DNA Extraction Research Articles

Non Invasive Preimplantation Testing for Aneuploidies in Assisted Reproduction: A SWOT Analysis.

The implementation of non-invasive PGT-A offers a new strategy to genetically assess the preimplantation embryo and to enhance IVF results. The extraction of DNA from the embryo culture medium has been sufficiently demonstrated, and the ability to obtain chromosomal information as a result is particularly interesting. As morphological criteria have proven to have a weak correlation with embryo ploidy status, this technique emerges as a promising alternative for embryo selection. It also appears reasonable that avoiding biopsy may enhance further embryo development. However, there are growing concerns regarding several aspects of this technique, such as the origin of this cell free DNA, the degree of representativeness of the whole embryo, the need for extended culture or the absence of standardized protocols. Despite the published data on good prognosis couples are promising, niPGT-A is yet to be considered a substitute for trophectoderm biopsy. The current SWOT analysis aims to summarize both resolved and unresolved issues, as well as limiting aspects of niPGT-A.

Applying nanopore sequencing technology in Paracoccidioides sp.: a high-quality DNA isolation method for next-generation genomic studies.

Paracoccidioidomycosis is a severe systemic endemic mycosis caused by Paracoccidioides spp. which mainly affects individuals in Latin America. Progress in Paracoccidioides genomics has been slow, as evidenced by the incomplete reference databases available. Next-generation sequencing is a valuable tool for epidemiological surveillance and genomic characterization. With the ability to sequence long reads without the need for prior amplification, Oxford Nanopore Technology (ONT) offers several advantages, but high-quality and high-quantity DNA samples are required to achieve satisfactory results. Due to the low concentration of Paracoccidioides DNA in clinical samples and inefficient culture isolation methods, DNA extraction can be a significant barrier to genomic studies of this genus. This study proposes a method to obtain a high-coverage de novo genome assembly for Paracoccidioides using an improved DNA extraction method suitable for sequencing with ONT. The assembly obtained was comparable in size to those constructed from available data from Illumina technology. To our knowledge, this is the first genome assembly of Paracoccidioides sp. of such a large size constructed using ONT.

Methodological approaches in 16S sequencing of female reproductive tract in fertility patients: a review.

The female genital tract microbiome has become a particular area of interest in improving assisted reproductive technology (ART) outcomes with the emergence of next-generation sequencing (NGS) technology. However, NGS assessment of microbiomes currently lacks uniformity and poses significant challenges for accurate and precise bacterial population representation. As multiple NGS platforms and assays have been developed in recent years for microbiome investigation-including the advent of long-read sequencing technologies-this work aimed to identify current trends and practices undertaken in female genital tract microbiome investigations. Areas like sample collection and transport, DNA extraction, 16S amplification vs. metagenomics, NGS library preparation, and bioinformatic analysis demonstrated a detrimental lack of uniformity. The lack of uniformity present is a significant limitation characterised by gap discrepancies in generation and interpretation of results. Minimal consistency was observed in primer design, DNA extraction techniques, sample transport, and bioinformatic analyses. With third-generation sequencing technology highlighted as a promising tool in microbiota-based research via full-length 16S rRNA sequencing, there is a desperate need for future studies to investigate and optimise methodological approaches of the genital tract microbiome to ensure better uniformity of methods and results interpretation to improve clinical impact.

A morphological and high throughput sequencing workflow to identify Australian ants (Hymenoptera, Formicidae): a new tool for biosecurity and biodiversity

Ants are often the most ubiquitous and ecologically influential components of terrestrial systems, exhibiting an exceptionally high level of endemic diversity. Simultaneously, exotic ants can be amongst the most environmentally and economically devastating biological invaders. Distinguishing between exotic and related natives is essential for early detection and to reduce the chance of establishment, but at the species-level there remains a great deal of uncertainty among several of the most diverse, widespread and ecologically dominant genera. This taxonomic impediment negatively impacts the use of molecular techniques relying on reference databases, due to the poor state of ant DNA sequences in publicly available repositories, with many species not represented at all and others incorrectly identified. As a result, biosecurity screening for targeted exotic ants and assigning ant species-level identifications for biodiversity remain two separate areas of focus in Australia. Here we propose a workflow for the identification of ants that enables simultaneous processing of large numbers of “bulk” ant samples each containing many individuals, increasing resolution for taxonomic assignment, and generating a curated database linked to voucher specimens. We use a non-destructive DNA extraction method and compare two high throughput sequencing (HTS) metabarcoding platforms – MiSeq (Illumina) and MinION (Oxford Nanopore Technologies) – processing up to 180 bulk mixed ant samples per run. This approach allowed for the acquisition of curated DNA sequences from voucher specimens morphologically examined by expert taxonomists. This work highlights the advantages and current limitations of DNA-based identifications, the needs for standardisation, as well as the importance of a taxonomy-based curation of DNA database for both biodiversity and biosecurity.

Association of HPA Antigens with Immune Thrombocytopenia: A Case-Control Study by PCR-SSP Method

Background: Human platelet antigens (HPAs) play a clinically significant role in alloimmunization and the development of immune-mediated disorders such as immune thrombocytopenia (ITP), fetal and neonatal alloimmune thrombocytopenia (FNAIT), and post-transfusion purpura (PTP). Understanding the genetic profiles of HPAs is critical for preventing and treating these conditions. Given the limitations of serological methods in determining HPA genotypes, this study aims to investigate the association between the genotypes of HPA1, HPA2, HPA3, HPA4, and HPA15 antigens and autoimmune thrombocytopenia in Lorestan Province, utilizing the PCR-SSP method. Materials and Methods: This case-control study involved 80 individuals diagnosed with ITP and 120 healthy controls. DNA samples were extracted using a commercial DNA extraction kit, with concentrations quantified via a Nanodrop spectrophotometer. Genotyping was performed using the PCR-SSP method with specific primers for each HPA gene. The genotype data were verified using previously established sample sets. The frequencies of each HPA genotype were recorded, and a comparative analysis was conducted between the patient and control groups to evaluate the study hypothesis. Results: The results revealed that individuals carrying the HPA2b allele had a 5.31-fold increased risk of developing ITP, a statistically significant finding (P < 0.05, OR = 5.31). Similarly, the presence of the HPA15b allele was associated with a 6.54-fold increased risk (P < 0.05, OR = 6.54). Conclusion: These findings, in conjunction with previous studies, suggest the need for larger-scale investigations across different populations. Such research could aid in the early diagnosis and prediction of thrombocytopenia severity, inform treatment strategies, and facilitate the removal of pathogenic antibodies from circulation.

Association of IL-6 G-174C (rs1800795) variant with the susceptibility to hepatocellular carcinoma in patients with chronic hepatitis

AimAn ineffective immune response resulting from dysregulation of cytokine production might encourage viral persistence and cause chronic viral hepatitis to worsen. This study examined the relationship between alterations in interleukin-6 (IL-6) levels and the IL-6 − 174 G > C (rs1800795) polymorphism, as well as how this polymorphism affects the development and progression of chronic hepatitis brought on by hepatitis B (HBV) and hepatitis C (HCV) into hepatocellular carcinoma (HCC).Patients and methodsWhole blood samples from 126 Egyptian patients with HCC (111 with HCV and 15 with HBV), as well as 126 age- and sex-matched healthy individuals, were used to extract DNA. Using PCR-based allele-specific amplification (ASA), the existence of the IL-6 G-174C polymorphism was investigated. Additionally, each participant's serum IL-6 levels were determined using an enzyme-linked immunosorbent assay (ELISA).ResultsThe primary observations revealed that HCC patients had greater serum levels of IL-6 compared to the control groups (p < 0.001). Patients with the variant (CG and GG) genotype in the HCC group were found to have more disease severity indicated by higher levels of alpha-fetoprotein (AFP) and a higher ascites grade, as well as increased inflammatory activity as defined by higher levels of IL-6 and C-reactive protein (CRP) (p < 0.001 for both) in comparison to patients with the wild-type (CC) genotype (p < 0.001 and p = 0.002, respectively).ConclusionThe rs1800795 SNP in the IL-6 gene was associated with increased inflammatory activity and high levels of IL-6, indicating that this SNP may play a role in the development of HCC in Egyptian patients with chronic viral hepatitis.

Metagenome-based microbial community analysis of urine-derived fertilizer.

Phosphorus is essential for food production and its supply is limited. Urine is an excellent source of phosphorus and one way to produce fertilizer is through conversion of urine to struvite (MgNH3PO4.6H2O). The present study aimed to understand the bacterial portion of the microbial community composition and dynamics of plasmid-mediated antimicrobial resistant genes during the optimized process of struvite production from composite human urine. Samples for DNA extraction was collected from fresh urine, stored urine and struvite during the process of struvite production. Shotgun metagenomic analysis was employed to understand the bacterial community. The most dominant phyla in the fresh and stored urine samples were Pseudomonadata, which comprised of 60% and 43% respectively, followed by Bacillota, comprised of 25% and 39% respectively. The struvite sample was dominated by the phylum Bacilliota (61%), Pseudomonadota (18%) and bacteroidota (12%). Members of the above phyla persisted in dominating each sample accordingly. Member of the family Morganellaceae was dominant in the fresh sample while the stored urine and struvite samples were dominated by the family Clostridiaceae. A decrease of members of the class Gammaproteobacteria was observed from the fresh to the struvite sample though not statistically significant. The genus Pseudomonas remained to be the most dominant member of Gammaproteobacteria in the fresh and stored urine sample with OTU count of 12,116 and 6,155 with a marked decrease by half in the stored sample. On the other hand, members of the genera Clostridium, Enterococcus, Bacteroides in the stored samples and Clostridium, Alkaliphilus and Pseudomonas in the struvite samples were dominant. 96% of the identified genera were shared in all the samples and the antimicrobial resistance genes (ARGs) identified in the fresh urine were shared by the struvite but not by the stored urine (e.g. sul, cat, aph and aac members). The presence of high abundance of ARGs in struvite needs attention in the persistence and transmissibility of the ARGs before application for agriculture.

Detection of exposure to sprout-inhibiting radiation doses in onions via 5,6-dihydrothymidine quantification using ultrasonication-mediated DNA extraction

Detection of exposure to sprout-inhibiting radiation doses in onions via 5,6-dihydrothymidine quantification using ultrasonication-mediated DNA extraction

Assessing the burden of submicroscopic Plasmodium infections in a pre-elimination malaria setting in sub-Saharan Africa, Guinea-Bissau.

Submicroscopic Plasmodium infections can be a source of persistent malaria transmission. The aim of this study was to assess their frequency, distribution, morbidity and associated factors in a pre-elimination malaria setting in sub-Saharan Africa, Guinea-Bissau, where the Plasmodium falciparum is the predominant Plasmodium species. Dried fingerprick whole blood samples from 601 participants in the 2017 national, household-based, cross-sectional survey to estimate malaria prevalence were subjected to DNA extraction. The DNA was used in nested end-point PCR assays targeting genus- and species-specific regions of the Plasmodium 18S rRNA genes. Statistical analysis of socio-demographic, clinical and molecular data was carried out using the Statistical Package for the Social Sciences, version 29. Factors associated with submicroscopic P. falciparum infections and their magnitude were sought using Chi-square test and multiple logistic regression models, respectively. Statistically significant level was considered at P-value < 0.05. Nested PCR assays detected submicroscopic P. falciparum infections in 20.3% (95% CI = 16.8-23.8) of individuals microscopically negative for Plasmodium species in the general population and in 21.4% (95% CI = 9.9-36.5) of microscopically negative pregnant women. Submicroscopic Plasmodium malariae infections were also detected as co-infections in 3.0% individuals who were microscopically positive only for P. falciparum. Infections with other Plasmodium species were not detected. Submicroscopic P. falciparum infections were not associated with age, sex, or the presence of fever. A logistic regression model adjusted for ethnicity and health region showed that individuals from the Balanta and Bijagos ethnic groups, most of whom live in the low malaria-transmission areas of Quinara and Bissau, and the Bijagos archipelago, respectively, were less likely to have submicroscopic P. falciparum infections than individuals from the large Fula ethnic group, most of whom live in the high malaria-transmission area of Gabu. Submicroscopic P. falciparum infections were not associated with anaemia in children under 5years of age. The results obtained highlight the contribution of asymptomatic and submicroscopic P. falciparum infections to malaria transmission in high malaria-transmission areas and the need for molecular-based tools to detect submicroscopic Plasmodium species.

Elucidating genetic variability between randomly bred domestic cats and Persian domestic cats from different geographical locations using microsatellite markers.

The domestic cat (Felis catus) is a newly evolved species in the family Felidae that has developed some great features among mammals. It is critical to conserve these species and prevent inbreeding from reducing their genetic diversity by understanding their genetic relationships and applying the information to breeding management. The diverse population was an excellent choice for studying genetic diversity and inbreeding phenomena. To conduct this research, 128 individuals from 8 populations, including Azerbaijan, Persian, Ahar, Uermia, Tehran, Karaj, Turkish and Shop cat (both genders), were randomly selected from different geographical regions. We selected eight STR markers with different chromosomal locations based on polymorphism and observed allele numbers in the next step. DNA extraction was performed using tail hair root, PCR and electrophoresis, and gel staining was performed according to routine laboratory protocol. For statistical analysis, CONVERT versions POPGENE, ARLEQUIN GenAlEx and R script analysis. Remarkably, our results showed that 23 alleles were identified in 128 samples. The highest number of alleles belonged to the FCa096 locus (eight alleles) in the Persian population, followed by FCa045 (seven alleles) in the Persian and Ahar populations. Another new finding is that the lowest number of alleles belonged to the 35 and FCa77 locus (two alleles). In addition, pairwise differentiation between and within populations was examined using the genetic distance index. Overall, the results showed that the degree of differentiation within the population is high in the Turkish population compared to other population groups and lower in the Azerbaijan population. In addition, principal component discriminant analysis-based analysis based on the ADAGENET package shows the distribution of samples by geographical location. The results show that genetic mixing between populations is high. On this basis, we conclude that randomly bred domestic cats have a higher level of diversity than Persian domestic cats. This is an interesting topic for future work.

Comparison of Urine and Cervical Samples for Genotyping Via HPV DNA Test

&amp;lt;i&amp;gt;Background&amp;lt;/i&amp;gt;: Human papillomavirus (HPV) is a key factor in cervical cancer development. This study aimed to compare the efficacy of urine and cervical samples for HPV genotyping, evaluating their diagnostic performance in detecting high-risk HPV genotypes. &amp;lt;i&amp;gt;Methods&amp;lt;/i&amp;gt;: This comparative cross-sectional study conducted over one year at the Department of Gynaecological Oncology, BSMMU, Dhaka, the study enrolled 74 women aged 30-60 years with positive visual inspection with acetic acid (VIA) results or abnormal Pap test findings. Urine samples (20 ml) and cervical samples were collected from each participant. The samples were analyzed using multiplex real-time PCR to amplify high-risk HPV types (16, 18, and others). DNA was extracted using the Qiagen viral DNA extraction kit. Sensitivity and specificity of HPV detection in urine samples were compared to cervical sampling, the gold standard. Data were analyzed with SPSS 22.0, and agreement was assessed using the Kappa index. &amp;lt;i&amp;gt;Result&amp;lt;/i&amp;gt;: Cervical samples detected HPV in 17.56% of participants, while urine samples identified HPV in 5.40%. The agreement between urine and cervical samples was moderate, with a kappa value of 0.743. Among 74 cases, 5 cases were detected as HPV 16 and HR (co-injection) in both cervical and urine sample, 2 cases as HPV 16 in both cervical and urine samples, 2 cases as only HR type in both cervical and urine sample. The kendall’s correlation of agreement was 0.361 and a significance of 0.002. &amp;lt;i&amp;gt;Conclusion&amp;lt;/i&amp;gt;: Cervical samples are more reliable for HPV detection compared to urine samples, though urine testing shows high sensitivity.

Streamlined boiling lysis DNA extraction for Gram-positive aquaculture pathogen Streptococcus agalactiae.

Accurate genetic analysis is essential for the detection of pathogens as it necessitates suitable DNA extraction methods tailored to specific microorganisms such as Gram-positive bacteria. This study examined several commercial and simplified DNA extraction methods for their suitability in isothermal downstream applications. Extracted DNA was assessed using spectrophotometry, electrophoresis, polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) while its stability was inspected after five months of storage. The findings revealed variations in DNA yield, purity and integrity among the extraction methods. While extraction kits demonstrated high yield and purity, the in-house extraction techniques showed incoherent correlation between yield and purity, yet showed promise for a streamlined extraction process. The DNA acquired from all methods yielded positive amplification in PCR and LAMP. DNA extracted by kits exhibits prolonged stability than those obtained via boiling lysis. Both methods offer distinct advantages, with commercial kits providing longer stability and high-quality DNA while boiling lysis stands out for its simplicity, with shorter handling and processing periods. This study emphasizes selecting ideal extraction methods for Streptococcus agalactiae, in the prospect of aquaculture settings. In particular, successful LAMP reaction suggests that boiled extracts are feasible enough for detection, and suited for point-of-care (POC) testing where prompt detection of aquatic pathogens is often critical. Ultimately, the choice of method should be contemplated on a case-by-case basis such as the study goals, intended settings, and type of samples.

Incidence of Occult Hepatitis B Infection (OBI) and hepatitis B genotype characterization among blood donors in Cameroon.

Occult hepatitis B infection (OBI) is characterized by the presence of hepatitis B virus (HBV) DNA at low levels in serum (<200 IU/mL) with a negative hepatitis B surface antigen (HBsAg) test. OBI remains a major challenge to blood safety, particularly in HBV-endemic regions like Cameroon, where HBV detection relies solely on HBsAg testing. This cross-sectional study aimed to investigate the actual incidence and genotype characteristics of OBI in Cameroonian blood donors. Between March and June 2023, samples were collected from 288 HBsAg-negative blood donors aged 18 to 55 years and analysed for antibodies against the HBV core (anti-HBc) and surface antigens (anti-HBs). Following DNA extraction from the serum samples, qualitative nested PCR and quantitative real-time PCR were used to detect HBV viral DNA and viral load respectively. For positive samples, sequencing of a fragment of the S gene was performed to identify the circulating HBV genotypes. The findings revealed that 58% (n = 167/288) of blood donors tested positive for anti-HBc, 29% (n = 83/288) tested positive for anti-HBs, and 26% (n = 75/288) being positive for both anti-HBc and anti-HBs. Occult hepatitis was confirmed in 4.5% of the blood donors, all of whom belonged to either HBV genotypes A or E, which are predominant in Cameroon. The amino acid substitution sA184V associated with HBsAg detection failure in genotype E was observed in 70% of OBI sequences, and the HBsAg immune escape variants (sT131N and sS143L) implicated in OBI were also observed. The mutation rtN139K in the reverse transcriptase (RT) domain of the overlapping HBV polymerase (P) gene was present in 17% of OBI-positive sequences of genotype E, likely contributing to masking HBsAg secretion. The results suggest a considerable risk of transfusion-transmitted HBV in this region. Therefore, to ensure blood safety, nucleic acid testing (NAT) is recommended, as relying solely on HBsAg assays is insufficient to eliminate this risk.

Genetic association and computational analysis of MTHFR gene polymorphisms rs1801131 and rs1801133 with breast cancer in the Bangladeshi population

Methylenetetrahydrofolate reductase (MTHFR) plays a crucial role in regulating one-carbon metabolism. Polymorphisms within the MTHFR gene have been found to increase the risk of breast cancer in different populations. In this study, we evaluated the association of polymorphisms of the MTHFR gene (rs1801133 and rs1801131) with the risk of breast cancer in the Bangladeshi population. This case‒control study included 202 breast cancer patients and 104 healthy controls. After the organic extraction of DNA, genotyping was performed via the PCR-RFLP method. Sanger sequencing was performed to validate the RFLP data. Statistical analyses were performed to evaluate the associations of the polymorphisms. Different computational tools were used to predict the structural and functional consequences of the SNPs. Our study revealed that the MTHFR gene polymorphism rs1801131 is associated with an increased risk of developing breast cancer (p < 0.001, OR = 3.85, 95% CI = 2.06–7.25 for the AC genotype and p < 0.001, OR = 7.82, 95% CI = 2.69–22.05 for the CC genotype). An association was also observed in the dominant model (AC + CC) (p < 0.001, OR = 4.19, 95% CI = 2.28–7.78). For rs1801131, premenopausal status was significantly associated with breast cancer risk (p < 0.001). For rs1801133, no significant association was found with breast cancer risk (p > 0.05, OR = 1.57, 95% CI = 0.90–2.74 for the CT genotype; p > 0.05, OR = 1.35, 95% CI = 0.36–4.92 for the TT genotype). Computational analyses predicted rs1801131 to be tolerated and rs1801133 to be deleterious. Structural analyses demonstrated no significant changes in protein structure but revealed alterations in neighboring interactions according to both bond distances and angles. In conclusion, rs1801131 but not rs1801133 is significantly associated with breast cancer risk in the Bangladeshi population. Moreover, in silico analyses demonstrated changes in the interaction pattern of polymorphic residues with adjacent amino acids.

Nanopore ultra-long sequencing and adaptive sampling spur plant complete telomere-to-telomere genome assembly

The pursuit of complete telomere-to-telomere (T2T) genome assembly in plants, challenged by genomic complexity, has been advanced by Oxford Nanopore Technologies (ONT), which offers ultra-long, real-time sequencing. Despite its promise, sequencing length and gap filling remain significant challenges. This study optimized DNA extraction and library preparation, achieving DNA lengths exceeding 485 kb; average N50 read lengths of 80.57 kb, reaching up to 440 kb; and maximum reads of 5.83 Mb. Importantly, we demonstrated that combining ultra-long sequencing and adaptive sampling can effectively fill gaps during assembly, evidenced by successfully filling the remaining gaps of a near-complete Arabidopsis genome assembly and resolving the sequence of an unknown telomeric region in watermelon genome. Collectively, our strategies improve the feasibility of complete T2T genomic assemblies across various plant species, enhancing genome-based research in diverse fields.

Bottleneck Effect Analysis in Indigenous Poonchi Chicken Located Near International Border of India and Pakistan

Background: Poonch is the remotest district of Jammu, India located near to line of control with Pakistan. Poonchi chicken is the first native poultry breed of Jammu region which is being characterized and registered with National Bureau of Animal Genetic Resources. Present study is the first study on genetic bottleneck assessment in Poonchi chicken population using recommended microsatellite markers. This study in Poonchi chicken would be of immense use in tracing evolutionary origin and formulating effective conservation strategies for genetic diversity within breeds. Methods: Blood samples were collected randomly from Poonch and Rajouri districts of Jammu for DNA extraction and analysis. Microsatellite markers selected for the analysis were ADL0268, MCW0206, MCW0081, ADL0278, MCW0069, MCW0248, MCW0111, MCW0222, MCW0016 and LEI0094. To evaluate the Poonchi chicken for mutation drift equilibrium, Bottleneck software was used. Infinite allele model (IAM), two phase model of mutation (TPM) and stepwise mutation model (SMM) tests were done. Mode shift test was also used as another method of qualitative test for allele frequency. Result: Total number of alleles observed were 72, with the total number alleles ranged from 6 to 9. The results of all three models (IAM, TPM, SMM) in Sign test depicted the heterozygosity was more in the expected number of loci in Poonchi chicken as 5.97 (P less than 0.05), 5.93 (P less than 0.05), 5.90 (P less than 0.05) respectively. Whereas for Wilcoxon rank test the IAM, TPM and SMM revealed significant values of (P less than 0.05) deviation indicating that all the loci deviate from mutation-drift equilibrium. Mode shift test graph obtained depicted a slight shift from normal L-shaped curve indicating recent bottleneck effect in Poonchi chicken. This might have resulted in the loss of number of effective alleles which suggests a need of planning and implementing an effective breeding policy in order to preserve and promote this native breed.

Chitinase and proteinase K treatments enhance the DNA yield of microsporidium Ecytonucleospora hepatopenaei spores

Microsporidium Ecytonucleospora hepatopenaei (EHP) spores were purified from the hepatopancreas of Penaeus vannamei infected with EHP by percoll density gradient centrifugation and differential centrifugation. The EHP spores contain a thick chitin wall and might not rupture using the routine DNA extraction protocol. In this study, three enzymes were used, including chitinase, proteinase K, and DNase I. Chitinase or proteinase K digestions caused weakened fluorescence of chitin showing by a blurred edge of EHP spores stained with calcofluor white under a fluorescence microscope. Different combinations of these enzymes followed by DNA extraction with phenol–chloroform from EHP spores showed significant increases in the copy number of the EHP SSU gene per spore. The combination of the chitinase and proteinase K treatments resulted 4.46 ± 1.07 copies/spore detected, which is 31.6 ± 20.7 folds of no treatment groups, accounting to (55.7 ± 13.4)% of the total copies of the gene in the spore. The additional treatment with chitinase to the conventional extraction protocol with a proteinase K digestion step for feces and hepatopancreas samples of P. vannamei resulted in a significant difference in EHP copies in the DNA of (83.8 ± 64.1)% and (55.3 ± 88.0)% increases. The study proved that chitinase and proteinase K treatment enhance the DNA extraction from microsporidian spores resulting in high yield.

Portable DNA extraction and loop-mediated isothermal amplification (LAMP) for the on-site detection of white spot syndrome virus (WSSV)

White spot disease (WSD) has been the most severe and lethal threat to the global shrimp industry for the past three decades. WSD caused by white spot syndrome virus (WSSV) can result in severe economic losses in shrimp culture. The disease can cause rapid and mass mortality of 90–100 % within 3–10 days. Currently, there are no effective treatment measures available for the control of WSD. Strict biosecurity, possible elimination, and early diagnosis can prevent the spread of the pathogen. Several advanced diagnostic methods have been standardised for the early and rapid detection of WSD. However, the field deployable diagnostic methods are limited. LAMP is a cost-effective, alternative nucleic acid amplification method that operates under isothermal conditions. In this study, a novel field-portable LAMP was developed for the diagnosis of WSSV. The WSSV amplification was optimized at an isothermal condition of 65 °C and a reaction time of 55 min. In this closed-tube amplification, the end results were easily visualized with the naked eye by observing the simple color change. This assay is highly sensitive and can detect up to 6 copies of WSSV plasmid DNA and 0.1 fg of WSSV genomic DNA. Additionally, this diagnostic method is highly specific and did not cross-react with other shrimp pathogens such as IHHNV, IMNV, EHP and Vibrio parahaemolyticus or host DNA of Penaeus vannamei, P. monodon, and P. indicus. Furthermore, a field-deployable DNA (FDD) extraction method has been standardised with a simple rotor and dry bath. This visual LAMP-coupled FDD extraction did not require any sophisticated equipment or technical expertise. Thus this robust LAMP assay can potentially be used for routine surveillance, point-of-care testing, early detection, and timely intervention of WSD.

NANOPORE-BASED INTRA-OPERATIVE CLASSIfiCATION OF CENTRAL NERVOUS SYSTEM TUMOURS IN THE NHS

Abstract AIMS The 2021 WHO classification of CNS tumours necessitates the use of molecular testing in order to reach a confident diagnosis of many tumour types. At present, this is achieved using array-based technology delivered by a centralised model. This approach has inherent delays that mean it is often several weeks before the treating clinical team is informed of diagnosis, delaying the start of adjuvant therapy and causing significant distress for patients. As such, there is an urgent need to rethink this pathway and improve time to diagnosis. Nanopore longread sequencing technology has the potential to enable a paradigm shift in diagnostic and therapeutic pathways, offering rapid, comprehensive molecular diagnosis that can be performed in the intra-operative setting. We aimed to test the hypothesis that intra-operative methylation-based classification can be performed using nanopore sequencing and thereby expediently inform surgical and oncological decision-making. METHOD Oxford Nanopore Technology (ONT) is a native-strand, long-read sequencing platform that can perform copy- number profiling along with parallel single-gene methylation, structural variant, mutational and methylation analyses. Capital and consumable costs are relatively low, and testing can be performed in the local setting on single samples. We have optimised DNA extraction and library preparation for rapid tissue processing. We have developed a robust bioinformatic pipeline which is able to produce intraoperative classification within minutes of sequencing and full molecular profiling after 24 hours. RESULTS We demonstrate the feasibility of using ONT to deliver intra-operative methylation-based classification of CNS tumours in ‘realtime’ within an NHS hospital setting and will discuss the challenges of implementing this testing and the opportunities to inform neuro-oncological practice. CONCLUSION By giving a summary of our novel protocol of rapid DNA extraction, library preparation and bioinformatic analysis, we will show that this approach can revolutionise the diagnosis of brain tumours and has the potential for rapid adoption by non-expert laboratories.

Development of an RPA-CRISPR/Cas12a Assay for Rapid and Sensitive Diagnosis of Plant Quarantine Fungus Setophoma terrestris.

Setophoma terrestris is an important phytopathogenic fungus listed by China as a harmful fungus subject to phytosanitary import control. This pathogen is a threat to a wide range of plants, particularly as the causal agent of onion pink root rot, one of the most severe diseases of onions. In order to provide rapid identification and early warning of S. terrestris and prevent its spread, we have developed a rapid, accurate, and visually intuitive diagnostic assay for this pathogen, by utilizing recombinase polymerase amplification (RPA), coupled with CRISPR/Cas12a cleavage and fluorescence-based detection systems or paper-based lateral flow strips. The developed RPA-CRISPR/Cas12a assay exhibited remarkable specificity for the detection of S. terrestris. Moreover, this protocol can detect the pathogen at a sensitivity level of 0.01 pg/μL, which significantly outperforms the 1 pg/μL sensitivity achieved by the existing qPCR-based detection method. The entire diagnostic procedure, including DNA extraction, the RPA reaction, the Cas12a cleavage, and the result interpretation, can be accomplished in 40 min. Furthermore, the successful application of the assay in infected plant samples highlighted its potential for rapid and accurate pathogen detection in agricultural settings. In summary, this RPA-CRISPR/Cas12a diagnostic method offers a potentially valuable technological solution for quarantine and disease management.