Polymerase Chain Reaction Protocol Research Articles

A quantitative polymerase chain reaction (qPCR) protocol predictive of treatment outcome in cutaneous leishmaniasis caused by Leishmania braziliensis

Abstract Leishmania braziliensis is the most prevalent agent causing cutaneous leishmaniasis (CL) in Brazil. While inflammation is a hallmark of CL, few parasites are found at the lesion site, leading to challenges regarding diagnosis. Using L. braziliensis kDNA and human 18S rRNA as targets, the present study developed a qPCR assay to determine parasite load in biopsies from CL patients residing in an endemic area in northeastern Brazil. In addition, we investigated whether parasite load correlated with clinical outcome and observed that patients with higher parasite load were more likely to fail therapy. Moreover, CL patients in the early phase of infection presented higher levels of parasite transcripts than individuals in later phases. Thus, our results suggest that parasite load as determined by qPCR may constitute a valuable prognostic tool to aid in both the determination of disease severity and treatment outcome.

Association of C4 Null Alleles and Persistently Low C4 in Asian Indian Patients With Systemic Lupus Erythematosus.

The association between heterozygous C4 deficiency and systemic lupus erythematosus (SLE) is unclear. There is a lack of data in South Asian Indians on any possible association of C4A and C4B null alleles with lupus. We aimed to study the prevalence of C4A and C4B null alleles in a cohort of SLE patients with persistently low C4 levels compared to healthy controls (HC). Patients with SLE and HC were recruited for this prospective observational study. C4 (C4AQ and C4BQ) polymorphisms were tested using a touch-down polymerase chain reaction protocol. One hundred and three SLE patients and 103 HC were included in the study. Persistently low C4 levels were observed in 25 (23.6%) of SLE. The frequency of C4A and C4B null alleles was similarly distributed across SLE and HC (4% and 3.8%, respectively). Univariate analysis showed a low age, higher proportion of elevated dsDNA, and higher positive anti-SSA (Sjogren's syndrome-related antigen A) antibodies at presentation were associated in SLE patients with the null allele group on comparison without the null allele group. However, these associations did not persist in the multivariate analysis. In conclusion, C4 null allele frequency was similar in SLE and HC. No characteristic associations were observed in SLE with C4 null alleles was observed. Therefore, C4 null allele is an unlikely explanation for persistently low C4 in South Indian Asian patients with lupus.

Conservation Genetics and Breeding using Molecular Genetic Markers in Japanese Quail (Coturnix japonica).

The Japanese quail (Coturnix japonica) is a small migratory bird whose main habitats are located in East Asia, Russia, China, Japan, Korea, and India. The Japanese quail was first introduced into the Iraqi research sector in the early 1980s. This investigation aimed to identify the genetic divergence between the available genetic lines of the Japanese quail in Iraq as a first step to conducting further conservation and breeding, benefiting from studying the genetic diversity related to productivity, adaptation, and immune susceptibility. In this study, we harnessed the random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) protocol to investigate the genetic structure, diversity, and differentiation of seven distinct genetic lines of these birds with white, brown, wild grey, and yellow plumage guided by 20 molecular genetic markers. Our findings showed a relatively high polymorphism level of these 20 markers, which was adequate for describing genetic variation within and between the quail lines under study. Overall, the pairs of the White male and Brown male, Brown female and Grey male, and Grey female and Grey male lines were the most genetically distant. Conversely, the White male and Grey male lines were the most similar. The genetic differences established between these lines can enable us to suggest recommendations for further conservation genetics and breeding of this species. In particular, we propose that choosing animals (lines) with the greatest genetic distances, i.e., the least genetic similarities, can help preserve the highest genetic variability within the population. This proposed approach mirrors many contemporary conservation strategies, and information derived directly from this study can potentially be used to improve breeder selection regimes for additional conservation initiatives ex situ.

Screening for STIs: Results of a Health-Promotion Programme in a Portuguese University.

Sexually Transmitted Infections (STIs) are an important and growing public health concern. Implementation of screening programmes and awareness campaigns are crucial to mitigate this problem. A university in the central region of Portugal has devised a health-promotion programme, named Protection+, specifically directed towards the sexual health of the university community. The present study aimed to evaluate the results of the different actions undertaken as part of the health-promotion programme during the 2023-2024 academic year. Chlamydia, gonorrhoea, trichomoniasis and infection with Mycoplasma genitalium were assessed through real-time polymerase chain reaction protocols. Syphilis, infection with HIV, HBV and HCV were assessed through immunological assays. The adherence to the health-promotion programme after the awareness campaigns was also evaluated. STIs have been diagnosed in 13.7% of the 475 screened participants. Chlamydia was the STI most frequently diagnosed (8.4%), followed by infection with M. genitalium (2.3%), T. pallidum (2.0%) and N. gonorrhoeae (1.1%). HIV, HBV and HCV were diagnosed in a residual number of cases, and T. vaginalis was not detected in any of the screened participants. At the time of diagnosis, more than half of the infected patients were asymptomatic. After the implementation of awareness campaigns, an increase in the adherence to STI screening was observed, with the expected simultaneous increase in STI diagnoses. The high prevalence of STIs, particularly chlamydia, in the university population, along with the asymptomatic nature of these infections, demonstrated the importance of STI screenings and the implementation of campaigns that raise awareness on the prevention and consequences of untreated STIs.

Impact of the Seasons on the Evolution of Tomato-infecting Geminivirus and Identification of Plant Reservoirs Harboring Geminivirus in Burkina Faso

Geminiviruses are known as responsible for huge damage to vegetable crops in many tropical, subtropical, and temperate regions. In Burkina Faso, tomato was reported as the most infected vegetable crop with the involvement of several geminivirus species despite its socioeconomic importance. This suggests and underlines the pressing need for additional information on virus population evolution (diversity, prevalence, and host plant reservoirs). Thus, to address this problem, surveys and sample collection were carried out in three localities in Burkina Faso (Goué, Léguéma, and Toussiana). Tomato leaves and those of cultivated and non-cultivated plants around the tomato fields were collected. The total DNA extracted from these samples was subjected to diagnosis based on the polymerase chain reaction (PCR) protocol with or without amplicon sequencing. Results showed that depending on the season and locality, the prevalence of the disease varied from 0 to 53.33% and that of the virus species varied from 0 to 76.67%. The highest prevalence was observed in the dry season. In addition, PepYVMLV was the virus that was widely detected in all three localities in the rainy (18.89%) and dry (52.22%) seasons. The DNA-B molecule associated with this virus was associated with other begomoviruses (ToLCMLV). In addition, out of 200 samples collected in the vicinity of the tomato fields, only 63 were positive based on PCR diagnosis. Amplicon sequencing yielded 63 partial sequences of virus from ten plant species, including six non-cultivated species. Based on phylogenetic analysis, the 63 partial sequences belonged to three phylogenetic groups (the ToLCV group, the PepYVMLV group, and the CLCuGV group). This study allowed a better understanding of the evolution of tomato leaf curl/yellow disease in Burkina Faso and the diversity of plant species serving as reservoirs for involved viruses. This constitutes an important step in the search for adequate control methods.

Scalable solutions for global health: the SalivaDirect model.

The COVID-19 pandemic caught the world unprepared. Large-scale testing efforts were urgently needed, and diagnostic strategies had to rapidly evolve in response to unprecedented worldwide demand. However, the rollout of diagnostic testing and screening for SARS-CoV-2 was often impeded by logistical challenges, including regulatory delays, workforce shortages, laboratory bottlenecks, and supply chain disruptions. Recognizing these hurdles early on, we developed a testing approach that supported frequent, repeat testing, particularly as communities reopened. We hypothesized and experimentally demonstrated that saliva was a suitable specimen for the detection of SARS-CoV-2. This finding was advanced into the development of open-source, extraction-free reverse transcription polymerase chain reaction protocols using readily available, "off-the-shelf" reagents and equipment for the direct detection of SARS-CoV-2 in saliva ("SalivaDirect''). Working with the US Food and Drug Administration (FDA), we established a novel regulatory framework wherein the FDA granted Emergency Use Authorization to Yale University to offer the SalivaDirect test protocol to high-complexity diagnostic laboratories (as designated by the Clinical Laboratory Improvement Amendments) with quality oversight provided by Yale. This grew into a network of more than 200 labs across the United States that, as of May 2024, resulted in over 6.5 million SARS-CoV-2 tests. By making the protocol flexible and open-source, laboratories were able to rapidly and economically scale testing using a simple, self-collected saliva specimen. Additionally, fostering a national network of laboratories enabled real-time exchanges, problem solving, and the development of community best practices. Preparing for the next pandemic, or simply the next seasonal epidemic, the SalivaDirect model of deploying a readily available, expandable solution and accompanying network provides a proven method for the successful implementation of pathogen testing in the United States and globally.

Prevalence and genotype distribution of high-risk Human Papillomavirus infection among Vietnamese women in Ho Chi Minh City, Viet Nam: A population-based cross-sectional study

IntroductionPersistent infection with high-risk Human Papillomavirus (HR-HPV) genotypes are wellknown to increase the risk of cervical cancer significantly. This study aims to determine the prevalence and distribution of high-risk HR-HPV genotypes among women in Ho Chi Minh City (HCMC), Viet Nam. MethodsA population-based cross-sectional study was conducted between June 2020 and September 2020 in all 24 districts of HCMC, Viet Nam. Socio-demographic and behavioral data were collected from 2478 women aged 25 to 65 who had sexual intercourse using a self-administered questionnaire. Vaginal swab specimens from all participants were collected to identify HR-HPV genotypes using Polymerase Chain Reaction (PCR) protocols. ResultsThe prevalence of HR-HPV infection was 3.5 % (87 women, including 20 cases infected with multi-genotypes), 3.1 % in urban and 0.4 % in rural areas. The most detected HR-HPV genotypes among positive cases were 58 (25.3 %), 52 (21.8 %), 16 (21.8 %), 68 (10.3 %), 51 (10.34 %) and 18 (9.2 %). The prevalence of multi-type HR-HPV genotypes was 0.81 %, of which the most common co-infection genotypes were 52 and 58 (20.0 %), 16 and 56 (10.0 %), and 16 and 39 (10.0 %). The percentages of one-, two-, three-, and four-genotypes of HPV among positive cases were 77.0 %, 16.1 %, 4.6 % and 2.3 %, respectively. ConclusionsOur findings explore a low prevalence of HR-HPV infection in Vietnamese women, of which genotypes 52 and 58 are more popular than 16 and 18. Continuously updated data on the genotype distribution of HPV are helpful for vaccine development and planning preventive activities to prevent HPV-related cancers.

Identification and genotyping of Chikungunya virus using reverse transcription polymerase chain reaction and restriction fragment length polymorphism methods

INTRODUCTION. Chikungunya virus (CHIKV) genotyping involves sequencing fractions of genes encoding E1, E2, and nsP1 proteins or the entire genome of the virus. Available reagent kits or polymerase chain reaction protocols cannot be used for CHIKV genotyping, and nucleic acid sequencing requires expensive equipment and materials, which are not always available. Therefore, it seems promising to use a simpler and more cost-effective restriction fragment length polymorphism (RFLP) method, which has not previously been used for CHIKV genotyping.AIM. This study aimed to investigate the possibility of using reverse transcription polymerase chain reaction (RT-PCR) and RFLP for CHIKV identification and genotyping.MATERIALS AND METHODS. The experimental study used RNA from CHIKV strains of four genotypes, including the Asian, West African (WAf), and East/Central/South African (ECSA) genotypes, and the Indian Ocean Lineage of the ECSA genotype (ECSA-IOL). The study used RT-PCR followed by DNA restriction and restriction fragment length analysis.RESULTS. The nsP2 gene fragment of 648 bp in length (positions 3806 to 4453) contains recognition sites for the restriction endonucleases PspEI, PvuII, and DraI. The presence or absence of these sites generates a different combination specific to each of the four CHIKV genotypes. The authors designed primers for amplification of the selected gene region and performed RTPCR and RFLP.CONCLUSIONS. The RFLP method can be used for rapid CHIKV identification and genotyping. The method provides results within a few hours and does not require high-tech equipment.

B-026 Transforming PCR protocols in pandemic response with automated non-contact liquid dispensing

Abstract Background During the COVID-19 pandemic, effective Polymerase Chain Reaction (PCR) protocols were crucial for detecting SARS-CoV-2 in clinical samples, playing a key role in rapid infection control and easing the strain on healthcare services. However, with the critical need for fast and accurate pathogen surveillance, the manual execution of PCR workflows presented numerous challenges, including limited throughput, risk of human error and variability, and risk to human health. Several automated liquid handling systems from a range of vendors were considered to prepare extraction plates for this process. Ultimately, RFL decided on dragonfly® discovery, a non-contact positive displacement dispenser. Methods The analytical sensitivity for the UltraDx SARS-CoV-2 N1/N2/RP assay was evaluated by the RFL development team using RNA extracted from a panel of virus inputs from a Qnostics Analytical Q Panel, Product Number: SCV2AQP. Samples were extracted using a modified RNA extraction procedure for Thermo MagMax reagents and consumables with dragonfly discovery. dragonfly discovery was utilized to distribute a combination of inactivation buffer and beads into a 96-well kingfisher flex plate. This process was accomplished using a peristaltic pump and a 6-way Auto Feed Reservoir (AFR) to ensure a constant buffer flow. Simultaneously, the AFR ensured that the beads were kept in constant suspension, resulting in precise dispensing across all wells. The EPCR assay, UltraDx SARS-CoV-2 N1/N2/RP assay (LGC, Biosearch Technologies), utilized two CDC-characterized amplicon primer sets alongside two additional probes using fluorescence-based (FAM dye) detection. The input RNA extracted from clinical samples was combined with RT-PCR reaction mixes, before the reaction arrays were amplified. The reactions were then measured by a single scan on a multi-channel fluorescence reader. Results The UltraDx SARS-CoV-2 N1/N2/RP assay (when performed using the manufacturer method and in the absence of dragonfly discovery) has an LoD of 80 copies per reaction for the N1 assay, and 20 copies per reaction for the N2 assay, across 20 replicates for each copy number tested. Using the same/equivalent reagents and control materials, inclusion of dragonfly discovery showed an LoD of 70 copies/mL (0.9 copies/reaction) sensitivity when compared to the LoD suggested by the manufacture. Conclusions Integrating dragonfly discovery into the UltraDx SARS-CoV-2 N1/N2/RP assay led to an 80-fold reduction in the limit of detection for the N1 assay compared to the reagent manufacturer's recommendation. Some of this improvement can be attributed to dragonfly discovery's non-contact positive displacement dispensing, coupled with the AFR pump mechanism that ensures a continuous suspension of magnetic beads, enhancing accuracy and precision. dragonfly discovery's versatility supports a robust business-as-usual (BAU) process and is easily scalable for pandemic-level testing. Its user-friendly software interface requires zero computer programming knowledge, providing end-users with the flexibility to run multiple protocols on the same instrument, making it ideal for BAU assay processing. The instrument played a pivotal role in the UltraDx SARS-CoV-2 N1/N2/RP assay conducted by UKHSA during the pandemic, and SPT Labtech will continue to support UKHSA in their mission to protect communities from the impact of health threats.

Innovative Multiplex PCR Assay for Detection of tlh, trh, and tdh Genes in Vibrio parahaemolyticus with Reference to the U.S. FDA's Bacteriological Analytical Manual (BAM).

Vibrio parahaemolyticus is an important foodborne bacterium that causes severe gastroenteritis following the consumption of contaminated seafood. To identify V. parahaemolyticus and determine its pathogenicity, the U.S. Food and Drug Administration (FDA)'s Bacteriological Analytical Manual (BAM) recommends a multiplex polymerase chain reaction (PCR) protocol to simultaneously detect the species-specific thermolabile hemolysin (tlh) gene and the pathogenic thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes. However, this assay has shown two limitations: difficulty in separating the amplicons of the trh (486 bp) and tlh (450 bp) genes due to their highly similar sizes, and the weaker band exhibited by the tdh gene amplicon (270 bp). The present study aimed to improve the BAM's multiplex PCR assay by separating the three amplicons with similar intensity. A new primer set was applied for the tlh gene (369 bp) alongside the existing primers for the trh and tdh genes. The amplicons for the three genes were effectively separated by electrophoresis on a 2% tris-borate-EDTA (TBE) agarose gel within 45 min. Primer concentrations of 0.25 µM for three genes produced a significant amount of amplicons among various combinations of primer concentrations with 35 PCR cycles. This assay exhibited a detection limit of 10 pg of bacterial DNA, demonstrating its high sensitivity. It did not display amplicons from nine Vibrio species known to be human pathogens or from 18 well-documented foodborne pathogens. Therefore, the present multiplex PCR protocol could help overcome the limitations of existing assays and provide a more reliable method for detecting the three genes of V. parahaemolyticus.

Veterinary perspectives on the urbanization of leishmaniosis in Morocco

BackgroundLeishmaniosis caused by Leishmania infantum, L. major and L. tropica is endemic in Morocco. Growing evidence of both human and canine Leishmania infections in urban centres has been reported. Since many forms of the disease are zoonotic, veterinarians play an important role in leishmaniosis control by intervening at the parasite host level. This study aimed to bring together One Health principles to connect canine and feline leishmaniosis epidemiology within urban centres of Morocco (Rabat and Fez) and assess the level of awareness of Moroccan veterinarians about facing this threat.MethodsA molecular survey was conducted for Leishmania DNA detection in canine (n = 155) and feline (n = 32) whole-blood samples. Three conventional polymerase chain reaction (PCR) protocols were implemented. The first PCR aimed at identifying infected animals by targeting Leishmania spp. kinetoplast minicircle DNA (kDNA). The second and third PCR targeted the Leishmania internal transcribed spacer region (ITS-1) and the Leishmania small subunit ribosomal RNA (SSUrRNA) gene, respectively, aiming at identification of the infecting species after Sanger sequencing-positive amplicons. Total immunoglobulin G (IgG) against Leishmania spp. was evaluated in 125 dogs by enzyme-linked immunosorbent assays (ELISA) using an in-house protocol, including three Leishmania-specific antigens (SPLA, rKDDR and LicTXNPx). Sera from 25 cats were screened for total IgG to Leishmania spp. by an indirect immunofluorescence antibody test (IFAT). An online questionnaire was presented to Moroccan veterinarians addressing their knowledge and practices towards animal leishmaniosis.ResultsOverall, 19.4% of the dogs tested positive for Leishmania kDNA and ITS-1 and sequencing revealed infection with L. infantum among PCR-positive dogs. These animals presented a wide range of ELISA seropositivity results (16.7%, 34.9% and 51.6%) according to the tested antigens (rKDDR, SPLA and LicTXNPx, respectively). Use of kDNA-PCR revealed 12.5% cats positive to Leishmania spp. otherwise found to be seronegative by IFAT.ConclusionsA considerable prevalence of infection was identified in dogs from urban centres of Morocco. Additionally, this is the first report of feline infection with Leishmania spp. in this country and in urban settings. Moroccan veterinarians are aware that animal leishmaniosis is endemic in Morocco, representing a public health threat, and are knowledgeable about canine leishmaniosis diagnosis and treatment.Graphical

Culicoides biting midges feeding behaviour as a key for understanding avian Haemoproteus transmission in Lithuania.

Investigations of host feeding behaviour in haematophagous insects are critical to assess transmission routes of vector-borne diseases. Understanding if a certain species has ornithophilic or mammalophilic feeding behaviour can facilitate future studies focused on pathogens transmission to and from certain host species. Culicoides Latreille (Diptera: Ceratopogonidae) are vectors of several pathogens, which include arboviruses, bacteria and parasites to a considerable diversity of vertebrate hosts. However, most of the studies focused on feeding habits target Culicoides species that could transmit the Bluetongue virus, consequently with a mammalophilic feeding behaviour, leaving aside the Culicoides species that are involved in the transmission of vector-borne parasites to birds, such as Haemoproteus Kruse (Haemosporida: Haemoproteidae). This study aimed to investigate the source of blood meals of wild-caught Culicoides using molecular-based methods and to correlate our findings with the reports of Haemoproteus parasites in Culicoides species. Engorged Culicoides females were collected using ultraviolet (UV)-light traps at seven different localities in Lithuania in 2021-2023. Biting midges were dissected, and the abdomens of engorged females were used for molecular investigation of the blood meal source. A polymerase chain reaction (PCR) protocol that amplifies a fragment of the Cytochrome B gene of vertebrates was used. Obtained sequences were compared to available information in GenBank database to confirm the source of the blood meal. In total, 258 engorged Culicoides females, representing nine different species, were analysed. The source of blood meal was identified in 29.1% of them with most of the insects having fed on birds (74.7%) and the remaining on mammals (25.3%). Culicoides segnis Campbell, Pelham-Clinton was the only species to feed exclusively on birds; Culicoides from the Obsoletus group, C. pallidicornis Kieffer and C. punctatus Latreille were found to feed exclusively on mammals; C. festivipennis Kieffer, C. kibunensis Tokunaga and C. pictipennis Staeger had an opportunistic feeding behaviour, with the first two preferably feeding on birds. Due to their feeding behaviour and the presence of Haemoproteus parasites reported in the literature, C. festivipennis, C. kibunensis, C. pictipennis, and C. segnis play an important role in the transmission of those avian vector-borne parasite in the wild. These Culicoides species were already confirmed as being able to support the development of several Haemoproteus species and lineages. Future studies focused on understanding the epidemiology of avian pathogens transmitted by Culicoides should target these species.

Resilience of DNA chains to molecular fracture after PCR heating cycles and implications on PCR reliability.

Soon after its introduction in 1987, polymerase chain reaction (PCR) has become a technique widely employed in diagnostic medical devices and forensic science with the intention of amplifying genetic information. PCR prescribes that each of its cycles must include a heating subprocess at 95 °C or more (denominated DNA denaturation and provided for allowing a claimed orderly separation of the two complementary nucleotides strands), which can produce significant damage to DNA, caused by high-speed collisions with surrounding molecules. Since such disruption should be prevented in order to reliably employ PCR, a study of the mechanics of such loss of structural integrity is herein presented, preceded by a review of the fundamental literature which has elucidated the effects of molecular agitation on DNA fragmentation. The main conclusion of this retrospective survey is that the body of examined theoretical and experimental evidence consistently and redundantly confirms scarce resilience and significant loss of structural integrity when DNA is heated at temperatures above 90 °C, even for 1minute. Such conclusion contradicts the claimed paradigm of PCR fidelity and raises the concern that, at least for long sequences, if PCR can amplify some information, such amplified information may be unreliable for diagnostic or forensic applications, since it originates from sequences of nucleotides subjected to random fragmentation and reaggregation. Such a low-reliability scenario should be preventively considered in the various fields where DNA amplification methodologies are employed which provide for high-temperature heating under conditions equal to or similar to those prescribed by the PCR protocols reviewed in this study.

Variant-specific BCR::ABL1 quantification discrepancy in chronic myeloid leukemia.

Accurate quantification of the BCR::ABL1 fusion gene in whole blood is pivotal for the clinical management of chronic myeloid leukemia (CML) patients. The fusion protein encoded by BCR::ABL1 can vary in size, depending on the BCR and/or ABL1 gene breakpoint. The vast majority of CML patients have a p210 BCR::ABL1 fusion gene (M-BCR), which can be attributed to the presence of either e14a2 (b3a2) or e13a2 (b2a2) mRNA transcript junctions. Twenty-five CML samples were analyzed in two different ISO15189-accredited centers that both use an Europe Against Cancer-based quantitative polymerase chain reaction (qPCR) protocol. Reanalysis of the sample set with transcript-specific standard curves and digital droplet PCR (ddPCR) were performed. qPCR quantification revealed a significant (up to 1 log) difference specifically for the e13a2 transcript variant in contrast to e14a2 transcripts (Hodges-Lehman 4.29; p < 0.001). Reanalysis of the sample set with transcript-specific standard curves abolishes the initial transcript-specific difference (Hodges-Lehman 0.003; p = 0.8192). Comparison of transcript-specific qPCR results of both centers with ddPCR, an absolute quantification method, showed a statically significant association, especially in the lower range, indicating the clinical utility of transcript-specific or absolute quantification methods. Our data show that differences between transcript-specific quantification might exist between centers, leading to potential clinical impact on the follow-up of CML patients. The use of transcript-specific standard curves for qPCR quantification, or absolute quantification, can significantly reduce these differences. Specific attention should be applied to the interpretation of quantification differences of CML patients that switch between diagnostic centers.

Characterisation of Pasteurella multocida Strains from Different Lesions in Rabbits.

Pasteurellosis, a disease caused by Pasteurella multocida, is responsible for economic losses in rabbit industrial farms due to rhinitis, conjunctivitis, pneumonia, metritis, mastitis, orchitis, subcutaneous abscesses, otitis, encephalitis, and septicaemic forms. Although the occurrence of the disease is conditioned by predisposing factors that affect the rabbit immune response, the strains of P. multocida involved in the infection may have a different pathogenic ability. Therefore, typing of strains spread among the rabbits is important to assess their pathogenic potential. The aim of this study is to investigate the P. multocida strains responsible for disease in rabbit industrial farms. A total of 114 strains identified from different lesions were serotyped. Additionally, the presence of virulence-associated genes was investigated using three PCR (polymerase chain reaction) protocols. Capsular type A was prevalently found in strains from respiratory lesions while types D and F in those from metritis, mastitis, and other lesions. Different associations between some virulence-associated genes and both capsular type and lesions found in rabbits were detected. The presence of 8 virulence-associated genes seems to increase the occurrence of metritis. In addition, strains belonging to capsular type A and responsible for respiratory disorders especially, were found equipped with 10 and 11 virulence-associated genes. Nevertheless, the presence of strains responsible only for rhinitis was also detected among the latter, suggesting that the pathogenic ability of the bacteria depends on the expression rather than the presence of a gene.

ProtoCode: Leveraging large language models (LLMs) for automated generation of machine-readable PCR protocols from scientific publications

Protocol standardization and sharing are crucial for reproducibility in life sciences. In spite of numerous efforts for standardized protocol description, adherence to these standards in literature remains largely inconsistent. Curation of protocols are especially challenging due to the labor intensive process, requiring expert domain knowledge of each experimental procedure. Recent advancements in Large Language Models (LLMs) offer a promising solution to interpret and curate knowledge from complex scientific literature. In this work, we develop ProtoCode, a tool leveraging fine-tune LLMs to curate protocols into intermediate representation formats which can be interpretable by both human and machine interfaces. Our proof-of-concept, focused on polymerase chain reaction (PCR) protocols, retrieves information from PCR protocols at an accuracy ranging 69–100 % depending on the information content. In all tested protocols, we demonstrate that ProtoCode successfully converts literature-based protocols into correct operational files for multiple thermal cycler systems. In conclusion, ProtoCode can alleviate labor intensive curation and standardization of life science protocols to enhance research reproducibility by providing a reliable, automated means to process and standardize protocols. ProtoCode is freely available as a web server at https://curation.taxila.io/ProtoCode/.

A novel TaqMan qPCR system for the detection of Diaporthe citri, the causative agent of citrus melanose

Citrus melanose, caused by Diaporthe citri, is one of the most economically significant fungal diseases in China. Given that the application of fungicides is the main technique used for the control of citrus melanose, many studies have focused on enhancing the effectiveness and efficiency of the use of fungicides for the control of this economically significant disease. Here, we developed a TaqMan quantitative polymerase chain reaction (qPCR) protocol for monitoring citrus melanose via detection of suspected infected leaves and airborne conidia of D. citri. A primer pair and TaqMan probe targeting D. citri were designed to amplify a region of the fungal β-tubulin gene. The limits of detection and quantification of our TaqMan qPCR method were 159.57 and 683 copies/μL, respectively. The method detected the DNA equivalent to 47 conidia when fungal conidia were cultured in vitro. To evaluate the feasibility of our TaqMan qPCR method, symptomatic and asymptomatic leaf samples, airborne conidia samples were collected from a citrus orchard and tea garden, respectively. D. citri was detected in leaves with citrus melanose symptoms in an orchard; airborne fungal conidia of D. citri were also detected. These findings suggested that this novel TaqMan qPCR method could be used to detect D. citri both in vivo and in vitro. Our methodology could be applied as an alternative method for monitoring the changes in the incidence of D. citri in orchards.

Preliminary study on buffy coat smear and molecular detection of microfilaria in domestic chickens (Gallus gallus domesticus) raised in Southern Thailand

Background and Aim: Filarial nematode typically produces a larval stage (microfilariae) in the bloodstream of vertebrate hosts, where microfilariae reside in the blood or subcutaneous tissues. Filarial nematodes cause human diseases, such as river blindness and elephantiasis, which are widely studied. However, in avian species, they are overlooked because they are nonpathogenic. In Thailand, microfilaria can be found in wild birds and domestic chickens. Recently, an increase in the number of blood samples submitted to veterinary diagnostic laboratories may have increased the number of microfilariae. Therefore, knowledge about filarial species and reliable detection methods are important. Therefore, this study aimed to investigate the efficacy of buffy coat smear and polymerase chain reaction (PCR)-based methods for the detection of microfilaria in domestic chickens. In addition, parasites were identified using the sequence of the cytochrome c oxidase subunit 1 (COX1) gene. Materials and Methods: Giemsa-stained buffy coat smears from a previous study were reanalyzed. These available buffy coat smears were prepared from 55 domestic chickens raised as backyard free-ranging in Southern Thailand. Fifty-seven frozen genomic DNA extracted from chicken blood were used to detect the presence of the COX1 gene in Onchocercidae nematodes. The nested PCR protocol for amplification of the OnchoCOI_ R2-OnchoCOI_ R2 fragment of the COX1 gene was applied from a previous report. Sequences of COX1 were analyzed to identify Onchocercidae nematodes and if they were single or mixed infections. We constructed Bayesian phylogenetics to identify parasites and assessment of the relationship between filarial nematodes in avian species and other vertebrate hosts. Results: Buffy coat smears from 15 samples revealed microfilaria. Of these 15 samples, only eight were positive for COX1 nested-PCR amplification. The other two buffy coat-negative samples were also positive for nested-PCR. Sequencing of these 11 nested PCR-positive samples revealed that almost all of them were Onchocercidae nematodes. Bayesian phylogenetic analysis showed that chicken Onchocercidae spp. were grouped with other avian filarial nematodes. However, all chickens Onchocercidae spp. showed a double peak in the sequencing chromatogram, indicating mixed filarial infection (species or haplotypes). Therefore, no chicken Onchocercidae sequence was deposited on National Center for Biotechnology Information, GenBank. Conclusion: Giemsa-stained buffy coat smear was a reliable method for the detection of chicken microfilaria in routine veterinary diagnostic laboratories. Development of a new PCR-based method is necessary. This method may provide greater sensitivity and specificity of detection. In addition, the PCR method allowed us to access the genetic characteristics of nematodes, which helped us maximize our knowledge of nematodes. Further investigations, such as the pathogenicity of filarial nematodes in chickens and their potential vectors, are required. Keywords: Buffy coat smear, Chickens, Cytochrome c oxidase I, Microfilaria, Nested-polymerase chain reaction.

Characterization of genetic diversity in Curcuma longa L. cultivars using Start Codon Targeted (SCoT) markers

Curcuma longa L. (Family- Zingiberaceae), commonly called turmeric, is one of the most important commercial spices. Being a center of turmeric's diversity, India boasts of more than fifty cultivars with variable agronomic characters and curcumin content. However, the quest for calibrating their genetic variability remains debatable as popular molecular marker-based studies have yielded inconsistent results. A promising PCR-based approach, known as the Start Codon Targeted (SCoT)-PCR system has been established to be more efficient than random DNA marker systems because this technique can generate more information on genetic polymorphism patterns. Hence, the present study proposes to use Start Codon Targeted (SCoT) markers to assess inter-cultivar genetic diversity of turmeric. We report an orthogonal L16 (45) design-based approach to investigate the influence of five crucial factors (dNTPs, Taq DNA polymerase, primer, template DNA and annealing temperature of primer) on the efficiency of the polymerase chain reaction (PCR) for development of an optimized Start Codon Targeted (SCoT) amplification protocol using twenty-seven different cultivars of C. longa. The optimized PCR protocol yields best results with a 20 uL reaction mixture, containing 10×PCR Buffer (with 15 mM MgCl2), 2.5 mM dNTPs, 10 mM SCoT primer, Taq polymerase (2 U), template DNA (50 ng), which was verified by using twenty-five SCoT primers. This standardized rapid protocol of SCoT-PCR reported here was employed to examine genetic diversity among twenty-seven Indian cultivars of turmeric, revealing high range of polymorphism ranging from 81.25 % to 100 %. Unweighted pair group method of arithmetic averages (UPGMA) cluster analysis of the SCoT marker data segregated the twenty-seven cultivars into two main clusters. The first cluster comprised of nine cultivars (36 % of the cultivars) which was further divided into three sub-clusters and the second one consisted of eighteen cultivars (64 %) which further divided into four sub-clusters. From the results of this study, it can be inferred that the employment of SCoT markers can be an effective tool for analysing genetic variability, phylogenetic relationships and develop a rich genetic resource management system for C. longa cultivars.

Development of a real-time PCR protocol for the detection of chicken DNA in meat products

Food falsification is a pressing issue in today’s food industry, with fraudulent substitution of costly ingredients with cheaper alternatives occurring globally. Consequently, developing straightforward and efficient diagnostic systems to detect such fraud is a top priority in scientific research. The aim of the work was to develop a test system and protocol for polymerase chain reaction (PCR) to detect in food products of animal origin the substitution of expensive meat raw materials for by-products of poultry processing. For this, real-time polymerase chain reaction (RT-PCR) was used, which allows determining the qualitative and quantitative substitution in raw and technologically prepared products. Other methods for detecting falsification – enzyme immunoassay (ELISA/ELISA) or express methods in the form of a lateral flow immunoassay are less informative. The extraction of nucleic acids for real-time polymerase chain reaction depends on the source matrix, with higher concentrations obtained from germ cells and parenchymal organs. Extraction from muscle and plant tissues is more challenging, but thorough grinding of these samples improves nucleic acid concentration by 1.5 times using DNA extraction kits. The selection of primers and fluorescent probes through GenBank and PCR Primer Design/DNASTAR software enables efficient amplification and identification of target chicken DNA fragments in various matrices.