End-point Polymerase Chain Reaction Research Articles

Detection Limits of Insect Fragments in Spiked Whole Wheat Flour Using Multiplex Polymerase Chain Reaction (PCR)

The need for a sensitive molecular method to detect specific species of insect contaminants in food products remains a significant challenge in the food industry. This study evaluated the detection limit of a multiplex end-point PCR assay for detecting insects in food. The assay amplifies two fragments of the cytochrome oxidase subunit I gene (COI-Fa and COI-Fb) and one fragment of the protein-coding wingless (wg) gene found in insects. Five insect species, comprising three vectors of foodborne pathogens (the housefly, Musca domestica, the American cockroach, Periplaneta americana, and the pharaoh ant, Monomorium pharaonis) and two storage insect pests (the red flour beetle, Tribolium castaneum and the Indian meal moth, Plodia interpunctella), were spiked separately and in combination at levels of 1, 0.1, 0.01, and 0.001% in whole wheat flour. At spike levels greater than 0.01%, amplicon bands of expected sizes were seen in 100% of samples containing fragments from distinct insect species. At least 25% of spiked samples at the lowest spike level had amplicon bands, except for samples spiked with M. domestica. Results showed an 18.9% probability (with 11.3% and 30% lower and upper confidence limits, respectively) of detecting insect fragments at the lowest spike level (0.001%, corresponding to 3–22 fragments), which is far below the FDA’s regulatory level of less than 75 fragments per 50 g of wheat flour. The intensity of amplicon bands in the gel images was higher at higher spike levels. However, this method is not quantitative enough to extrapolate the intensity of the amplicon bands to the number of insect fragments present in a sample. This multiplex assay was also evaluated in a variety of market food samples derived from plants and animals, showing its potential use in various food types. Overall, the sensitivity and specificity of this molecular approach suggest that it could be used in the future as a screening tool for detecting insect contaminants in food.

Nested Multiplex Endpoint Polymerase Chain Reaction: An Alternative Method for Human Papilloma Virus Genotyping in Resource-limited Settings

Nested Multiplex Endpoint Polymerase Chain Reaction: An Alternative Method for Human Papilloma Virus Genotyping in Resource-limited Settings

Cor1 Gene: A Suitable Marker for Identification of Opium Poppy (Papaver somniferum L.).

This paper discusses the development of rapid, reliable, and accurate polymerase chain reaction (PCR) assays for detecting opium poppy (Papaver somniferum L.) in food. Endpoint, quantitative, and digital PCRs were compared based on the amplification of a newly developed DNA marker targeting the NADPH-dependent codeinone reductase (COR) gene. Designed assays were shown to be highly specific and sensitive in discriminating opium poppy from other plant species, even in heat-treated and food samples. Digital PCR was the most sensitive, with a detection limit of up to 5 copies, i.e., approximately 14 pg of target DNA per reaction. Quantitative and digital PCR further allowed the quantification of opium poppy in up to 1.5 ng and 42 pg (15 copies) of target DNA in a sample, respectively. In addition, two duplex PCRs have been developed for the simultaneous detection of opium poppy DNA and representatives of (i) the Papaveraceae family or (ii) the Plantae kingdom. Finally, all designed assays were successfully applied for analysis of 15 commercial foodstuffs; two were suspected of being adulterated. The study results have an important impact on addressing food fraud and ensuring the safety and authenticity of food products. Beyond food adulteration, the study may also have significant implications for forensics and law enforcement.

Identification of 'Candidatus Liberibacter asiaticus', the Huanglongbing Bacterium, in Citrus from Colombia.

'Candidatus Liberibacter' spp. are the most prevalent microorganisms in the citrus plant, associated with citrus huanglongbing, which are transmitted by psyllid vectors. In Colombia, the vector Diaphorina citri Kuwayama has been reported in different regions, but 'Ca. Liberibacter asiaticus' (CLas) has only been detected in insect vectors, not in citrus host plants. To identify the presence and quantify the pathogen in citrus tissues, we employed a combined strategy that involved three techniques based on polymerase chain reaction (PCR). First, we used endpoint PCR with specific primers for CLas (OI1/OI2c) to confirm the infection. Second, we used qPCR with specific primers CIT295a/CIT298 designed on 16S rDNA gene regions to quantify the pathogen load. Finally, we used droplet digital PCR (ddPCR) to determine the copy number of the pathogen in citrus tissues using the β-subunit of the ribonucleotide reductase gene (nrdB) that is specific to CLas. We identified the presence of CLas in citrus plants for the first time in Colombia and quantified its titer in the plant tissue. We employed ddPCR and qPCR to provide crucial information for the country's disease management, control strategies, and general crop health.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Candida Variety in the Oral Cavity of Mexican Subjects with Type 2 Diabetes Mellitus and TLR2 Gene Expression.

The aim was to diagnose Candida in the oral cavity of subjects with type 2 diabetes mellitus (T2DM) using a genotyping technique and compare the results with those from conventional diagnosis by Papanicolaou (Pap) staining. Palatal mucosa smears were performed on 18 dental care patients diagnosed with T2DM and grade I, II, and III prosthetic stomatitis who met the inclusion criteria; 18 healthy control subjects were also included in the study. Hemoglobin A1c (HbA1c) levels were determined from total blood. Using exfoliative cytology, the Pap staining technique was used to diagnose candidiasis. Exfoliative cytology was also used for molecular diagnosis; DNA was obtained for Candida genotyping, and RNA was used for gene expression studies. Clinical patterns indicated that all subjects were positive for Candida; however, Pap analysis revealed only three positive subjects, whereas end-point polymerase chain reaction (PCR) analysis revealed 15 subjects with some type of Candida. The most common Candida species found were Candida guilliermondii (38.8%), Candida krusei (33.3%), Candida tropicalis, and Candida lusitaniae (22.2%). Interestingly, the coexpression of different species of Candida was found in various patients. In all patients, HbA1c levels were increased. Gene expression analysis showed a significant decrease (p ≤ 0.05) in TLR2 expression in positive subjects, whereas TLR4 expression did not differ significantly among patients. The end-point PCR technique showed better sensitivity for the diagnosis of Candida when compared with the diagnosis by Pap staining. T2DM subjects showed an increased presence of C. guilliermondii that was correlated with decreased TLR2 expression.

Evaluation of Phenotypic Tests for Carbapenemase Detection in Enterobacteriaceae in Tunisia.

Introduction: Resistance to carbapenems in Enterobacteriaceae is a challenge for public health. Carbapenemase production is the leading mechanism. This work aims to evaluate four phenotypic methods for carbapenemase detection in comparison with a molecular method. Materials and Methods: Thirty-seven nonrepeating Enterobacteriaceae strains with decreased susceptibility to ertapenem were included. Imipenem MIC, Modified Hodge Test (MHT), Neo-Rapid Carb Kit® and KPC, MBL, and OXA-48 Confirm Kit® were performed. Isolates were tested for blaOXA-48, blaNDM, and blaVIM genes by end-point polymerase chain reaction. The results of the molecular study were used as a reference test to determine the performances of the phenotypic tests. Results: Imipenem resistance does not seem to be a good marker for carbapenemase production with a sensitivity of 54% (95% CI: 38-71). MHT showed 82% sensitivity (95% CI: 65-91). Overall, the enzymatic test showed the best performances for carbapenemase detection with 100% sensitivity (95% CI: 89-100) and the best turnaround time. The characterization of carbapenemases classes by the combined discs test demonstrated 88% overall sensitivity (95% CI: 72-95). Conclusion: The results of this study support the combination of the enzymatic and the combined disc tests for carbapenemase detection in Enterobacteria.

Progress in polymerase chain reaction technology and its contribution to the tuberculosis diagnostic ecosystem in India: A mini review

The advent of Polymerase Chain Reaction (PCR) has revolutionized the human molecular diagnostic scenario. Its ability to exponentially generate desired stretch of DNA from chromosomal or complimentary DNA has laid the foundation for sensitive and specific detection of a pathogen or an aberrant stretch of DNA. Importantly, PCR technology has helped generate diagnostic immunity from phenotypic variation of pathogenic targets. The technological evolution of conventional end-point PCR to its real time version further added clinically important features such as enhanced sensitivity and quantification of target DNA molecules. Two important chemistries became prominent in the real-time PCR domain. The intercalating dye-based chemistry comes with convenience, economy and feature such as melt curve analysis but also carries the disadvantage of overestimation of signal due to unwanted signal from primer dimers. Dual labelled probe on the other hand provides unprecedented sensitivity and specificity. Isothermal method of genome nucleic acid amplification has also evolved as a parallel and convenient method of amplification-based detection of nucleic acid targets. Tuberculosis diagnostics has immensely benefitted from these technologies. However, unmet need exists in sputum handling methods and in its adaptation for molecular diagnostics in resource limited settings. Significant dependence for diagnostic needs on the western world is a matter of concern in India. The country will benefit from rapid and indigenous research and development of molecular diagnostic solutions by way of formulating kits for rapid extraction of DNA and RNA from various clinical sources and developing detection technologies that can compete international products in its category. A solution to address sputum transportation, storage and DNA extraction that has enhanced advantages compared to current methods will also be helpful in the efforts to make India free from tuberculosis.

Genotyping Protocols for Genetically Engineered Mice.

Historically, the laboratory mouse has been the mammalian species of choice for studying gene function and for modeling diseases in humans. This was mainly due to their availability from mouse fanciers. In addition, their short generation time, small size, and minimal food consumption compared to that of larger mammals were definite advantages. This led to the establishment of large hubs for the development of genetically modified mouse models, such as the Jackson Laboratory. Initial research into inbred mouse strains in the early 1900s revolved around coat color genetics and cancer studies, but gene targeting in embryonic stem cells and the introduction of transgenes through pronuclear injection of a mouse zygote, along with current clustered regularly interspaced short palindromic repeat (CRISPR) RNA gene editing, have allowed easy manipulation of the mouse genome. Originally, to distribute a mouse model to other facilities, standard methods had to be developed to ensure that each modified mouse trait could be consistently identified no matter which laboratory requested it. The task of establishing uniform protocols became easier with the development of the polymerase chain reaction (PCR). This chapter will provide guidelines for identifying genetically modified mouse models, mainly using endpoint PCR. In addition, we will discuss strategies to identify genetically modified mouse models that have been established using newer gene-editing technology such as CRISPR. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Digestion with proteinase K followed by purification of genomic DNA using phenol/chloroform Alternate Protocol: Digestion with proteinase K followed by crude isopropanol extraction of genomic DNA for tail biopsy and ear punch samples Basic Protocol 2: Purification of genomic DNA using a semi-automated system Basic Protocol 3: Purification of genomic DNA from semen, blood, or buccal swabs Basic Protocol 4: Purification of genomic DNA from mouse blastocysts to assess CRISPR gene editing Basic Protocol 5: Routine endpoint-PCR-based genotyping using DNA polymerase and thermal cycler Basic Protocol 6: T7E1/Surveyor assays to detect insertion or deletions following CRISPR editing Basic Protocol 7: Detecting off-target mutations following CRISPR editing Basic Protocol 8: Detecting genomic sequence deletion after CRISPR editing using a pair of guide RNAs Basic Protocol 9: Detecting gene knock-in events following CRISPR editing Basic Protocol 10: Screening of conditional knockout floxed mice.

The host specificity of pilus gene traA in Escherichia coli and its use in tracking human fecal pollution

A reliable and accurate fecal source tracking (FST) approach is important in water quality management and preventing foodborne and waterborne diseases. In this study, a genetic marker of Escherichia coli (E. coli) was identified and utilized to differentiate between human and animal sources of fecal contamination. Nucleotide polymorphisms of 14 genes coding for cellular surface proteins, mainly fimbriae, were analyzed using the 22 draft genomes of E. coli strains from human and three domestic animal sources in Japan. A signature sequence, traAh, within the pilin gene traA, was found to be highly associated with E. coli of human origin. Subsequently, an end-point polymerase chain reaction (PCR) assay, namely PCR-Htra, was developed, specifically targeting traAh. The high association between traAh and E. coli of human origin was validated through the PCR-Htra amplification. This encompassed 1045 E. coli strains isolated from surface water, human feces or sewages, and feces from 12 animal species, including domestic and wild animals in the states of Missouri and Virginia in the United States of America (USA). The data suggested that the sensitivity and specificity of PCR-Htra assay were 49.0 % and 99.5 % respectively in distinguishing human-origin E. coli from nonhuman-source ones. Furthermore, the result of our in silico analysis of GenBank® data suggests that traAh may have a global distribution as the sequence was found in human-origin E. coli isolated from at least 14 countries around the world. Thus, the PCR-Htra may provide a new FST tool for rapid and accurate detection of human-origin E. coli, serving as a means to identify human fecal contamination in water.

Monitoring the Spread of Grapevine Viruses in Vineyards of Contrasting Agronomic Practices: A Metagenomic Investigation.

This study investigated the transmission of grapevine viruses, specifically grapevine red blotch virus (GRBV) and grapevine Pinot gris virus (GPGV), in vineyards in Niagara Region, Ontario, Canada. Forty sentinel vines that were confirmed free of GRBV and GPGV by both high-throughput sequencing (HTS) and endpoint polymerase chain reaction (PCR) were introduced to two vineyards (one organic and one conventional) that were heavily infected with both GRBV and GPGV. Four months post-introduction, the sentinel vines were relocated to a phytotron. The HTS results from 15 months post-introduction revealed a widespread infection of GPGV among the sentinel vines but did not detect any GRBV. The GPGV infection rate of sentinel vines in the organic vineyard (13/18) was higher than in the conventional vineyard (1/19). The possibility of an alternative viral reservoir was assessed by testing the most abundant plants in between rows (Medicago sativa, Trifolium repens, Cirsium arvense and Taraxacum officinale), perennial plants in border areas (Fraxinus americana, Ulmus americana, Rhamnus cathartica) and wild grape (unknown Vitis sp.). The HTS result showed that cover crops and perennial plants did not harbor any grapevine viruses, while 4/5 wild grapes tested positive for GPGV but not GRBV. A pairwise sequence identity analysis revealed high similarities between the GPGV isolates found in the established vines on the vineyard and the newly contracted GPGV isolates in the sentinel vines, implicating a recent transmission event. This work provides novel insights into the spread of grapevine viruses in Niagara Region and is also the first direct proof of the spread of GPGV in natural vineyard conditions in North America.

Loop-mediated isothermal amplification identifies nematode Leidynema in the hindgut of non-pest cockroach

Cockroach microbiome studies generally focus on pest cockroach species belonging to the Blattidae and Ectobiidae families. There are no reports characterizing the gut microbiome of non-pest cockroach species Blaberus discoidalis (family Blaberidae), which is commonly used as a food source for insectivorous animals. We discovered the parasitic nematode Leidynema appendiculata in the B. discoidalis hindgut during initial work characterizing the gut microbiome of this organism. To determine the proportion of the B. discoidalis colony that was colonized by L. appendiculata, 28 S rDNA was amplified using two Methods: endpoint polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP). B. discoidalis colonies were raised on three diet types (control, high fibre, and high fat and salt) for 21 days before dissection. Each individual was sexed during dissection to identify potential sex-based effects of colonization. Data collected were analysed to determine if diet and sex impacted parasite colonization patterns. LAMP detected a higher proportion of parasite positive samples when compared to endpoint PCR. No sex- or diet-based differences in L. appendiculata colonization were found. This study adds to the limited existing knowledge of the B. discoidalis gut microbiome.

First identification and characterization of ovine gammaherpesvirus type 2 in horses and artiodactyla from an outbreak of malignant catarrhal fever in Mexico.

Ovine gammaherpesvirus 2 (OvHV-2), a member of the genus Macavirus, causes sheep-associated malignant catarrhal fever (SA-MCF), a fatal lymphoproliferative disease affecting a wide variety of ungulates in addition to horses. This study described an outbreak of SA-MCF in Mexico and the identification of the OvHV-2 virus in primary rabbit testis cultures through the generation of intranuclear inclusion bodies, syncytia, immunofluorescence (IF), immunocytochemistry (ICC), immunohistochemistry (IHC), endpoint polymerase chain reaction (PCR), and partial sequencing of the ORF75 gene. The animals involved in this outbreak showed mucogingival ulcers in the vestibule of the mouth and tongue, hypersalivation, corneal opacity, reduced food consumption, and weight loss of variable severity. These clinical signs and the histopathological findings suggested the diagnosis of SA-MCF. Buffy coat fractions from the anticoagulated blood samples of ill animals were collected and analyzed by PCR. Positive buffy coats were used to inoculate the primary cell cultures of rabbit testis to identify the virus. Small clusters of refractile cytomegalic cells, characteristic of viral cytopathic effects, were observed between 48 and 72 h post-infection. Furthermore, intranuclear acidophilic inclusion bodies (IBs) were identified in the inoculated primary culture cells, and the cytoplasm showed immunoreactivity with hyperimmune rabbit serum against OvHV-2. Moreover, in the liver histological sections from sick deer, immunoreactive juxtanuclear IBs were identified with the same rabbit hyperimmune serum. The obtained sequences were aligned with the OvHV-2 sequences reported in GenBank and revealed a nucleotide identity higher than 98%. Based on the evidence provided in this study, we conclude that the outbreak of SA-MCF in the municipality of Tequisquiapan in the state of Queretaro, Mexico, was caused by OvHV-2. This is the second study reporting that horses are susceptible to OvHV-2 infection and can develop SA-MCF. We identified for the first time in Mexico, the presence of OvHV-2 in buffy coats from horses and Artiodactyla.

Emergence of Raoultella ornithinolytica in human infections from different hospitals in Ecuador with OXA-48-producing resistance.

The purpose of this study was to highlight the clinical and molecular features of 13 Raoultella ornithinolytica strains isolated from clinical environments in Ecuador, and to perform comparative genomics with previously published genomes of Raoultella spp. As Raoultella is primarily found in environmental, clinical settings, we focused our work on identifying mechanisms of resistance that can provide this bacterium an advantage to establish and persist in hospital environments. We analyzed 13 strains of Raoultella ornithinolytica isolated from patients with healthcare associated infections (HAI) in three hospitals in Quito and one in Santo Domingo de Los Tsáchilas, Ecuador, between November 2017 and April 2018. These isolates were subjected to phenotypic antimicrobial susceptibility testing, end-point polymerase chain reaction (PCR) to detect the presence of carbapenemases and whole-genome sequencing. Polymerase chain reaction revealed that seven isolates were positive isolates for blaOXA-48 and one for blaKPC-2 gene. Of the seven strains that presented the blaOXA-48 gene, six harbored it on an IncFII plasmid, one was inserted into the bacterial chromosome. The blaKPC gene was detected in an IncM2/IncR plasmid. From the bioinformatics analysis, nine genomes had the gene blaOXA-48, originating from Ecuador. Moreover, all R. ornithinolytica strains contained the ORN-1 gene, which confers resistance for β-lactams, such as penicillins and cephalosporins. Comparative genome analysis of the strains showed that the pangenome of R. ornithinolytica is considered an open pangenome, with 27.77% of core genes, which could be explained by the fact that the antibiotic resistance genes in the ancestral reconstruction are relatively new, suggesting that this genome is constantly incorporating new genes. These results reveal the genome plasticity of R. ornithinolytica, particularly in acquiring antibiotic-resistance genes. The genomic surveillance and infectious control of these uncommon species are important since they may contribute to the burden of antimicrobial resistance and human health.

Low-cost molecular methods to characterise gastrointestinal nematode co-infections of goats in Africa

BackgroundVeterinary diagnostics aid intervention strategies, track zoonoses, and direct selective breeding programs in livestock. In ruminants, gastrointestinal nematode (GIN) parasites are a major cause of production losses, but morphologically similar species limit our understanding of how specific GIN co-infections impact health in resource-limited settings. To estimate the presence and relative abundance of GINs and other helminths at the species level, we sought to develop a low-cost and low-resource molecular toolkit applied to goats from rural Malawi smallholdings.MethodsGoats were subjected to health scoring and faecal sampling on smallholdings in Lilongwe district, Malawi. Infection intensities were estimated by faecal nematode egg counts with a faecal subsample desiccated for DNA analysis. Two DNA extraction methods were tested (low-resource magbead kit vs high-resource spin-column kit), with resulting DNA screened by endpoint polymerase chain reaction (PCR), semi-quantitative PCR, quantitative PCR (qPCR), high-resolution melt curve analysis (HRMC), and ‘nemabiome’ internal transcribed spacer 2 (ITS-2) amplicon sequencing.ResultsBoth DNA isolation methods yielded comparable results despite poorer DNA purity and faecal contaminant carryover from the low-resource magbead method. GINs were detected in 100% of samples regardless of infection intensity. Co-infections with GINs and coccidia (Eimeria spp.) were present in most goats, with GIN populations dominated by Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus axei, and Oesophagostomum columbianum. Both multiplex PCR and qPCR were highly predictive of GIN species proportions obtained using nemabiome amplicon sequencing; however, HRMC was less reliable than PCR in predicting the presence of particular species.ConclusionsThese data represent the first ‘nemabiome’ sequencing of GINs from naturally infected smallholder goats in Africa and show the variable nature of GIN co-infections between individual animals. A similar level of granularity was detected by semi-quantitative PCR methods, which provided an accurate summary of species composition.Assessing GIN co-infections is therefore possible using cost-efficient low-resource DNA extraction and PCR approaches that can increase the capacity of molecular resources in areas where sequencing platforms are not available; and also open the door to affordable molecular GIN diagnostics. Given the diverse nature of infections in livestock and wildlife, these approaches have potential for disease surveillance in other areas.Graphical

HLA-DR Association of Anti-dsDNA and Anti-Sm Autoantibodies in Bangladeshi Patients with Systemic Lupus Erythematosus

Background: Rheumatic disorders are one of the largest health problems in the world in both developed and developing countries. Among systemic rheumatic disorders, systemic lupus erythematosus (SLE) is very much common. This debilitating disease most commonly affects females, especially at a young age. Though the exact etiology for the development of SLE still remains vague but genetic factors especially, HLA-DR plays an important role particularly in the development of autoantibodies in SLE cases. Objective: This study was undertaken to find out the association of HLA-DR with anti-dsDNA and anti-Sm autoantibodies among patients with SLE. Methods: Buccal swabs for HLA-DR typing and blood samples for detection of anti-dsDNA and anti-Sm were collected from 46 SLE cases. HLA-DR typing was carried out by end point polymerase chain reaction (PCR) with sequence specific primers. Autoantibodies were detected by using ELISA. Results: Out of 46 cases with SLE 44 (95.65%) were female and 2 (4.35%) were male with female: male ratio was 22: 1. Their mean age at study entry was 27.05 ± 8.17 years (mean ± SD), ranging from 12.5-45 years. AntidsDNA was positive in 38 (82.61%) cases and negative in 8 (17.39%) cases. Anti-Sm was positive in 19 (41.30%) cases and negative in 27 (58.70%) cases. The most frequently identified HLA-DR was DR2 (86.96%). When Anti-dsDNA positive cases were compared with Anti-dsDNA negative cases significant association was found with HLA-DR2 (94.73% vs 50%, p value = 0.0044, pc = 0.044, RR = 18.0000). No positive association of HLA-DR was found with anti-Sm autoantibody in this study. The above data suggest that HLA-DR2 has a role in anti-dsDNA production in Bangladeshi patients with SLE. Bangladesh Medical Res Counc Bull 2022; 48: 138-145

Are bacteria, fungi, and archaea present in the midtrimester amniotic fluid?

This study was conducted to determine whether bacteria, fungi, or archaea are detected in the amniotic fluid of patients who underwent midtrimester amniocentesis for clinical indications. Amniotic fluid samples from 692 pregnancies were tested by using a combination of culture and end-point polymerase chain reaction (PCR) techniques. Intra-amniotic inflammation was defined as an interleukin-6 concentration >2,935 pg/mL. Microorganisms were detected in 0.3% (2/692) of cases based on cultivation, 1.73% (12/692) based on broad-range end-point PCR, and 2% (14/692) based on the combination of both methods. However, most (13/14) of these cases did not have evidence of intra-amniotic inflammation and delivered at term. Therefore, a positive culture or end-point PCR in most patients appears to have no apparent clinical significance. Amniotic fluid in the midtrimester of pregnancy generally does not contain bacteria, fungi, or archaea. Interpretation of amniotic fluid culture and molecular microbiologic results is aided by the assessment of the inflammatory state of the amniotic cavity. The presence of microorganisms, as determined by culture or a microbial signal in the absence of intra-amniotic inflammation, appears to be a benign condition.

Recycling of Polymerase Chain Reaction (PCR) Kits.

Polymerase chain reaction (PCR) kits have been used as common diagnosing tools during the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, with daily worldwide usage in the millions. It is well known that at the beginning of the pandemic, there was a shortage of PCR kits. So far, the ecosystem of a PCR kit is linear use; that is, kits are produced, used once, and disposed of as biolab waste. Here, we show that to mitigate the risk of future shortages, it is possible to envision recyclable PCR kits based on a more sustainable use of nucleic acid resources. A PCR kit is mainly composed of primers, nucleotides, and enzymes. In the case of a positive test, the free nucleotides are polymerized onto the primers to form longer DNA strands. Our approach depolymerizes such strands, keeping the primers and regenerating the nucleotides, i.e., returning the nucleic acid materials to the original state. The polymerized long DNA strands are hydrolyzed into nucleotide monophosphates that are then phosphorylated into triphosphates using a method that is developed from a recent publication. We used oligonucleotides with a 3'-terminal phosphorothioate (PS) backbone modification as nonhydrolyzable PCR primers, which are able to undergo the recycling process unchanged. The nuclease resistance of oligonucleotides with a ribose sugar modification was also evaluated, which showed worse recycling efficiency than PS-modified oligonucleotides. We successfully recycled both PCR primers and nucleotide monomers (∼75% yield). We demonstrate that the method allows for the direct reuse of PCR kits. We also show that the recycled primers can be isolated and then added to endpoint or quantitative PCR. This recycling approach provides a new path for circularly reusing nucleic acid materials in PCR kits.

Development of a validated molecular analytical method to determine the viral safety of F(AB´)2 products: A novel application for a well-known technique

The immunotherapy agents derived from horses are biological products that allow the neutralization of clinically relevant immunogens, such as the SARS-CoV-2 virus that causes COVID-19, or the neutralization of toxins present in the venoms of snakes, spiders, and other poisonous animals. Due to their importance, detecting adventitious viruses in equine hyperimmune serum (raw material in industrial processes) is a critical step to support the safety of products for human use, and, in consequence, it is a requirement for commercialization and distribution. The safety of the finished product is based on three complementary approaches: (i) testing of the source material (horse serum) donations, (ii) release of the starting material (i.e., pool of horse serum) based on non-reactivity for a range of human infectious or pathogenic viruses, and (iii) validate (selected) steps of the manufacturing process for their capacity to inactivate and/or remove a wide range of viruses potentially present in the starting material. Orthogonal approaches to reduce viral contamination risk include implementing a reliable and validated system for detecting adventitious viruses. Thus, it is necessary to establish trustworthy and sufficiently sensitive analytical methods to evidence the lack of viruses to assure the safety of the therapeutic product. Therefore, in this research, an analytical method based on end-point Reverse Transcription Polymerase Chain Reaction (RT-PCR) was developed, implemented, and validated in hyperimmune equine serum samples to detect Venezuelan equine encephalitis virus, West Nile virus, and Rabies virus.

Application of Nucleic Acid-Based Strategies to Detect Infectious Pathogens in Orthopaedic Implant-Related Infection.

Implant-associated infection in orthopaedic surgery remains an enormous and largely unsolved clinical problem with a high rate of persistent or recurrent infection. This may be due, at least in part, to the potential for underdiagnosis by traditional microbial culture or the potential for culture to incompletely identify the microbial species present. Nucleic acid-based diagnostic techniques, focused on using the diagnostic information contained in DNA or RNA to identify microbial species, have been developing rapidly and have garnered escalating interest for both clinical and research applications. Commonly applied techniques include end-point polymerase chain reaction (PCR), quantitative PCR, Sanger sequencing, and next-generation sequencing. Understanding the specific strengths and weaknesses of each technique is critical to understanding their utility, applying the correct assessment strategy, and critically understanding and interpreting research. The best practices for interpreting nucleic acid-based diagnostic techniques include considering positive and negative controls, reads per sample, detection thresholds (for differentiating contaminants from positive results), and the primer set or targeted regions.

Integrated Approach from Sample-to-Answer for Grapevine Varietal Identification on a Portable Graphene Sensor Chip.

Identifying grape varieties in wine, related products, and raw materials is of great interest for enology and to ensure its authenticity. However, these matrices' complexity and low DNA content make this analysis particularly challenging. Integrating DNA analysis with 2D materials, such as graphene, offers an advantageous pathway toward ultrasensitive DNA detection. Here, we show that monolayer graphene provides an optimal test bed for nucleic acid detection with single-base resolution. Graphene's ultrathinness creates a large surface area with quantum confinement in the perpendicular direction that, upon functionalization, provides multiple sites for DNA immobilization and efficient detection. Its highly conjugated electronic structure, high carrier mobility, zero-energy band gap with the associated gating effect, and chemical inertness explain graphene's superior performance. For the first time, we present a DNA-based analytic tool for grapevine varietal discrimination using an integrated portable biosensor based on a monolayer graphene field-effect transistor array. The system comprises a wafer-scale fabricated graphene chip operated under liquid gating and connected to a miniaturized electronic readout. The platform can distinguish closely related grapevine varieties, thanks to specific DNA probes immobilized on the sensor, demonstrating high specificity even for discriminating single-nucleotide polymorphisms, which is hard to achieve with a classical end-point polymerase chain reaction or quantitative polymerase chain reaction. The sensor was operated in ultralow DNA concentrations, with a dynamic range of 1 aM to 0.1 nM and an attomolar detection limit of ∼0.19 aM. The reported biosensor provides a promising way toward developing decentralized analytical tools for tracking wine authenticity at different points of the food value chain, enabling data transmission and contributing to the digitalization of the agro-food industry.