Performed Polymerase Chain Reaction Research Articles

Peculiarities of Systemic Hemodynamics and Autonomic Status in Medical Personnel of PCR-Laboratories with Different Emotional Intelligence

INTRODUCTION: The ongoing pandemic of a new coronavirus infection COVID-19 (COronaVIrus Disease 2019) places high demands on the activity of PCR-laboratories (laboratories performing polymerase chain reaction). The medical personnel experience overloads due to stay in the red zone wearing the individual protection means, and also in association with a high personal responsibility for the result of their work. One of the criteria that is proposed for professional selection with the aim of increasing the effectiveness of the work of personnel of PCR-laboratories, is the emotional quotient (EQ).
 AIM: To analyze the state of systemic hemodynamics and heart rate variability in medical workers of PCR-laboratories with different emotional intelligence working in condition of COVID-19 pandemic using means of individual protection against the biological factor.
 MATERIALS AND METHODS: The extent of health risk and tension of adaptation mechanisms during the work were evaluated by parameters of systemic hemodynamics (tonometry, pulse oximetry) and heart rate variability (cardiorhythmography). Thirty three individuals with the working period in a PCR-laboratory from 1 to 33 months were examined. The average age of the participants at the time of inclusion in the study was 32.4 1.44 years. In 33.7%, EQ corresponded to the medium level (81120 points), and in 66.7% to the high level (121168 points).
 RESULTS: Before the start of the work shift, increased blood supply to tissues in personnel with high EQ was achieved by increased heart activity: cardiac output (CO) 3938.9 ml/min, interquartile range (IQR) = 3404.24230.2 ml/min with the underlying reduction of vessel tone: total peripheral vascular resistance (TPVR) 1922.8 dyn s/cm5, IQR = 1636.52085.8 dyn s/cm5. After the end of the shift, to these compensatory mechanisms (CO 4284.7 ml/min, IQR = 3935.44688.8 ml/min; TPVR 1692.4 dyn s/cm5, IQR = 1584.61903.1 dyn s/cm5), increased myocardial oxygen consumption (MOC; 7917.5 RU, IQR = 7430.28811.0 RU) added as a reaction to the muscular component of labor activity. In individuals with average EQ, these mechanisms were less pronounced in considerable mental stress: before the shift CO 3722.0 ml/min, IQR = 3163.13739.0 ml/min, p = 0.013; TPVR 2303.1 dyn s/cm5, IQR = 2058.12402.1 dyn s/cm5, p = 0.016; after the shift CO 3375.0 ml/min, IQR = 3372.04402.0 ml/min, p = 0.010; TPVR 2010.5 dyn s/cm5, IQR = 1972.62351.9 dyn s/cm5, p = 0.017; MOC 7450.0 RU, IQR = 6653.37867.3 RU, p = 0.023.
 CONCLUSION: The extent of tension of mechanisms of self-regulation of systemic circulation, apart from characteristics of used technical means and the working regime, significantly depends on the level of emotional intelligence. With this, personnel with high emotional intellect are preferable for the work in the red zone because of lower tension of regulatory mechanisms.

Amdoparvovirus-associated disease in red pandas (Ailurus fulgens).

The roster of amdoparvoviruses (APVs) in small carnivores is growing rapidly, but in most cases, the consequences of infection are poorly understood. Red panda amdoparvovirus (RPAV) is highly prevalent in zoo-housed red pandas and has been detected in both healthy and sick animals. Clarifying the clinical impact of RPAV in this endangered species is critical, and zoological collections offer a unique opportunity to examine viral disease association in carefully managed populations. We evaluated the potential impact of RPAV in captive red pandas with a combination of prospective and retrospective analyses. First, we collected feces from 2 healthy animals from one collection over a 6-year period and detected virus in 72/75 total samples, suggesting that RPAV can be a long-term subclinical infection. We next investigated the infections using a retrospective study of infection status and tissue distribution in a cohort of necropsied animals. We performed polymerase chain reaction and in situ hybridization on 43 necropsy cases from 4 zoo collections (3 from the United States, 1 from Europe, 1997-2022). RPAV was present in these populations for at least 2 decades before its discovery and is detectable in common and significant lesions of zoo-housed red pandas, including myocarditis (3/3 cases), nephritis (9/10), and interstitial pneumonia (2/4). RPAV is also detectable in sporadic lesions, including multisystemic pyogranulomatous inflammation, oral/pharyngeal mucosal inflammation, and dermatitis. The colocalization of virus with lesions supports a role in causation, suggesting that despite the apparently persistent and subclinical carriage of most infections, RPAV may have a significant impact in zoo collections.

Pathogenic bacteria associated with outbreaks of respiratory disease in Iranian broiler farms.

Multi-causal respiratory infections are more commonly observed than uncomplicated cases with single agents in the commercial poultry industry. Recently, increased mortality rates associated with respiratory clinical signs have been reported in Iranian broiler farms. The present study aimed to determine the spectra of avian mycoplasmas (Mycoplasma gallisepticum, MG and Mycoplasma synoviae, MS) and Ornithobacterium rhinotracheale (ORT) in the broiler farms with the multi-causal respiratory disease (MCRD) from 2017 to 2020. Trachea and lung tissue samples were collected from 70 broiler flocks presenting increased mortality and acute respiratory disease. MG, MS, and ORT were detected by performing polymerase chain reaction with primers complementary to the 16S rRNA, vlhA, and 16S rRNA genes, respectively. Genetic materials of MG, MS, and ORT were detected in five, three, and five of the 70 flocks. Based on the phylogenetic analysis of the complete mgc2 coding sequences, all MG strains formed a distinct cluster along with other Iranian MG isolates. According to the phylogenetic analysis of the partial vlhA gene of MS strains, two isolates were located along with Australian and European strains. In addition, one of them displayed an out-group association with MS isolates from Jordan. Phylogenetic analysis of Iranian ORT strains using a partial sequence of the 16S rRNA gene showed a distinct group among the other ORT strains. The results indicate that MG, MS, and ORT are not predominantly responsible for the MCRD. However, continuous monitoring of poultry flocks could be significant for obtaining valuable information related to different MG, MS, and ORT strains and designing effective control strategies.

Toll-like Receptor 3 c.1377C/T and -7C/A Polymorphisms Associated with COVID-19 and COVID-19 Severity

Chinese officials have reported the novel coronavirus to the world health organization, which is called the SARS-CoV-2. Toll-like receptor 3 (TLR3) induces antiviral immune responses via the production of type I interferons and inflammatory cytokines. In this study, we aimed to examine TLR3 c.1377C/T and -7C/A polymorphisms in COVID-19 and the association between some clinical parameters. We investigated the frequencies of TLR3 (c.1377C/T and -7C/A) polymorphisms in 150 patients with COVID-19 and 171 healthy individuals as controls. We performed polymerase chain reaction (PCR) based on restriction fragment length polymorphism (RFLP). We also investigated whether TLR3 c.1377C/T and-7C/A were associated with the severity of COVID-19. In addition, CHAID tree-based classification algorithm was created to investigate the severity of the patients in our study. TLR3 c.1377C/T TT genotype frequencies were statistically significant between cases and controls (p= 0.02). For TLR3 -7C/A polymorphism, the findings showed a statistically significant difference in A allele frequencies (p= 0.03). There was a statistically significant difference in the distribution of TLR3 -7C/A CA genotype frequency (p= 0.04). Our findings suggest that TLR3 c.1377C/T and -7C/A polymorphisms may be important on susceptibility or clinical course of COVID-19.

Smartphone-operated affordable PCR thermal cycler for the detection of antimicrobial resistant bacterial genes.

Antimicrobial resistance (AMR) is a global public health threat. Surveillance of AMR requires affordable, rapid, and user-friendly diagnostic methods. Our aim was to develop a low-cost thermocycler to perform polymerase chain reaction (PCR). We developed a smartphone-operated PCR thermal cycler using locally available recycled materials. The thermal cycler was used for the amplification for three bacterial genes-bla-TEM, bla-CTXM and 16s rRNA in human urine samples. The performance of custom-built thermal cycler was compared with commercial thermal cycler. The thermal cycler was portable (<1kg weight), required 12 V power supply, 25 μL of solution, and cost only USD50.0. Temperature and time conditions were instructed using a custom-built smartphone application. The ramping rate of was 0.23°C for heating and 0.43°C for cooling. The reported temperatures were within ± 0.5°C of set temperature. The human urine samples were highly resistance and multi-resistant. Nearly 46% (n = 54) E. coli isolates were positive in ESBL screening test. The custom-built thermocycler was able to accurately predict the presence of bla-TEM, bla-CTXM genes, and 16s rRNA (n = 6). We developed and demonstrated a portable, low-cost, easy-to-use, and smartphone-operated PCR thermal cycler. Since it is portable, it can be used in remote location and field settings, including places without stable power supply. The use of the thermal cycler system can be extended, beyond the detection of AMR genes, e.g., in clinical diagnosis, genetics, forensic analysis, and environmental protection.

Genetic association between Q192R polymorphism in the paraoxonase 1 gene and female infertility in the Saudi women: Validated using DNA sequencing analysis

ObjectiveInfertility may also describe the inability to conceive whether a woman continues to have miscarriages. Female infertility is a complex condition which can suffer from a range of conditions and one-third of all infertility cases occur in women. Obesity is correlated by various complex pathways with infertility among women. Paraoxonase (PON) is an antioxidant and oxidant enzyme that plays an important role in a variety of conditions, including inflammation, oxidative stress, and lipid metabolism. Paraoxonase1 (PON1) gene variants can influence oxidative stress and thus female infertility susceptibility. However, no molecular studies have been conducted in Saudi infertile women and the Q192R polymorphism. The purpose of this study was to investigate into the relationship between the Q192R polymorphism in the PON1 gene and the risk of infertility in Saudi women. Study designThis case-control study was carried out with 122 female infertile women and 100 fertile women. Genomic DNA was extracted from 222 participants, quantified performed polymerase chain reaction, agarose gel run, and RFLP analysis. Finally, Sanger sequencing was validated in 10% of the samples obtained. Excel was used to collect clinical and genomic data, which was then analyzed statistically. ResultsAnthropmetric and clinical data was assessed using both the cases and control women (p < 0.05). Hardy Weinberg equilibrium was in accordance within the Q192R polymorphism (p = 0.001). Q192R polymorphism confirms the allele (p = 0.003) and genetic association (QR vs QQ; p = 0.001) with different mode of inheritances including dominant and co-dominant models (p = 0.003). ANOVA analysis revealed a positive association between weight and Q192R polymorphism (p = 0.04). ConclusionThis study concludes as Q192R polymorphism was positively associated in this case-control study performed in the Saudi women diagnosed with female infertility. However, obesity factor might also be playing a role in the infertile women.

BRAF V600E and Novel Somatic Mutations in Thyroid Cancer of Libyan Patients

Thyroid cancer (TC) is a common endocrine malignancy that frequently harbours the oncogenic V600E BRAF mutation. This mutation has received considerable attention in recent years for its potential utility in the risk stratification and management of TC. This study aims to investigate BRAF mutational status in thyroid cancer of Libyan patients and their association with clinicopathological factors. 44 thyroid tissue samples were analysed for mutations in exon 15 of the BRAF gene by performing polymerase chain reaction and Sanger sequencing. The results of BRAF mutation screening were correlated to clinical and pathological characteristics of the studied thyroid cancer patients. Statistical analyses were performed using SPSS. The BRAF exon 15 mutations were detected in 19 (43.2%) of the thyroid cancer cases. The V600E was the most frequent one found in 15/44 (34.1%) cases. We also detected 6 other variants in 7 patients (15.9%), the S616F, the W619R and the T599S. Three mutations were associated with V600E, the L584I, the D587Y and the synonymous L597L. None of these mutations were reported previously in thyroid cancers. No statistical association was found between BRAF mutations and clinicopathological factors except with papillary thyroid cancer type (p= 0,032). Novel BRAF mutations and V600E were frequently detected in thyroid cancer of Libyan patients; this suggests a potential role of these novel mutations in carcinogenesis and in anti-EGFR therapy resistance.

Role of the Q36R polymorphism in the KISS1 gene in female infertility

BackgroundInfertility has evolved into a worldwide reproductive health concern that is distinctively constituted within various socio-cultural contexts. Female infertility is a global health concern ailment in couples who have been unable to conceive for a year. Kisspeptin (KISS1) is a peptide hormone that is important in women's health difficulties such as infertility. Based on previous studies, single nucleotide polymorphisms (SNPs) in KISS1 gene can be a cause for infertility. The objective of this study is to explore into the Q36R SNP in the KISS1 gene in Saudi infertile women. MethodsIn this case-control study, 96 cases of female infertile and 96 control women was selected. Deoxyribonucleic acid (DNA) was extracted to perform polymerase chain reaction and followed by restriction fragment length polymorphism analysis. All DNA products were run on 2% agarose and obtained data was imported into excel to perform the statistical analysis using SPSS software between cases and controls. ResultsThe anthropometric analysis (p > 0.05), genotype (OR-3.60; 95 %CI:0.96–13.55; p = 0.04), allele (OR-2.49; 95 %CI: 0.86–7.21; p = 0.08) and different genetic models (AG + GG vs AA; OR-1.00; 95 %CI: 0.01–7.02; p = 0.005) showed negative association and fertility histories showed significant association (p < 0.0001). Anova analysis showed the positive association with weight (p = 0.01) and BMI (p = 0.003). ConclusionThis study found no connection between the Q36R SNP in the KISS1 gene and female infertility in Saudi Arabia.

Evaluating the relationship between Ex3 vNTR and rs3758653 polymorphisms in DRD4 genes in children with attention deficit hyperactivity disorder with respect to the dose of methylphenidate

Background. Attention deficit hyperactivity disorder (ADHD) is one of the most common psychiatric disorders starting at an early age with symptoms of attention deficit hyperactivity disorder and impulsivity. This study aimed to investigate the molecular alterations of the DRD4 gene, which have been linked to ADHD in previous studies with respect to the dose of methylphenidate. Methods. The study population included all ADHD patients needing drug therapy aged 5-18 years referred to Bozorgmehr psychiatric clinic in Tabriz in 2017 for one year. After diagnosis, symptoms severity was assessed using the Conners Scale for ADHD Assessment. Then, 4 ml of blood was collected from each individual and DNA was extracted using salting out method. After performing polymerase chain reaction (PCR), the products were separated using specific restriction enzymes and then polymorphisms of Ex3 VNTR and rs3758653 of DRD4 gene were analyzed in 2% agarose gel. The last dose to achieve the therapeutic response was recorded in mg/kg. Then, we investigated the relationship between the doses of drug used to achieve the therapeutic response and the existence of the mentioned polymorphisms. Results. There was no significant relationship between treatment response and type of polymorphism. Also, there was no significant relationship between the average dose of drug and the polymorphisms of three replicates, four replicates, six replicates, and more than six replicates in EX3VNTR in DRD4 gene. In addition, we witnessed no statistically significant relationship between the mean drug dose and CC, TC, and TT genotypes in DRD4rs3758653. Conclusion. There was no significant relationship between treatment response and type of polymorphism. There was no significant relationship between the average dose of drug and the polymorphisms of three replicate, four replicates, six replicates and more than six replicates in EX3VNTR in DRD4 gene. Between mean drug dose and CC, TC and TT genotypes in DRD4rs3758653, there was no statistically significant relationship. Practical Implications. Determining the reduction of ADHD symptoms by taking methylphenidate and evaluating the relationship between presence or absence of DRD4 polymorphisms and the dosage of drug to achieve a better response to treatment can collect enough evidence to determine the dose of methylphenidate according to the molecular analysis of DRD4 gene in patients with ADHD. This can reduce doctor visits, save time, and decrease the drug side effects.

Intuitive Remote Robotic Nasal Sampling by Orientation Control With Variable RCM in Limited Space

When performing polymerase chain reaction testing, collecting nasopharyngeal swabs from patients is necessary. Hence, the development of a noncontact specimen collection system is desired to avoid the risk of secondary infection from direct contact between the medical personnel and patients. This study aims to develop a master-follower-type remote specimen collection system to achieve infection protection through adequate isolation. A teleoperated robot is used for specimen collection work instead of a human; the teleoperated robot moves remotely, and a cotton swab is grasped and inserted into the nostril by the robot. A remote center of motion (RCM) mechanism was implemented to prevent the cotton swabs from making excessive contact with the nostril during specimen collection. Further, a variable RCM constraint that combines safety and operability is proposed as the conventional RCM control modification. The evaluation results indicated that the variable RCM restraint allowed a quicker and safer collection of specimens than the conventional RCM constraint.

Authentication of Rusa timorensis DNA Using Sequencing Assay with Modified Extraction Method

A technique to isolate DNA from deer meat was established to authenticate the deer species and was successfully extracted from deer meat. The method was based on the Epicentre MasterPureTM Complete DNA and RNA Purification Kit extraction kit with slight modification by increasing the volume of Proteinase K and RNase from 5 µL to 20 µL each. The recovery of deer DNA was then analysed by Polymerase Chain Reaction using cytochrome b gene oligonucleotide primers for the deer family, which targeted the mitochondrial DNA. The increasing volume of Proteinase K and RNase resulted in clean DNA and no smearing after performing Polymerase Chain Reaction. Furthermore, the amplified DNA product was extended to sequence assay to authenticate deer species above 95% maximum identification. The result showed that the increasing volume of Proteinase K and RNase offered a high yield of DNA recovery, which provided more than 100 ng per μL and was useful in validating animal-based sources, especially in deer products.

Data driven health monitoring of Peltier modules using machine-learning-methods.

Thermal cyclers are used to perform polymerase chain reaction runs (PCR runs) and Peltier modules are the key components in these instruments. The demand for thermal cyclers has strongly increased during the COVID-19 pandemic due to the fact that they are important tools used in the research, identification, and diagnosis of the virus. Even though Peltier modules are quite durable, their failure poses a serious threat to the integrity of the instrument, which can lead to plant shutdowns and sample loss. Therefore, it is highly desirable to be able to predict the state of health of Peltier modules and thus reduce downtime. In this paper methods from three sub-categories of supervised machine learning, namely classical methods, ensemble methods and convolutional neural networks, were compared with respect to their ability to detect the state of health of Peltier modules integrated in thermal cyclers. Device-specific data from on-deck thermal cyclers (ODTC®) supplied by INHECO Industrial Heating & Cooling GmbH (Fig 1), Martinsried, Germany were used as a database for training the models. The purpose of this study was to investigate methods for data-driven condition monitoring with the aim of integrating predictive analytics into future product platforms. The results show that information about the state of health can be extracted from operational data - most importantly current readings - and that convolutional neural networks were the best at producing a generalized model for fault classification.

Tracking the turnover of SARS-CoV-2 VOCs Gamma to Delta in a Brazilian state (Minas Gerais) with a high-vaccination status.

The emergence and global dissemination of Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2) variants of concern (VOCs) have been described as the main factor driving the Coronavirus Disease 2019 pandemic. In Brazil, the Gamma variant dominated the epidemiological scenario during the first period of 2021. Many Brazilian regions detected the Delta variant after its first description and documented its spread. To monitor the introduction and spread of VOC Delta, we performed Polymerase Chain Reaction (PCR) genotyping and genome sequencing in ten regional sentinel units from June to October 2021 in the State of Minas Gerais (MG). We documented the introduction and spread of Delta, comprising 70 per cent of the cases 8 weeks later. Comparing the viral loads of the Gamma and Delta dominance periods, we provide additional evidence that the latter is more transmissible. The spread and dominance of Delta did not culminate in the increase in cases and deaths, suggesting that the vaccination may have restrained the epidemic growth. Analysis of 224 novel Delta genomes revealed that Rio de Janeiro state was the primary source for disseminating this variant in the state of MG. We present the establishment of Delta, providing evidence of its enhanced transmissibility and showing that this variant shift did not aggravate the epidemiological scenario in a high immunity setting.

COVID-19 infection control protocol and acceptance in an ART in vitro fertilization hospital

PurposeIn Tokyo, where the highest coronavirus disease 2019 (COVID-19) infection rates have been reported nationally, we introduced and performed polymerase chain reaction (PCR) testing on the patients prior to them coming for oocyte retrieval (OR) or embryo transfer (ET) procedures. In addition, we recommended that patients self-inject ovarian stimulation drugs to reduce the number of hospital visits required. We aimed to assess the patient acceptance of these measures and the change of treatment number.MethodsWe conducted a retrospective study examining the patients coming for OR or ET, from the first time a state of emergency was declared in Japan, May 2020, until September 2021.ResultsA total of 79 out of 94 (94%) patients complied with the measures. This may reflect that PCR universal screening was accepted by most patients as necessary for reducing infection spread. In addition, the number of patients receiving OR and ET increased. The widespread adoption of work-from-home practices during the pandemic has made outpatient visits more acceptable to the general public.ConclusionsUniversal screening and self-injection are accepted and effective infection measures in patients presenting for OR and ET.

IL-36 is Closely Related to Neutrophilic Inflammation in Chronic Obstructive Pulmonary Disease.

BackgroundInterleukin (IL)-36α, IL-36β, and IL-36γ belong to the IL-36 family and play an important role in the pathogenesis of many diseases. Chronic obstructive pulmonary disease (COPD) may be correlated with IL-36; however, the specific role of IL-36 in COPD is unclear. In this study, we aimed to clarify whether IL-36 could be an indicator for determining COPD severity and the specific nature of the pro-inflammatory effects of IL-36 in COPD.MethodsA total of 70 patients with COPD and 20 control subjects were included in this study. We collected peripheral blood samples from both the groups, analyzed the blood cell fractions by routine blood examination, and measured the serum levels of IL-36α, IL-36β, and IL-36γ by performing polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, the correlation between the number of neutrophils and eosinophils and the level of IL-36 was also analyzed.ResultsWe found that level of IL-36 in patients with COPD was positively correlated with the number of neutrophils but not with eosinophils, whereas the correlation was not found in the control group. Moreover, the level of IL-36 was negatively correlated with the level of lung function of patients with COPD, and the levels of IL-36α, IL-36β, and IL-36γ increased with advancing disease severity.ConclusionIn COPD, the pro-inflammatory effect of IL-36 is closely related to neutrophils, and hence, IL-36 might be considered a novel biomarker for determining COPD severity.

Phenotypic and genotypic detection of Enterobacter spp isolated from clinical cases

Enterobacter Spp an opportunistic bacteria that causes a variety of infections in hospitalized patients around the world, can resist -lactams primarily by generating the CTX-M enzymes and AmpC -lactamase enzyme. The spread of ESBL is a serious public health concern, and infections with ESBL-producing organisms have been linked to negative outcomes. We looked at the prevalence and screening criteria for extended-spectrum-lactamases (ESBLs) in Enterobacter spp. that produce AmpC. The 20 isolates (18 Enterobacter Cloacae Complex,2 Enterobacter Aerogenes) isolated from clinical cases . We performed polymerase chain reaction for Ampc and blaCTX-M. The Ampc was detected in 12 of 20 isolated, and the blaCTX-M was detected in 11 of 20 isolated.

Effect of the Progression of Fusobacterium nucleatum–induced Apical Periodontitis on the Gut Microbiota

IntroductionFusobacterium nucleatum, which is involved in the development of periodontal disease and apical lesions, can be transmitted to the colon and metastasize to colorectal cancer, suggesting a link between oral and systemic diseases. We analyzed the effects of F. nucleatum on bacterial flora in the gut and surrounding organs in a rat model of apical periodontitis and analyzed the infection route to the gut and distant organs. MethodsWe induced apical periodontitis in rat molars by infecting the dental pulp with F. nucleatum and then took X-ray images and performed histopathologic analyses. Next, we removed the maxilla, gut, heart, liver, and kidney from the rats at 0, 2, 4, and 8 weeks postsurgery and then extracted DNA samples and performed polymerase chain reaction and microbiome analyses using the Illumina MiSeq (Illumina Co, Tokyo, Japan). ResultsThe presence of inflammatory cell infiltration confirmed apical periodontitis from 2–8 weeks. Polymerase chain reaction and microbiome analyses revealed F. nucleatum in the rat gut from 2 weeks and in the kidney from 8 weeks. The rat gut, heart, liver, and kidney exhibited altered bacterial flora, including a marked decrease in Verrucomicrobia and an increase in Proteobacteria after 2 weeks and increases in Bacteroidetes and Firmicutes after 4 weeks. ConclusionsThe onset of F. nucleatum–induced apical periodontitis changed the bacterial flora in the rat gut, heart, liver, and kidney, with a confirmed progressing infection in the large intestines.

Troubleshooting Real‐time PCR Data Online and on Ground to Develop Critical Inquiry and Problem Solving Skills

One of the most important skills for molecular biology students upon entering the workforce is their ability to problem solve. From answering research questions to diagnosing and troubleshooting, learning to interpret data and draw pertinent conclusions is essential for scientists.In the last decade, we devised a series of laboratory experiences during which students extract RNA from their own cells, retro‐transcribe it, design expression specific primers for a house‐keeping transcript using tools freely available online, perform polymerase chain reaction (PCR), amplicon purification and quantification, serial dilutions of the amplicon to prepare real‐time‐PCR standards, run real‐time PCR experiments, and finally analyze their data. To maximize student engagement and discussion during the labs, each module is weaved though with a gaming experience awarding students points for answering questions by themselves or as part of a team.During the pandemic, the curriculum was adapted to run online as a simulation.The gamification approach significantly increased student participation, both online and on ground, and it also increased camaraderie on ground. The discussion‐focused report made a significant difference in student understanding; 85% of the students, as compared to 40% with a regular lab report, where able to draw pertinent conclusions from their dataset pinpointing errors to one or more of the following: standard preparation, addition of standards to the plate, inconsistent pipetting, or contamination.The robust laboratory modules described here have been improved for the past decade and can be adapted to study any gene of interest.

Performance improved fluorescence polarization for easy and accurate authentication of chicken adulteration

Rapid, accurate, and effective authentication of unlabeled meat components in commercial meat products is of great significance for guaranteeing food safety and human health. In this research, we have constructed a single-strand binding protein (SSB)-assisted performance improved fluorescence polarization (FP) protocol for authentication of chicken component in meat products. The SSB-assisted FP strategy involves the use of fluorescein isothiocyanate (FITC)-labeled primers to perform polymerase chain reaction (PCR). In addition, FP enhancement was accomplished by mixing the amplified product with SSB. Based on the designed mechanism, the free primers in the final amplified product can be tightly bound by SSB, which greatly restricts the rotation of FITC, thereby resulting a high FP signal. In the presence of chicken DNA, the transformation of single-stranded primers to double-stranded amplicons greatly hinders the combination of SSB and primers, leading to the decrease of FP signal. This method well couples the advantages of both PCR and FP technology. Through this new strategy, as low as 0.035% (wt. %) of chicken adulteration in meat samples can be well authenticated. Moreover, this method is also available for commercial meat products. We expect this SSB-assisted FP strategy will provide new insights into the construction of versatile analysis tools in the field of food safety detection. • Fluorescence polarization protocol for rapid sensing of chicken adulteration. • Fluorescence polarization adopted for analysis of PCR products. • Single strand binding protein adopted to improve detection limit of adulteration. • Successful authentication of commercial meat products with improved FP strategy.

Detection of Sugarcane Mosaic Diseases Using Deep Learning Architecture to Avoid Annealing Temperature of PCR Primer in Laboratory Testing

The sugarcane leaf diseases such as mosaic and streak mosaic are difficult to differentiate using Image processing techniques because both diseases show similar visual attributes such as pattern and color. To identify the type of diseases, we need to perform Polymerase Chain Reaction (PCR) testing which is used for the classification of diseases in laboratories. The accuracy of the PCR test depends on reaction mix preparation, reaction time, and DNA/RNA extraction. The major problem influencing the PCR test accuracy is the Annealing temperature of the primers and needs a standardized set of samples. In addition, it is a time-consuming process. In this paper, we proposed a Diversified Deep Learning Architecture (DDLA) which is developed with the input images after various pre-processing steps such as denoising using Discrete Wavelet Transform (DWT) and enhancing using histogram equalization in HSI color space to improve the similar pattern disease prediction accuracy. The performance of the proposed model is analyzed for a set of diseased leaves and the results are compared with the output of the popular pertained models such asVGG16, InceptionV3, ResNET50, Inception ResNET, and DenseNET201 with and without pre-processing. The training accuracy of the proposed model is 97% and the testing accuracy is 87%. The DDLA model produces ground truth test results with an accuracy of 88.7% for mosaic and 85.7% for streak mosaic with a less computational time of 152sec compared to the lab test duration of 6 hrs. The performance of the model is also measured in terms of Precision, F1 Score, Specificity, and Sensitivity. The Proposed DDLA model’s F1 score is higher than the pre-trained models with a minimum test loss of 1.167. Moreover, the DDLA structure occupies less memory space when compared to the pertained models.