displaying water-soaked chlorosis, followed by dry yellow shrinkage that gradually spread from the leaf tips to the entire plant. To identify the causal agent, 20 leaf lesions (4 mm2) collected from 20 plants were surface sterilized with 75% ethanol for 10 s, 5% NaOCl for 30 s, rinsed in sterile distilled water three times, transferred to potato dextrose agar (PDA) plates with lactic acid (0.125%) , and incubated at 28 °C in the dark. Four isolates (PP21 to PP24) with similar morphologies were obtained and purified by the hyphal-tip method. Colonies on PDA initially appeared white with cottony mycelium, later turning light-yellow on the underside. Septate hyphae were branched at right angles with a small constriction at the departure of the branch point and measured 4.16 to 7.93 µm in diameter. Binucleate cells within the septate hyphae were visualized using Giemsa staining (Servicebio, China). For molecular identification, the rDNA internal transcribed spacer (ITS), the second largest subunit of nuclear DNA-directed RNA polymerase II (RPB2), and ATP synthase subunit 6 (ATP6) were amplified from genomic DNA of the isolates extracted by Fungus Genomic DNA Extraction Kit (Bioflux, China) using primers ITS1/ITS4 (White et al. 1990), bRPB2-6F/bRPB2-7.1R (Matheny 2005; Reeb et al. 2004) and ATP61/ATP62 (Kretzer and Bruns 1999), respectively. The ITS, RBP2 and ATP6 of the four isolates were sequenced and deposited in GenBank. BLASTn search of sequenced ITS (PQ187050, PQ187051, PP728052, PQ187052), RBP2 (PQ202833, PQ202834, PP735921, PQ202835) and ATP6 (PQ202837, PQ202838, PP735922, PQ202839) revealed a >99% identity with the type strain of Ceratobasidium ramicola CBS133.82 (NR138368, DQ301708, and DQ301577). For phylogenetic analysis, concatenated sequences of ITS, RBP2, and ATP6 were employed using the maximum-likelihood method in MEGA-X. Based on the morphological and molecular analyses, the isolates were classified into the C. ramicola clade (Bandoni 1979; Samuels et al. 2012; Jeong et al. 2023). To test the pathogenicity of the isolate PP23, mycelial plugs (5 mm in diameter) were placed directly on 15 healthy leaves from 15 three-year-old plants after puncturing with sterile needles. Sterile PDA plugs served as controls. All plants were kept in a greenhouse with conditions of 25°C, 80% relative humidity and a photoperiod of 12 h. After 5 days, all infected plants developed leaf blight symptoms similar to those described above, whereas the control plants remained asymptomatic. The pathogenicity test was conducted three times. C. ramicolawas re-isolated from all symptomatic leaves and confirmed to be identical to the original isolate based on morphology and nucleotide sequences of ITS, RBP2, and ATP6. While C. ramicola is commonly reported to be isolated from diseased cacao, its pathogenicity to cacao remains unknown (Samuels et al. 2012). To our knowledge, this is the first report of C. ramicola causing leaf blight on P. polyphylla, a medicinal herb with significant economic importance in China.

Unraveling influenza sentinel surveillance in Pakistan 2008-2024: Epidemiological insights during the pre and post pandemic period of COVID-19.

The coronavirus pandemic has substantially influenced the transmission pattern of other respiratory viruses. However, screening and detecting other respiratory pathogens was unheeded during this time to combat the COVID-19 pandemic. High virulence and re assortment factors intensify the importance of influenza virus surveillance for effective disease management. Therefore, the present surveillance study was designed to determine the influenza positivity rate from 2008-24. This study will provide integral support in depicting a panoramic representation of two respiratory-pandemic periods, 2010-11 and 2019-2021, for influenza and COVID-19 pandemics, respectively. An inferential cross-sectional study was conducted from 2008 to 2024 by collecting influenza surveillance data from twelve sentinel sites in Pakistan. Clinical and demographic data was recorded at sample collection time. Specimens were collected through nasopharyngeal/throat swabs and stored in viral transport medium (VTM) at the sentinel site laboratory at 2-4°C. Viral RNA was isolated from the samples using KingFisher TM Flex Purification System and MagMAX™ Viral/Pathogen Nucleic Acid Extraction Kit. Within 16 years, 78118 samples were tested for influenza, of which 7999 (10.2%) appeared positive. The positivity rate appeared very low in recent years, with only a 3.5% positivity rate observed in 2020. Influenza A strain H1N1pdm09 seemed to be the prominent strain (n=3407, 42.6%), followed by influenza B (n=2125, 26.6%). The positivity of influenza samples was 10.2% and recorded in patients where typical clinical representation of influenza was absent. Fewer samples were reported during the coronavirus pandemic, which might be because influenza screening was hindered and overlooked to combat the SARS-CoV-2 virus, and the patient threshold was very high for COVID-19 virus screening.

A rapid quarantine approach for the pathogenic and invasive Phytophthora species associated with imported fruits in China.

Phytophthora spp. represent a pivotal genus of plant pathogens with a global distribution, exerting significant deleterious effects on food safety and forestry ecosystems. Numerous pathogenic and invasive Phytophthora species, introduced through imported fruits, have been frequently detected at Chinese ports. With the rise in global trade activities, the plant quarantine of imported fruits is becoming increasingly important but challenging. Fast, simple, and labor-saving techniques are necessary and anticipated. A polymerase chain reaction restriction fragment length polymorphism capillary electrophoresis (PCR-RFLP-CE) technology-based quarantine approach was developed for 16 Phytophthora species associated with the imported fruits in China. The Ypt1 gene, exhibiting abundant interspecific variations, was selected as the marker gene for PCR. The restriction endonuclease AluI was proven to be capable and compatible in simultaneously separating different Phytophthora species during CE. By combining with a fast and efficient DNA extraction kit, the developed PCR-RFLP-CE technique was successfully applied to identify Phytophthora species in artificially infested fruits. We provide a quick, practical, and high-throughput detection approach for hazardous and invasive Phytophthora species associated with imported fruits in China. This strategy can give good convenience and technological support for carrying out massive quarantine activities at Chinese ports. © 2024 Society of Chemical Industry.

Exposure to Hypoxic Conditions Up-regulates HER2 in Breast Cancer Cell Lines.

Tissue specimen quality is becoming increasingly important for basic research and routine clinical results. Warm ischemia time (WIT) affects human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) scores. However, the role of WIT on HER2 modulation remains unclear. We hypothesized that the WIT-mediated increase in HER2 IHC scores was caused by hypoxia. Therefore, this study aimed to determine the mechanism by which WIT mediates the increase in HER2. HER2 mRNA expression was measured in 4T1, SKBR3, and HCC1954 breast cancer cell lines using real-time PCR following hypoxia exposure. The membrane proteins were isolated and extracted using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA) or evaluated through non-permeabilized immunofluorescent analysis. Hypoxic conditions up-regulated GLUT1 mRNA expression but not HER2 expression. The HER2 membrane protein fraction increased in response to hypoxic conditions. Nonpermeabilized immunofluorescence analysis showed that membrane-bound HER2 was also promoted under hypoxic conditions. HER2 is not regulated at the mRNA level; however, the level of membrane-bound HER2 increases in response to hypoxia.

Comparative evaluation of PCR and loop-mediated isothermal amplification (LAMP) assays for detecting Pasteurella multocida in poultry

ABSTRACT Aims To develop a colourimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Pasteurella multocida in clinical poultry samples and compare the performance of this assay with PCR. A secondary aim was to evaluate a simple DNA extraction method that could enable LAMP-based testing in the field without the need for specialised laboratory equipment. Methods Primer sets for both LAMP and PCR were designed to amplify the KMT1 gene of P. multocida. DNA was extracted from 12 P. multocida isolates using a commercial extraction kit, and subjected to analysis using both LAMP and PCR. The analytical specificity of the LAMP assay was evaluated by testing it against a panel of 12 unrelated bacterial species, and the analytical sensitivity (limit of detection) was determined through testing of serial dilutions of the target DNA and compared to that of PCR. Subsequently, cloacal swabs (n = 40) from a commercial turkey flock were subjected to analysis using both LAMP and PCR assays, using a rapid DNA extraction method and a commercial extraction kit. Clinical sensitivity and specificity of the LAMP assay were calculated in comparison to PCR. Results A single DNA fragment of the expected size (∼ 200 base pairs), was amplified by PCR from 12 P. multocida isolates, which were also all positive by the LAMP assay. The identity of all PCR amplicons was confirmed by sequencing. Both PCR and LAMP showed similar analytical sensitivity, with a LOD of 20 pg of target DNA. As neither PCR nor LAMP assays produced positive results with 12 non-related bacterial species, the analytical specificity was assessed as 100%. However, LAMP demonstrated lower clinical specificity (94.74%) compared to PCR (100%) when 40 clinical samples were tested. None of the DNA samples extracted using the simplified DNA extraction method were amplified by either LAMP or PCR. Conclusion The LAMP assay developed in this study exhibits comparable performance to PCR in detecting P. multocida. Clinical relevance The use of a rapid and portable DNA extraction method, in conjunction with LAMP assays, could create opportunities for point-of-care testing for fowl cholera in field settings.

Optimization of RNA extraction protocol from Eucalyptus and Populus species for gene expression studies

ABSTRACT Extraction of high-quality RNA is complicated due to the presence of polysaccharides and polyphenols in woody plant species like Eucalyptus and Populus. To conduct gene expression studies, RNA must be of high quality and intact integrity. Although, RNA extraction kits make the process quick and easy but they might not consistently provide high-quality RNA from Eucalyptus and Populus. Therefore, the primary objective of this work is to optimize a repeatable and systematic CTAB method for RNA extraction of high quality from Populus and Eucalyptus leaves. According to the study results, the optimized CTAB approach is more suitable for these plant species than RNA extraction kits. The agarose gel electrophoresis demonstrated that the RNA samples retained their integrity due to the presence of distinct 28S and 18S rRNA bands. Additionally, the RNA integrity number was found to be between 7.2 and 8.0. As indicated by the A260/A280 ratios, the optimized method’s RNA extraction yielded minimal levels of polysaccharide and polyphenol contamination. For Eucalyptus, the absorbance values ranged from 1.98 to 1.99 while for Populus it was 1.97 to 1.98. Similarly, the A260/230 ratios, which represent the second indicator of nucleic acid purity, were found to be 2.01 to 2.08 and 2.02 to 2.09 for Eucalyptus and Populus, respectively. The high-quality RNA was shown to be appropriate for use in further processes, such as cDNA synthesis, gene amplification, and real-time quantitative PCR (RT-qPCR).