RT-PCR

Recently, a novel approach to a highly sensitive and quantitative detection of rare earth element (REE) ions including La3+, Eu3+ and Tb3+, by the polymerase chain reaction (PCR) technique, has been reported. The detection of REE ions is based on the catalytic nature of REE ions targeting the deoxyribonucleic acid (DNA), thus monitoring of the ions can be achieved by reading the level of intact DNA by PCR. Despite of its high sensitivity (at ppb to ppt levels), the conventional PCR-based REE detection protocol requires certain length of time (1-2 hours). In the present study, we modified the PCR-based REE detection protocols by employing the high-speed PCR, and performed the automated and rapid detection of La3+ in small-sized aqueous samples within 5min.

Throughout the years, the medical technologist's job role and capacities are ever-changing, from being a medical technologist to medical laboratory scientist and now a molecular medical technologist or analyst. The study aims to review the changing role of medical technologists, in context with the emerging job demands and with the uprise of a novel virus, the COVID-19, and the development of new and improved RT-PCR testing, and the increasing number of COVID-19 cases, the role of the medical technologist in this time of pandemic cannot be overemphasized.

BACKGROUND: Avian Metapneumovirus (AMPV) causes mild to acute contagious infection of the upper respiratory tract in turkey and chicken with different mortality rate. OBJECTIVES: This study was aimed to molecular detection and subtyping of AMPV infection in broiler flocks using RT-PCR method in Semnan province on samples from 2016 to2020. METHODS: Sampling was carried out from the upper part of the trachea, choana, and sinuses of broiler chickens from the 85 broiler flocks. All flocks were more than 3 weeks of age. In total 10 swabs were taken from each flock, while each 5 were pooled as one sample (total two samples per flock). The samples were transferred to the laboratory for RNA extraction and RT-PCR amplification. RESULTS: Out of 85 tested broiler flocks, 30 (35.3%) were positive for AMPV using the Nd/Nx primer set. In addition, 28 positive samples were found to be of subtype B using the Ga/G12 primer set and 2 remaining positive samples were non-subtype B, probably A, C or D subtypes. CONCLUSIONS: Since AMPV vaccination was not performed in Semnan province, it can be concluded that some cases were infected with the natural viruses. Therefore, vaccination could be effective in controlling AMPV-induced respiratory distress.

A B S T R A C T The SA44 isolate of Rotavirus A (RVA) was identified from a neonatal Peruvian alpaca presenting with diarrhea, and the full-length genome sequence of the isolate (designated RVA/Alpaca-tc/PER/SA44/2014/ G3P[40]) was determined. Phylogenetic analyses showed that the isolate possessed the genotype constellation G3-P[40]-I8-R3-C3-M3-A9-N3-T3-E3-H6, which differs considerably from those of RVA strains isolated from other species of the order Artiodactyla. Overall, the genetic constellation of the SA44 strain was quite similar to those of RVA strains isolated from a bat in Asia (MSLH14 and MYAS33). Nonetheless, phylogenetic analyses of each genome segment identified a distinct combination of genes. Several sequences were closely related to corresponding gene sequences in RVA strains from other species, including human (VP1, VP2, NSP1, and NSP2), simian (VP3 and NSP5), bat (VP6 and NSP4), and equine (NSP3). The VP7 gene sequence was closely related to RVA strains from a Peruvian alpaca (K'ayra/ 3368-10; 99.0% nucleotide and 99.7% amino acid identity) and from humans (RCH272; 95% nucleotide and 99.0% amino acid identity). The nucleotide sequence of the VP4 gene was distantly related to other VP4 sequences and was designated as the reference strain for the new P[40] genotype. This unique genetic makeup suggests that the SA44 strain emerged from multiple reassortment events between bat-, equine-, and human-like RVA strains.

Inflammatory episodes observed in multiple sclerosis are characterized by perivascular infiltration of central nervous system (CNS) tissue by circulating leucocytes directed by chemokines and activation of both microglia and astrocytes. An in vitro approach, using a three-dimensional aggregate culture system derived from foetal rat brain tissue, is being taken to study the effect of inflammatory mediators on CNS cells. These aggregates model the brain in vivo in terms of glial and neuronal development and cell-cell interactions. Aggregates cultures were treated with lipopolysaccharide (LPS) and the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFNγ). The effect of these treatments on the expression of a range of chemokine receptors (CCR1, CCR2, CCR5, CXCR3 and CX3CR1) and other markers of cell activation (MHC class I and II, CR3) was determined at the mRNA level by semiquantitative RT-PCR and at the protein level by confocal microscopy. Rat peritoneal macrophages were loaded with a fluorescent cell-tracer and their ability to infiltrate inflamed aggregates treated with IFNγ was analysed by FACS and confocal microscopy.

The sudden spike in the number of patients with COVID-19, a new respiratory virus, has put an unprecedented load on healthcare
systems across the world. In many countries, healthcare systems have already been overwhelmed. As there are limited kits available,
for diagnosis, along with limited hospital beds for admission of such patients, and limited personal protective equipment (PPE) for
healthcare personnel, it is thus very important to differentiate which patients with severe acute respiratory illness (SARI) could have
COVID-19 infection to efficiently utilize the limited resources. In this work, we propose the use of chest X-Ray to detect COVID19 infection in patients exhibiting symptoms of SARI. Our tool can classify a given X-Ray as one among the three classes: normal,
pneumonia, and COVID pneumonia

Gastric cancer is a major cause of cancer death worldwide, especially in developing countries. The incidence of gastric cancer varies from country to country, probably as a result of genetic, epigenetic, and environmental factors. H. pylori infection is considered as a major risk factor in the development of gastric cancer. However, the scenario varies in Asian countries, exhibiting a higher rate of H. pylori infection and low incidence of gastric cancer, which could be attributed to strain-specific virulence factors and host genetic makeup. In this review, we discuss the various virulence factors expressed by this bacterium and their interaction with the host factors, to influence pathogenesis.

A infecção pelo vírus da diarreia viral bovina (BVDV) foi avaliada em um rebanho bovino leiteiro de alta produção com histórico de problemas reprodutivos e de vacinação regular contra o BVDV. A identificação do vírus foi realizada por RT-PCR em soro sanguíneo e o perfil sorológico por vírus-neutralização. Inicialmente, 100% (n=692) dos animais do rebanho foram avaliados com relação à presença de infecção ativa pelo BVDV por meio da RT-PCR. Quatro meses após, todos os animais positivos (n=29) na primeira avaliação foram avaliados novamente pela RT-PCR, assim como todos os animais que nasceram (n=72) e os que apresentaram problemas reprodutivos (n=36) no intervalo entre a primeira e a segunda colheita de sangue. Os resultados finais do estudo possibilitaram identificar 27 animais transitoriamente infectados e três animais persistentemente infectados (PI). A sorologia, realizada apenas nos animais positivos na primeira avaliação pela RT-PCR e nas vacas que apresentaram problemas reprodutivos entre a primeira e a segunda RT-PCR, demonstrou grande flutuação nos títulos de anticorpos neutralizantes, além de soroconversão na maioria dos animais. Foram identificados aumentos nos títulos de anticorpos neutralizantes que variaram entre 3 e 8 log2, indicando infecção ativa no rebanho. A circulação viral no rebanho avaliado foi responsável pela expressão de sinais clínicos da esfera reprodutiva em animais com baixo título de anticorpos e consequente falha na proteção fetal. Os resultados demonstram que o controle da infecção pelo BVDV apenas por meio da vacinação regular em rebanhos com animais PI pode não ser eficaz na profilaxia dessa virose.

Population-scale and rapid testing for SARS-CoV-2 continues to be a priority for several parts of the world. We revisit the in vitro technology platforms for COVID-19 testing and diagnostics—molecular tests and rapid antigen tests, serology or antibody tests, and tests for the management of COVID-19 patients. Within each category of tests, we review the commercialized testing platforms, their analyzing systems, specimen collection protocols, testing methodologies, supply chain logistics, and related attributes. Our discussion is essentially focused on test products that have been granted emergency use authorization by the FDA to detect and diagnose COVID-19 infections. Different strategies for scaled-up and faster screening are covered here, such as pooled testing, screening programs, and surveillance testing. The near-term challenges lie in detecting subtle infectivity profiles, mapping the transmission dynamics of new variants, lowering the cost for testing, training a large healthcare workforce, and providing test kits for the masses. Through this review, we try to understand the feasibility of universal access to COVID-19 testing and diagnostics in the near future while being cognizant of the implicit tradeoffs during the development and distribution cycles of new testing platforms.

La leucemia es un tipo de cáncer que afecta a los leucocitos, caracterizado por un aumento exacerbado en la producción de células inmaduras provenientes de la médula ósea. Descrita por primera vez por Valpeu en 1847, adquiriendo el nombre de leucemia hasta 1845 por el patólogo alemán Rudolf Virchow llamando a esta "sangre blanca" por visualizar una "sustancia semejante al pus" en los vasos sanguíneos durante una necropsia convirtiéndose desde entonces en una línea de investigación médica prioritaria a nivel mundial debido a sus trágicas manifestaciones clínicas y alta incidencia e impacto en la niñez. La leucemia se divide en cuatro tipos principales, dependiendo la estirpe celular afectada y rapidez en el desarrollo de la enfermedad. En base a lo anterior se clasifican en: leucemia mieloide aguda, leucemia linfocítica aguda, leucemia mieloide crónica y leucemia linfocítica crónica. Las leucemias agudas se identifican por progresar muy rápidamente, tener una producción desregulada con acumulación de células inmaduras de las estirpes mieloides o linfoides provenientes de médula ósea, deteriorando la salud del individuo de forma súbita. Por otra parte, las leucemias crónicas se caracterizan por una evolución lenta donde las células proliferantes maduran más en comparación con las leucemias agudas, sintomatología inespecífica y variable afectando principalmente a adultos de edad avanzada. El diagnostico presuntivo de leucemia inicia con las manifestaciones clínicas (fatiga, disnea, artralgia y fiebre), la exploración física del paciente (hepatomegalia, esplenomegalia, palidez y hemorragias a nivel piel) junto a la determinación de una leucocitosis en el hemograma (>11,000 determinación de una leucocitosis en el hemograma (>11,000 leucocitos/mm3). Sin embargo, estos estudios no son suficientes para descartar o confirmar esta patología, ya que se requiere de una biopsia y/o aspirado de médula ósea, procedimientos altamente invasivos que pueden resultar en complicaciones. El dictamen se basa en la citopatología de células sanguíneas mutadas tanto de sangre periférica como de médula ósea que son observadas a través de frotis sanguíneo y se conjuntan con estudios de citogenética conven-cional pero solo detectan una pequeña parte de los casos debido a que se basa en análisis de metafases. Aunque hoy en día existe un gran avance en el entendimiento de las aberraciones genéticas, moleculares y genotípicas en los afectados, su comprensión es insuficiente a causa de su diagnóstico de rutina actual porque se basan en la experiencia de quien analiza obteniendo datos ambiguos aun cuando se pueden apoyar en algoritmos de diagnósticos como los presentes en la guía de práctica clínica IMSS-142-04, estos son propensos a errores en la correcta estimación diagnostico/tratamiento que pudieran ser fatales al momento de otorgar una valoración final. Dentro del diagnóstico de las leucemias agudas es necesario descubrir las alteraciones citogenéticas y moleculares para adecuar el tratamiento del paciente o en los que se les administrarán medicamentos específicos e identificar la enfermedad mínima residual y suministrar la medicación correspondiente.

Enteric viruses are excreted in large amount in the faeces of infected persons. These
pathogens can contaminate surface and ground water mainly via watewater. Enteroviruses
are responsible for many pathologies by faecal oral route. These viruses are very resistant
in environment. They are able to remain infectious after infiltration in soil. We analysed the
well water used in six public baths of Mohammedia to assess enterovirus contamination. In
this purpose water samples were concentrated by adsorption elution on cellulose acetate
membrane filter. Rna was then extracted using guanidinium thiocyanate. Primers from the
highly conserved polymerase region of enteroviruses were used to carry out Reverse
Transcription and nested PCR. More than 16% of the samples were found positive. The use
of such groundwater in public baths may lead to virus transmission to the users since
enteric virus infection have linked to recreational activities in contaminated waters.
200 Incidence of Enteroviruses in Groundwater from Public Baths of Mohammedia(

Background: Human Respiratory Syncytial virus (hRSV) is one of the major causes of viral respiratory tract infection in infants and young children. Aim of study: The aim of this study was to determine the risk factors associated with hRSV infection. Objective: This study included 100 hospitalized infants and young children with chest infection (39 female and 61 male) aged from (1) to (24) months, their mean age (6.87) months. Material and methods: Nasopharyngeal/throat swabs specimens were collected over a three-month winter period from January to April, 2017. hRSV was determined by reverse transcription polymerase chain reaction (RT-PCR). Results: The highest percentage of hRSV RNA (56.81%) was observed in the age group less than 6 months, followed by (27.27%) and (15.91%) in the age group 6-12 months and 12-24 months, respectively, which mean that (84%) of hRSV infections were under 12 months of age. Regarding the type of feeding, about (84%) of hRSV infections were positive in patients with bottle feeding which indicated that the role of breastfeeding in preventing hRSV infection and hospitalization is undisputed. The hRSV-RNA is equally distributed between patients exposed and not exposed to smoking (50%). Conclusion: This study showed that there were no statistical significant association between risk factors under study and the occurrence of hRSV infection (P˃ 0.05).

At the start of the pandemic, the Philippines had to send swab samples to the Victorian Infectious Diseases Reference Laboratory in Melbourne, Australia for COVID-19 confirmation. With the increasing number of suspected cases needing confirmatory diagnostic testing, there was a demand to rapidly expand the capacity for widescale testing. Remarkably, within 200 days from announcement of the first confirmed COVID-19 case in the Philippines in January 30, 2020, the country has been able to expand its testing capacity from one national reference laboratory, the Research Institute for Tropical Medicine (RITM), to more than 100 licensed reverse transcription-polymerase chain reaction (RT-PCR) and cartridge-based PCR laboratories across the country. Due to the shortage of a trained clinical laboratory workforce, diagnostic centers are forced to hire additional personnel who have limited experience and technical knowledge and skills of molecular assays, especially in processing specimens, interpreting the results, identifying errors, and troubleshooting, in order to meet the demand of increased testing. Thus, the vulnerability to diagnostic errors, including cross-contamination, is increased and with the tendency for generating false-positive results that can compromise the health of the patient and disrupt the efficacy of public health policies and public health response, surveillance programs, and restrictive measures for containing the outbreak. Hence, this review article aims to present the different sources of contamination in the laboratory setting where RT-PCR assays are conducted, as well as provide efficient, effective and feasible solutions to address these issues, most especially in low-and middle-income countries (LMICs) like the Philippines.

Legume plants have the ability to fix atmospheric nitrogen symbiotically with the association of Rhizobium that inhabits the root nodules. Cletoria ternatea L. is a legume, which grows naturally in dry and less fertile lands. It has the ability to fix nitrogen even under harsh environmental conditions. The present study was aimed at screening the Rhizobial strains of Cletoria ternatea L which have the ability to tolerate high salinity, high temperature, drought, and broad pH range and identifying and differentiating the best tolerant strains at molecular level using PCR amplified Enterobacterial Repetitive Intergenic Consensus (ERIC) profiling. Bacterial colonies were isolated from root nodules, which were collected from five different districts of Sri Lanka namely Jaffna (J), Trincomalee (T), Vavuniya (V), Mannar (M) and Kandy (K). Fresh nodules were collected and pure cultures were obtained by using ½ Lupin agar medium. Total of 25 pure colonies representing the five districts (5 from each district) were selected. These cultures were grown in ½ Lupin broth ranging the temperature from 25℃ to 60℃, NaCl concentrations 1-5%, pH from 2-10, and PEG from 10-30%, separately. Growth of the cultures were tested by measuring the optical density at 600 nm. From physiological characterisation, fourteen best high tolerant strains were selected for ERIC profiling. Among these 14 isolates, M3, and V3 isolates exhibited good tolerance to the temperature range from 25℃ to 60℃, NaCl concentration range from 1-5%, pH range from 2-10 and PEG concentration 10-30%. The isolates J1, T1, K1 and K3 were tolerant to the same temperature range, salinity and pH conditions. J2 and K3 exhibited high tolerance to 30% PEG concentration. Out of 14stress tolerant isolates, we were able to distinguish 5 different banding patterns using PCRamplified ERIC profiling. The high tolerant M3 and V3 were showing different polymorphic banding patterns suggesting that they are different strains. These high tolerant strains can be used for cross inoculation with commercially important legume crops to improve their yield under harsh environmental conditions and for biofertilizer production.

УБАВИ НОВОСТИ И УШТЕ ЕДНА НАГРАДА - WONDERFUL NEWS AND THANK YOU VERY MUCH! -Jury Special Section Winner/Official Selection, 5.The Gladiator Film Festival, Istanbul-Turkey, April 2022!
https://filmfreeway.com/TheGladiatorFilmFestival; https://communityofcinema.com/gladiator-film-festival

Along with an increasing population and global demand, the need for meat and meat products is gradually rising. In recent years, consumer preference is for safe and halal compliant food produce. Accordingly, this study investigated and compared the sensitivity of DNA-based real-time polymerase chain reaction (RT-PCR) and protein-based enzyme-linked immunosorbent analysis (ELISA) methods through the detection of 33 beef and 30 pork products. The identification success of both ELISA and real-time PCR methods was determined to be 100% (33/33) in the beef analysis. However, although real-time PCR yielded 100% positive results in pork analyses, only 63.7% (30/19) positive results were observed using ELISA. While all of the real-time PCR and ELISA results for the 33 Beef (salami, sausage and fermented sausage) samples were positive and the real-time PCR results for 30 pork samples were positive for all samples, ELISA was negative for 3 pork sausages, 5 pork salami and 3 pork ham samples. On the other hand, when both systems were evaluated in terms of time, cost and ease of use, it was seen that ELISA analyses were less time-consuming, easier and more economical, but real-time PCR testing was more sensitive. As a result, there is a need for methods that are both highly sensitive, inexpensive and easily applicable.

Prevalence of honeybee viral diseases has recently been causing major problems in the beekeeping industry, causing economic losses worldwide. Honeybees are sus‐ ceptible to a variety of diseases and various pathogens. Among these pathogens, prevalence viruses, along with other factors, are seriously threatening the health of bee species. In the present study, samples were collected from 80 Apis cerana cerana (A. c. cerana) colonies from three different locations, Cangshan, Fuan, and Yongtai, in the Fujian Province of China. All samples were screened using the reverse transcrip‐ tion polymerase chain reaction (RT‐PCR) method for detection of seven honeybee viruses, namely, Chinese sacbrood virus (CSBV), deformed wing virus (DWV), Isreal acute paralysis virus (IAPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), acute bee paralysis virus (ABPV), and Kashmir bee virus (KBV). Our results showed that CSBV was the most prevalent as it was detected in (90%), of the sam‐ ples, DWV was detected in (81.25%), and IAPV was detected in (26.25%). In contrast, insignificant prevalence results were obtained from all apiaries for BQCV, CBPV, APBV, and KBV, which were not detected in any sample. Here, we are providing the first report on the molecular detection of honeybee viruses, especially the preva‐ lence of IAPV, from different regions in the Fujian Province of China with a high prevalence of bee viruses, on A. c. cerana, and there is great concern for the presence of honeybee viruses in the population of the native honeybee (A. c. cerana) in China.

An immunocytochemical approach to the identification of neuroendocrine cells in the thymus of the chicken was taken based on a novel monoclonal antibody against turkey chromogranin A (CgA), a classic marker protein for neuroendocrine cells. CgA-immunoreactive cells were readily observed in the thymus, and were typically confined to the medullary side of the corticomedullary junction of the thymic lobules. Reversed transcription PCR confirmed local production of CgA in the thymus. The majority of CgA+ cells were small and round or oval in shape but some cells were larger and had conspicuous extensions. Immunofluorescent double staining experiments with antibodies against Neuron-specific enolase and with a neural crest marker (HNK-1) indicated no demonstrable overlap between the CgA-positive cells and either of the above cell populations, demonstrating the existence of three distinct neuronal/neuroendocrine cell populations in the avian thymus.