A General Protocol for Gene Expression Analysis in Chemically Mutant Coffee Plants in Response to Rust (Hemileia vastatrix Berk & Broome) Infection.

Coffea spp. is the source of one of the most widely consumed beverages in the world. However, the cultivation of this crop is threatened by Hemileia vastatrix Berk & Broome, a fungal disease, which reduces the productivity and can cause significant economic losses. In this protocol, coffee leaf segment derived from a chemical mutagenesis process are inoculated with uredospores of the pathogen. Subsequently, the gene expression changes are analyzed over the time (0, 5, 24, 48, and 120 h) using quantitative real-time polymerase chain reaction (RT-qPCR). The procedures and example data are presented for expression analysis in the CaWRKY1 gene. This procedure can be applied for quantitative analysis of other genes of interest to coffee breeders and scientists for elucidating the molecular mechanisms involved in the interaction between the plant and pathogen, potentially leading to the development of more efficient approaches for managing this disease.

A quantitative assay for the assessment of cutaneous human papillomaviruses and polyomaviruses over time: A proof-of-concept in two patients with atopic dermatitis and psoriasis.

The human skin virome, unlike commensal bacteria, is an under investigated component of the human skin microbiome. We developed a sensitive, quantitative assay to detect cutaneous human resident papillomaviruses (HPV) and polyomaviruses (HPyV) and we first used it to describe these viral populations at the skin surface of two patients with atopic dermatitis (AD) and psoriasis (PSO). We performed skin swabs on lesional and non-lesional skin in one AD and one PSO patient at M0, M1 and M3. After extraction, DNA was amplified using an original multiplex PCR technique before high throughput sequencing (HTS) of the amplicons (named AmpliSeq-HTS). Quantitative results were ultimately compared with monoplex quantitative PCRs (qPCRs) for previously detected viruses and were significantly correlated (R2 = 0.95, ρ = 0.75). Fifteen and 13 HPV types (mainly gamma and beta-HPVs) or HPyV species (mainly Merkel Cell Polyomavirus (MCPyV)) were detected on the skin of the AD and PSO patients, respectively. In both patients, the composition of the viral flora was variable across body sites but remained stable over time in non-lesional skin samples, mostly colonized with gamma-papillomaviruses. In lesional skin samples, beta-papillomaviruses and MCPyV were the major components of a viral flora more prone to vary over time especially with treatment and subsequent clinical improvement. We believe this method might be further used in extensive studies to further enhance the concept of an individual cutaneous viral fingerprint and the putative role of its alterations through various skin diseases and their treatments.

Microscopy detection and molecular characterisation of Giardia duodenalis infection in outpatients seeking medical care in Egypt.

Introduction: Giardiosis remains one of the most prevalent enteric parasitic infections globally. Earlier molecular-based studies conducted in Egypt have primarily focused on paediatric clinical populations and most were based on single genotyping markers. As a result, there is limited information on the frequency and genetic diversity of G. duodenalis infections in individuals of all age groups. Methods: Individual stool samples (n = 460) from outpatients seeking medical care were collected during January-December 2021 in Kafr El-Sheikh governorate, northern Egypt. Initial screening for the presence of G. duodenalis was conducted by coprological examination. Microscopy-positive samples were further confirmed by real-time PCR. A multilocus sequence typing approach targeted amplification of the glutamate dehydrogenase (gdh), beta-giardin (bg), and triose phosphate isomerase (tpi) genes was used for genotyping purposes. A standardised epidemiological questionnaire was used to gather basic sociodemographic and clinical features of the recruited patients. Results: Giardia duodenalis cysts were observed in 5.4% (25/460, 95% CI: 3.6-7.9) of the stool samples examined by conventional microscopy. The infection was more frequent in children under the age of 10 years and in individuals presenting with diarrhoea but without reaching statistical significance. Stool samples collected during the winter period were more likely to harbour G. duodenalis. All 25 microscopy-positive samples were confirmed by real-time PCR, but genotyping data was only available for 56.0% (14/25) of the isolates. Sequence analyses revealed the presence of assemblages A (78.6%, 11/14) and B (21.4%, 3/14). All assemblage A isolates were identified as sub-assemblage AII, whereas the three assemblage B sequences belonged to the sub-assemblage BIII. Patients with giardiosis presenting with diarrhoea were more frequently infected by the assemblage A of the parasite. Conclusion: This is one of the largest epidemiological studies evaluating G. duodenalis infection in individuals of all age groups in Egypt. Our molecular data suggest that G. duodenalis infections in the surveyed population are primarily of anthropic origin. However, because assemblages A and B are zoonotic, some of the infections identified can have an animal origin. Additional investigations targeting animal (domestic and free-living) and environmental (water) samples are warranted to better understand the epidemiology of giardiosis in Egypt.

Based on molecular docking and real-time PCR technology, the two-component system Bae SR was investigated on the mechanism of drug resistance in CRAB.

This study aimed to explore the role of the two-component system Bae SR in the mechanism of drug resistance in carbapenem-resistant A. baumannii (CRAB) using molecular docking and real-time polymerase chain reaction (PCR). The two-component system Bae SR of Acinetobacter baumannii was subjected to molecular docking with imipenem, meropenem, and levofloxacin. Antibacterial assays and fluorescence quantitative PCR were used to explore protein-ligand interactions and molecular biological resistance mechanisms related to CRAB. The analysis of the two-component system in A. baumannii revealed that imipenem exhibited the highest docking energy in Bae S at - 5.81 kcal/mol, while the docking energy for meropenem was - 4.92 kcal/mol. For Bae R, imipenem had a maximum docking energy of - 4.28 kcal/mol, compared with - 4.60 kcal/mol for meropenem. The highest binding energies for Bae S-levofloxacin and Bae R-levofloxacin were - 3.60 and - 3.65 kcal/mol, respectively. All imipenem-resistant strains had minimum inhibitory concentration (MIC) values of 16 µg/mL, whereas levofloxacin-resistant strains had MIC values of 8 µg/mL. The time-sterilization curve showed a significant decrease in bacterial colony numbers at 2 h under the action of 8 µg/mL imipenem, indicating antibacterial effects. In contrast, levofloxacin did not exhibit any antibacterial activity. Fluorescence quantitative PCR results revealed significantly increased relative expression levels of bae S and bae R genes in the CRAB group, which were 2 and 1.5 times higher than those in the CSAB group, respectively, with statistically significant differences. Molecular docking in this study found that the combination of Bae SR and carbapenem antibiotics (imipenem, meropenem) exhibited stronger affinity and stability compared with levofloxacin. Moreover, the overexpression of the two-component system genes in carbapenem-resistant A. baumannii enhanced its resistance to carbapenem, providing theoretical and practical insights into carbapenem resistance in respiratory tract infections caused by A. baumannii.

Expression of RAS and RAB interactor 1 (RIN1) in head and neck tumors at selected hospital in Ghana.

BACKGROUND: Head and neck tumors (HNT) are tumors of the paranasal sinuses, the salivary glands and the upper aerodigestive tract. RIN1 is a Ras effector protein regulating epithelial cell properties and has been implicated in a number of cancers. METHOD: The aim of this study was to investigate the expression of RIN1 in head and neck tumors. RIN1 expression was assessed using quantitative real-time PCR (qRT-PCR) and immunohistochemical staining on archival head and neck tissue samples between 2014 and 2020. RESULTS: RIN1 expression was low in tissue samples as compared with the normal head and neck tissues. High and low RIN1 levels were compared with ages ≤40, >40 in the head and neck tumors of p-value 0.02. There was a significant difference with p-values of 0.001 when poor and well-moderate malignant tumors were compared. CONCLUSION: Our data suggests that RIN1may play an important role in head and neck tumor progression and that its expression may provide baseline data to facilitate identification of new molecular targeted therapeutics.

Evaluation of different genomic regions of rotavirus B and rotavirus C for development of real-time RT‒PCR assays.

BACKGROUND: The causative agents of diarrhea, rotavirus B (RVB) and rotavirus C (RVC) are common in adults and patients of all age groups, respectively. Due to the Rotavirus A (RVA) vaccination program, a significant decrease in the number of gastroenteritis cases has been observed globally. The replacement of RVA infections with RVB, RVC, or other related serogroups is suspected due to the possibility of reducing natural selective constraints due to RVA infections. The data available on RVB and RVC incidence are scant due to the lack of cheap and rapid commercial diagnostic assays and the focus on RVA infections. The present study aimed to develop real-time RT‒PCR assays using the data from all genomic RNA segments of human RVB and RVC strains available in the Gene Bank. RESULTS: Among the 11 gene segments, NSP3 and NSP5 of RVB and the VP6 gene of RVC were found to be suitable for real-time RT‒PCR (qRT‒PCR) assays. Fecal specimens collected from diarrheal patients were tested simultaneously for the presence of RVB (n = 192) and RVC (n = 188) using the respective conventional RT‒PCR and newly developed qRT‒PCR assays. All RVB- and RVC-positive specimens were reactive in their respective qRT‒PCR assays and had Ct values ranging between 23.69 and 41.97 and 11.49 and 36.05, respectively. All known positive and negative specimens for other viral agents were nonreactive, and comparative analysis showed 100% concordance with conventional RT‒PCR assays. CONCLUSIONS: The suitability of the NSP5 gene of RVB and the VP6 gene of RVC was verified via qRT‒PCR assays, which showed 100% sensitivity and specificity. The rapid qRT‒PCR assays developed will be useful diagnostic tools, especially during diarrheal outbreaks for testing non-RVA rotaviral agents and reducing the unnecessary use of antibiotics.

Snapshot study of canine distemper virus in Bangladesh with on-site PCR detection and nanopore sequencing.

Canine distemper virus (CDV) is a highly contagious virus that affects domestic and wild animals, causing severe illness with high mortality rates. Rapid monitoring and sequencing can provide valuable information about circulating CDV strains, which may foster effective vaccination strategies and the successful integration of these into conservation programs. During two site visits in Bangladesh in 2023, we tested a mobile, deployable genomic surveillance setup to explore the genetic diversity and phylogenetic patterns of locally circulating CDV strains. We collected and analysed 355 oral swab samples from stray dogs in Rajshahi and Chattogram cities, Bangladesh. CDV-specific real-time RT-PCR was performed to screen the samples. Out of the 355 samples, 7.4% (10/135) from Rajshahi city and 0.9% (2/220) from Chattogram city tested positive for CDV. We applied a real-time RT-PCR assay and a pan-genotype CDV-specific amplicon-based Nanopore sequencing technology to obtain the near-completes. Five near-complete genome sequences were generated, with phylogenetic relation to the India-1/Asia-5 lineage previously identified in India. This is the first study to provide genomic data on CDV in Bangladesh and the first demonstration of a mobile laboratory setup as a powerful tool in rapid genomic surveillance and risk assessment for CDV in low resource regions.

Elimination of E. faecalis with NaOCl versus chlorhexidine gluconate from primary molar root canal systems: an ex vivo model study.

OBJECTIVES: This ex vivo human study aimed to evaluate the efficacy of NaOCl and chlorhexidine gluconate (CHG) irrigations in eliminating Enterococcus faecalis from the RCS of primary molars. MATERIALS AND METHODS: Disinfected extracted primary molars were inoculated with E. faecalis for 24 h. Then, the RCS samples were then irrigated with either 2.5% NaOCl, 0.2% and 2% CHG, or sham saline. The samples were collected immediately after irrigation; and 24 h later, the bacterial viability and counts were measured using blood agar and qRT-PCR, respectively. Histological sections were used to measure E. faecalis penetration and viability in dentin tubules using fluorescence microscopy. RESULTS: The recovery of viable E. faecalis after the irrigation of the primary molars showed more significant bactericidal effects of NaOCl and 0.2% and 2% CHG than of saline. Immediately after the irrigation, the NaOCl group showed the greatest reduction in E. faecalis; and 24 h later, all the groups had lower viable E. faecalis than the saline control. The bacterial penetration was also lowest in the NaOCl group, although there was no difference in bacterial viability in the tubules between the groups. CONCLUSION: In primary teeth, NaOCl and CHG showed similar degrees of bacterial elimination efficacy in terms of E.faecalis. CLINICAL RELEVANCE: Within the limitations of this study, NaOCl and CHG have the similar ability to perform endodontic irrigation of primary ex vivo teeth regarding the elimination of E.faecalis, but NaOCl penetrates dentin tubules better.

The evaluation of miR-1181 and miR-4314 as serum microRNA biomarkers for epithelial ovarian cancer diagnosis and prognosis.

AIM: Epithelial ovarian cancer (EOC) is the most ominous tumor of gynecological cancers due to its poor early detection rate and unfavorable prognosis. To date, there is no reliable screening method for the diagnosis of ovarian cancer at an early stage. MiRNAs are small non-coding RNA molecules, and their main function is to regulate gene expression. The present study compared the serum miR-1181 and miR-4314 levels in patients with EOC and healthy controls to measure the diagnostic and prognostic value as candidate biomarkers. MATERIALS AND METHODS: We collected serum samples from a total of 135 participants (69 patients with EOC and 66 healthy controls). Relative expressions of miR-1181 and miR-4314 were measured by quantitative real-time polymerase chain reaction assay (qPCR). RESULTS: The present study revealed that both serum miR-1181 and miR-4314 levels in patients with EOC were significantly increased compared to healthy controls for each marker. In addition, there was a significant relationship between miR-1181 and miR-4314 overexpressions and the stage and prognosis of the disease. Finally, patients with high expression levels of miR-1181 and miR-4314 had significantly shorter survival rates than those with low expression levels. CONCLUSION: The current study proposed that serum miR-1181 and miR-4314 could discriminate the EOC patients from healthy controls. In addition, both miR-1181 and miR-4314 may be predictive biomarkers for ovarian cancer prognosis. Further studies are needed to confirm the findings of the present study.

Molecular epidemiological study of Scrub Typhus in residence, farm and forest habitats from Yunnan Province, China.

The number of people suffering from scrub typhus, which is not of concern, is increasing year by year, especially in Yunnan Province, China. From June 1, 2021 to August 15, 2022, a total of 505 mammalian samples were collected from farm, forest, and residential habitats with high incidence of scrub typhus in Yunnan, China, for nPCR (nested PCR) and qPCR (quantitative real-time PCR) detection of Orientia tsutsugamushi. A total of 4 orders of murine-like animals, Rodentia (87.52%, n = 442), Insectivora (10.29%, n = 52), Lagomorpha (1.79%, n = 9) and Scandentia (0.40%, n = 2) were trapped. Comparing the qPCR infection rates in the three habitats, it was no significant difference that the infection rate of residential habitat (44.44%) and that of the farm habitat (45.05%, P>0.05), which is much larger than that of the forest habitat (3.08%) (P<0.001). Three genotypes (Karp-like, Kato-like and TA763-like) of O. tsutsugamushi were found from Yunnan, China in this study.

Experimental investigation of confinement effect in single molecule amplification via real-time digital PCR on a multivolume droplet array SlipChip.

BACKGROUND: Digital polymerase chain reaction (digital PCR) is an important quantitative nucleic acid analysis method in both life science research and clinical diagnostics. One important hypothesis is that by physically constraining a single nucleic acid molecule in a small volume, the relative concentration can be increased therefore further improving the analysis performance, and this is commonly defined as the confinement effect in digital PCR. However, experimental investigation of this confinement effect can be challenging since it requires a microfluidic device that can generate partitions of different volumes and an instrument that can monitor the kinetics of amplification. (96). RESULTS: Here, we developed a real-time digital PCR system with a multivolume droplet array SlipChip (Muda-SlipChip) that can generate droplet of 125 nL, 25 nL, 5 nL, and 1 nL by a simple "load-slip" operation. In the digital region, by reducing the volume, the relative concentration is increased, the amplification kinetic can be accelerated, and the time to reach the fluorescence threshold, or Cq value, can be reduced. When the copy number per well is much higher than one, the relative concentration is independent of the partition volume, thus the amplification kinetics are similar in different volume partitions. This system is not limited to studying the kinetics of digital nucleic acid amplification, it can also extend the dynamic range of the digital nucleic acid analysis by additional three orders of magnitude by combining a digital and an analog quantification algorithm. (140). SIGNIFICANCE: In this study, we experimentally investigated for the first time the confinement effect in the community of digital PCR via a new real-time digital PCR system with a multivolume droplet array SlipChip (Muda-SlipChip). And a wider dynamic range of quantification methods compared to conventional digital PCR was validated by this system. This system provides emerging opportunities for life science research and clinical diagnostics. (63).

EndoGeneAnalyzer: A tool for selection and validation of reference genes.

The selection of proper reference genes is critical for accurate gene expression analysis in all fields of biological and medical research, mainly because there are many distinctions between different tissues and specimens. Given this variability, even in known classic reference genes, demands of a comprehensive analysis platform is needed to identify the most suitable genes for each study. For this purpose, we present an analysis tool for assisting in decision-making in the analysis of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) data. EndoGeneAnalyzer, an open-source web tool for reference gene analysis in RT-qPCR studies, was used to compare the groups/conditions under investigation. This interactive application offers an easy-to-use interface that allows efficient exploration of datasets. Through statistical and stability analyses, EndoGeneAnalyzer assists in the select of the most appropriate reference gene or set of genes for each condition. It also allows researchers to identify and remove unwanted outliers. Moreover, EndoGeneAnalyzer provides a graphical interface to compare the evaluated groups, providing a visually informative differential analysis.

[Comparison of DNA Extraction Methods for Processed Foods Containing Soybean or Maize].

Processed foods containing soybean or maize are subject to labeling regulations pertinent to genetically modified (GM) foods in Japan. To confirm the reliability of the labeling procedure of GM foods, the Japanese standard analytical methods (standard methods) using real-time PCR technique have been established. Although certain DNA extraction protocols are stipulated as standard in these methods, the use of other protocols confirmed to be equivalent to the existing ones was permitted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or corn was conducted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or maize was conducted. The silica membrane-based DNA extraction kits, GM quicker 4 and DNeasy Plant Maxi Kit (Maxi Kit), as an existing method were compared. GM quicker 4 was considered to be equivalent to or better than Maxi Kit.

Development of a novel sequence based real-time PCR assay for specific and sensitive detection of Burkholderia pseudomallei in clinical and environmental matrices.

BACKGROUND: Melioidosis, caused by the category B biothreat agent Burkholderia pseudomallei, is a disease with a high mortality rate and requires an immediate culture-independent diagnosis for effective disease management. In this study, we developed a highly sensitive qPCR assay for specific detection of Burkholderia pseudomallei and melioidosis disease diagnosis based on a novel target sequence. METHODS: An extensive in-silico analysis was done to identify a novel and highly conserved sequence for developing a qPCR assay. The specificity of the developed assay was analyzed with 65 different bacterial cultures, and the analytical sensitivity of the assay was determined with the purified genomic DNA of B. pseudomallei. The applicability of the assay for B. pseudomallei detection in clinical and environmental matrices was evaluated by spiking B. pseudomallei cells in the blood, urine, soil, and water along with suitable internal controls. RESULTS: A novel 85-nucleotide-long sequence was identified using in-silico tools and employed for the development of the highly sensitive and specific quantitative real-time PCR assay S664. The assay S664 was found to be highly specific when evaluated with 65 different bacterial cultures related and non-related to B. pseudomallei. The assay was found to be highly sensitive, with a detection limit of 3 B. pseudomallei genome equivalent copies per qPCR reaction. The detection limit in clinical matrices was found to be 5 × 102 CFU/mL for both human blood and urine. In environmental matrices, the detection limit was found to be 5 × 101 CFU/mL of river water and 2 × 103 CFU/gm of paddy field soil. CONCLUSIONS: The findings of the present study suggest that the developed assay S664 along with suitable internal controls has a huge diagnostic potential and can be successfully employed for specific, sensitive, and rapid molecular detection of B. pseudomallei in various clinical and environmental matrices.

The development, evaluation, performance, and validation of micro-PCR and extractor for the quantification of HIV-1 &-2 RNA.

HIV infection has been a global public health threat and overall reported ~ 40 million deaths. Acquired immunodeficiency syndrome (AIDS) is attributed to the retroviruses (HIV-1/2), disseminated through various body fluids. The temporal progression of AIDS is in context to the rate of HIV-1 infection, which is twice as protracted in HIV-2 transmission. Q-PCR is the only available method that requires a well-developed lab infrastructure and trained personnel. Micro-PCR, a portable Q-PCR device, was developed by Bigtec Labs, Bangalore, India. It is simple, accurate, fast, and operationalised in remote places where diagnostic services are inaccessible in developing countries. This novel micro-PCR determines HIV-1 and HIV-2 viral load using a TruePrep™ extractor device for RNA isolation. Five ml blood samples were collected at the blood collection centre at AIIMS, New Delhi, India. Samples were screened for serology, and a comparison of HIV-1/2 RNA was done between qPCR and micro-PCR in the samples. The micro-PCR assay of HIV-RNA has compared well with those from real-time PCR (r = 0.99, i < 0.002). Micro-PCR has good inter and intra-assay reproducibility over a wide dynamic range (1.0 × 102-1.0 × 108 IU/ml). The linear dynamic range was 102-108 IU/ml. The clinical and analytical specificity of the assay was comparable, i.e., 100%. Intra-assay and inter-assay coefficients of variation ranged from 1.17% to 3.15% and from 0.02% to 0.46%, respectively. Moreover, due to the robust, simple, and empirical method, the Probit analysis has also been done for qPCR LODs to avoid uncertainties in target recoveries. The micro-PCR is reliable, accurate, and reproducible for early detection of HIV-1 and HIV-2 viral loads simultaneously. Thus, it can easily be used in the field and in remote places where quantification of both HIV-1/2 is not reachable.

Development of a real-time recombinase-aided amplification assay for rapid and sensitive detection of Edwardsiella piscicida.

Edwardsiella piscicida, a significant intracellular pathogen, is widely distributed in aquatic environments and causes systemic infection in various species. Therefore, it's essential to develop a rapid, uncomplicated and sensitive method for detection of E. piscicida in order to control the transmission of this pathogen effectively. The recombinase-aided amplification (RAA) assay is a newly developed, rapid detection method that has been utilized for various pathogens. In the present study, a real-time RAA (RT-RAA) assay, targeting the conserved positions of the EvpP gene, was successfully established for the detection of E. piscicida. This assay can be performed in a one-step single tube reaction at a temperature of 39°C within 20 min. The RT-RAA assay exhibited a sensitivity of 42 copies per reaction at a 95% probability, which was comparable to the sensitivity of real-time quantitative PCR (qPCR) assay. The specificity assay confirmed that the RT-RAA assay specifically targeted E. piscicida without any cross-reactivity with other important marine bacterial pathogens. Moreover, when clinical specimens were utilized, a perfect agreement of 100% was achieved between the RT-RAA and qPCR assays, resulting a kappa value of 1. These findings indicated that the established RT-RAA assay provided a viable alternative for the rapid, sensitive, and specific detection of E. piscicida.

Development and application of one-step multiplex Real-Time PCR for detection of three main pathogens associated with bovine neonatal diarrhea.

Introduction: Neonatal calf diarrhea (NCD) is one of the most common diseases in calves, causing huge economic and productivity losses to the bovine industry worldwide. The main pathogens include bovine rotavirus (BRV), bovine coronavirus (BCoV), and Enterotoxigenic Escherichia coli (ETEC) K99. Since multiple infectious agents can be involved in calf diarrhea, detecting each causative agent by traditional methods is laborious and expensive. Methods: In this study, we developed a one-step multiplex Real-Time PCR assay to simultaneously detect BRV, BCoV, and E. coli K99+. The assay performance on field samples was evaluated on 1100 rectal swabs of diseased cattle with diarrhea symptoms and compared with the conventional gel-based RT-PCR assay detect BRV, BCoV, and E. coli K99+. Results: The established assay could specifically detect the target pathogens without cross-reactivity with other pathogens. A single real-time PCR can detect ~1 copy/µL for each pathogen, and multiplex real-time PCR has a detection limit of 10 copies/µL. Reproducibility as measured by standard deviation and coefficient of variation were desirable. The triple real-time PCR method established in this study was compared with gel-based PT-PCR. Both methods are reasonably consistent, while the real-time PCR assay was more sensitive and could rapidly distinguish these three pathogens in one tube. Analysis of surveillance data showed that BRV and BCoV are major enteric viral pathogens accounting for calves' diarrhea in China. Discussion: The established assay has excellent specificity and sensitivity and was suitable for clinical application. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and calf diarrhea research. ​.

Bacteriological evaluation and advanced SYBR-green multiplex real-time PCR assay for detection of minced meat adulteration.

Background: Minced meat is a valuable source of nutrients, but it is vulnerable to contamination by microorganisms commonly present in the environment. In addition, there is a risk of adulteration with cheaper meat sources, which can be harmful to consumers. Aim: It is crucial to identify meat adulteration with distinct microbiological analysis for legal, economic, religious, and public health purposes. Methods: A total of 100 minced meat samples were collected from several markets in Sharkia Governorate, Egypt. These samples were then subjected to bacteriological testing and an advanced multiplex PCR method. This method enables the detection of bovine, equine, porcine, and dog species in meat samples with just one step. Results: The adulterated samples had a higher total bacterial count and pH values compared to pure bovine meat. These differences in bacterial count and pH values were statistically significant, with p-values of 0.843 (log10) and 0.233, respectively. The frequency of Escherichia coli occurrence was 13%, and the O111 serotype was predominant in the adulterated samples. Listeria monocytogenes and Staphylococcus aureus were isolated with prevalence rates of 3% and 29%, respectively. Besides, the SYBR-green multiplex real-time PCR assay used in this study detected adulteration with dog, equine, and porcine meats in the examined samples at rates of 9%, 5%, and 4%, respectively. Conclusion: This method provides a sensitive and specific approach to detect issues related to well-being and safety.

Analysis of Suspected Measles Cases with Discrepant Measles-Specific IgM and rRT-PCR Test Results, Japan.

We investigated clinically suspected measles cases that had discrepant real-time reverse transcription PCR (rRT-PCR) and measles-specific IgM test results to determine diagnoses. We performed rRT-PCR and measles-specific IgM testing on samples from 541 suspected measles cases. Of the 24 IgM-positive and rRT-PCR--negative cases, 20 were among children who received a measles-containing vaccine within the previous 6 months; most had low IgG relative avidity indexes (RAIs). The other 4 cases were among adults who had an unknown previous measles history, unknown vaccination status, and high RAIs. We detected viral nucleic acid for viruses other than measles in 15 (62.5%) of the 24 cases with discrepant rRT-PCR and IgM test results. Measles vaccination, measles history, and contact history should be considered in suspected measles cases with discrepant rRT-PCR and IgM test results. If in doubt, measles IgG avidity and PCR testing for other febrile exanthematous viruses can help confirm or refute the diagnosis.

High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh.

TPO (Thyroid Peroxidase) is known to be one of the major genes involved in congenital hypothyroid patients with thyroid dyshormonogenesis. The present study aims to validate high-resolution melting (HRM) curve analysis as a substitute method for Sanger sequencing, focusing on the frequently observed non-synonymous mutations c.1117G>T, c.1193G>C, and c.2173A>C in the TPO gene in patients from Bangladesh. We enrolled 36 confirmed cases of congenital hypothyroid patients with dyshormonogenesis to establish the HRM method. Blood specimens were collected, and DNA was extracted followed by PCR and Sanger sequencing. Among the 36 specimens, 20 were pre-sequenced, and variants were characterized through Sanger sequencing. Following pre-sequencing, the 20 pre-sequenced specimens underwent real-time PCR-HRM curve analysis to determine the proper HRM condition for separating the three variations from the wild-type state into heterozygous and homozygous states. Furthermore, 16 unknown specimens were subjected to HRM analysis to validate the method. This method demonstrated a sensitivity and specificity of 100 percent in accurately discerning wild-type alleles from both homozygous and heterozygous states of c.1117G>T (23/36; 63.8%), c.1193G>C (30/36; 83.3%), and c.2173A>C (23/36; 63.8%) variants frequently encountered among 36 Bangladeshi patients. The HRM data was found to be similar to the sequencing result, thus confirming the validity of the HRM approach for TPO gene variant detection. In conclusion, HRM-based molecular technique targeting variants c.1117G>T, c.1193G>C, and c.2173A>C could be used as a high throughput, rapid, reliable, and cost-effective screening approach for the detection of all common mutations in TPO gene in Bangladeshi patients with dyshormonogenesis.