Combination Of Polymerase Chain Reaction Research Articles

Mesenteric fibrosis in patients with small intestinal neuroendocrine tumors is associated with enrichment of alpha-smooth muscle actin-positive fibrosis and COMP-expressing stromal cells.

Neuroendocrine tumors of the small intestine (SI-NETs) often develop lymph node metastasis (LNM)-induced mesenteric fibrosis (MF). MF can cause intestinal obstruction as well as ischemia and render surgical resection technically challenging. The underlying pathomechanisms of MF are still not well understood. We examined mesenteric LNM and the surrounding stroma compartment from 24 SI-NET patients, including 11 with in situ presentation of strong MF (MF+) and 13 without MF (MF-). Differential gene expression was assessed with the HTG EdgeSeq Oncology Biomarker Panel comparing MF+ with MF- within LNM and paired stromal samples, respectively. Most interesting differentially expressed genes were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in combination with validation of associated protein levels utilizing immunohistochemistry (IHC) staining of MF+ and MF- formalin-fixed, paraffin-embedded (FFPE) patient samples. Overall, 14 genes measured with a 2549-gene expression panel were differentially expressed in MF+ patients compared to MF-. Of those, nine were differentially expressed genes in LNM and five genes in the stromal tissue (>2-fold change, p < .05). The top hits included increased COMP and COL11A1 expression in the stroma of MF+ patients compared to MF-, as well as decreased HMGA2, COL6A6, and SLC22A3 expression in LNM of MF+ patients compared to LNM of MF- patients. RT-qPCR confirmed high levels of COMP and COL11A1 in stroma samples of MF+ compared to MF- patients. IHC staining confirmed the enrichment of α-smooth muscle actin-positive fibrosis in MF+ compared to MF- patients with corresponding increase of COMP-expressing stromal cells in MF+. Since COMP is associated with the known driver for fibrosis development transforming growth factor beta and with a cancer-associated fibroblasts enriched environment, it seems to be a promising new target for MF research.

Ultrasensitive electrochemiluminescence biosensor based on polymethylene blue nanoparticles and DNA network for Staphylococcus aureus detection

A novel bipolar electrode (BPE)-electrochemiluminescence (ECL) device was constructed for the ultra-sensitive detection of Staphylococcus aureus (S. aureus) by combining polymerase chain reaction (PCR) amplification and DNA network-loaded polymethylene blue nanoparticles (pMB NPs). The presence of target triggered the dissociation of double-stranded DNA on Fe3O4 NPs and the release of T strand, which initiated the PCR. The PCR product contains two protruding single-stranded DNA fragments that serve as bridges to connect Au NPs labeled probes. The PCR-Au products were captured by the probes on cathode of BPE to form three-dimensional DNA networks, which offer multiple adsorption sites for pMB NPs, leading to the remarkable enhancement of ECL intensity. Under optimal circumstances, a wide linear range from 10 to 108 CFU/mL and a low detection limit of 0.78 CFU/mL were achieved. This research opens new horizons for the application of PCR-based biosensors for the accurate and sensitive measurement of pathogenic bacteria.

Quick preculture method for determining low concentration Vibrio spp. in contaminated seafood and its verification

This study aimed to present a new approach for detecting pathogenic Vibrio spp., including V. parahaemolyticus, V. vulnificus, and V. cholerae in contaminated seafood. This method combined polymerase chain reaction (PCR) with a concise preculturing step and a simplified DNA extraction procedure. The effectiveness of the method was confirmed using contaminated mussel samples. To validate this proposed method, 15 different shellfish samples were analyzed and the results were compared with the results obtained using a conventional method, most-probable-number-PCR (MPN-PCR). These results confirm the efficiency of this new approach in detecting pathogenic Vibrio spp. This method takes approximately 6 h for results and offers significant time saving with a reduction of 75% compared with MPN-PCR. Additionally, it demonstrated high sensitivity for the efficient Vibrio spp. detection, and the simplification of the DNA extraction process using boiling makes it accessible to non-experts and contributes to overall time savings in the sample preparation stage. The proposed method in this study suggests a time-efficient and simplified alternative approach for detecting Vibrio spp. in seafood products, providing an in-situ application for improving safety measures in the food industry.

Regulation of Tumor Apoptosis of Poriae cutis-Derived Lanostane Triterpenes by AKT/PI3K and MAPK Signaling Pathways In Vitro.

Poria cocos is traditionally used as both food and medicine. Triterpenoids in Poria cocos have a wide range of pharmacological activities, such as diuretic, sedative and tonic properties. In this study, the anti-tumor activities of poricoic acid A (PAA) and poricoic acid B (PAB), purified by high-speed counter-current chromatography, as well as their mechanisms and signaling pathways, were investigated using a HepG2 cell model. After treatment with PAA and PAB on HepG2 cells, the apoptosis was obviously increased (p < 0.05), and the cell cycle arrested in the G2/M phase. Studies showed that PAA and PAB can also inhibit the occurrence and development of tumor cells by stimulating the generation of ROS in tumor cells and inhibiting tumor migration and invasion. Combined Polymerase Chain Reaction and computer simulation of molecular docking were employed to explore the mechanism of tumor proliferation inhibition by PAA and PAB. By interfering with phosphatidylinositol-3-kinase/protein kinase B, Mitogen-activated protein kinases and p53 signaling pathways; and further affecting the expression of downstream caspases; matrix metalloproteinase family, cyclin-dependent kinase -cyclin, Intercellular adhesion molecules-1, Vascular Cell Adhesion Molecule-1 and Cyclooxygenase -2, may be responsible for their anti-tumor activity. Overall, the results suggested that PAA and PAB induced apoptosis, halted the cell cycle, and inhibited tumor migration and invasion through multi-pathway interactions, which may serve as a potential therapeutic agent against cancer.

HPV prevalence and genotype distribution in 2,306 patients with cervical squamous cell carcinoma in central and eastern China.

To explore the positivity rate and genotype distribution of human papillomavirus (HPV) in cervical squamous cell carcinoma (CSCC) tissues in central and eastern China and to provide theoretical basis for cervical cancer screening and prophylactic HPV vaccine development in China. DNA was extracted from paraffin-embedded tissues of CSCC samples and exfoliated cervical cells of cervical cancer screening populations. 23 HPV genotypes were detected by combining polymerase chain reaction (PCR) and reverse dot hybridized gene chip detection technology in 2,306 CSCC tissues and 10,245 cervical cancer screening populations. The genotype distribution of HPV infection was analyzed. The overall infection rate of HPVs in 2,306 CSCC patients was 92.71%. The frequency of single-type HPV infection and multiple-type HPV infection were 86.48% and 13.51%, respectively. The most common HPV genotypes detected in Chinese CSCC tissues were HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, HPV-58, and HPV-59. The overall positivity rate of these eight high-risk HPV (HR-HPV) genotypes in HPV-positive CSCC was as high as 96.91%. Of which the positivity rate of seven HR-HPV genotypes related to nine-valent HPV vaccines in HPV-positive CSCC was 95.09%. Meanwhile, the overall infection rates of HR-HPV and low-risk HPV (LR-HPV) in female aged 35-64 years who underwent cervical cancer screening were 13.16% and 1.32%, respectively. The high-frequency HR-HPV genotypes in cervical cancer screening women were HPV-52, HPV-58, HPV-16, HPV-53, HPV-68, HPV-39, HPV-51, and HPV-56, with positivity rates of 2.25%, 1.60%, 1.31%, 1.22%, 0.93%, 0.92%, 0.78%, and 0.74%, respectively. Among women screened for cervical cancer in China, detecting the 8 high-frequency HR-HPV genotypes can reduce technical difficulty and reagent costs, while also improving the efficiency and effectiveness of cervical cancer screening. HPV genotyping assists gynecologists in assessing the risk of HR-HPV-positive cervical intraepithelial neoplasia and guiding them in implementing appropriate interventions. Furthermore, HPV genotyping is helpful for doctors to follow up HR-HPV-positive women and to evaluate the protective effect of HPV vaccine.

Rapid and visual detection of specific bacteria for saliva and vaginal fluid identification with the lateral flow dipstick strategy.

Identification of body fluids is critical for crime scene reconstruction, and a source of investigation source of investigative leads. In recent years, microbial DNA analysis using sequencing and quantitative real-time polymerase chain reaction have been used to identify body fluids. However, these techniques are time-consuming, expensive, and require complex workflows. In this study, a new method for simultaneous detection of Streptococcus salivarius and Lactobacillus crispatus using polymerase chain reaction (PCR) in combination with a lateral flow dipstick (LFD) was developed to identify saliva and vaginal fluid in forensic samples. LFD results can be observed with the naked eye within 3min with a sensitivity of 0.001ng/µL DNA. The PCR-LFD assay was successfully used to detect S. salivarius and L. crispatus in saliva and vaginal fluid respectively, and showed negative results in blood, semen, nasal fluid, and skin. Moreover, saliva and vaginal fluid were detectable even at an extremely high mixing ratio of sample DNA (1:999). Saliva and vaginal fluid were identified in various mock forensic samples. These results indicate that saliva and vaginal fluid can be effectively detected by identifying S. salivarius and L. crispatus, respectively. Furthermore, we have shown that DNA samples used to identify saliva and vaginal fluid can also provide a complete short tandem repeat (STR) profile when used as source material for forensic STR profiling. In summary, our results suggest that PCR-LFD is a promising assay for rapid, simple, reliable, and efficient identification of body fluids.

Rapid detection of insect pest with CRISPR‐Cas13a‐Based Lateral Flow Strip: Using Locusta migratoria manilensis

AbstractThe Clustered Regularly Interspaced Short Palindromic Repeats‐associated (CRISPR‐Cas) system has been used as a powerful diagnostic tool because of its trans‐cleavage ability to accurately recognize and cleave specific nucleic acid targets. However, its application in insect pest detection has rarely been reported. The aim of the current study was to develop a new molecular identification method for insect based on CRISPR‐Cas13a system. In this study, we have developed a highly specific and sensitive detection system that combines polymerase chain reaction, CRISPR‐Cas13a system and lateral flow dipstick (PCR‐Cas13a‐LFD) to rapidly detect the presence of Locusta migratoria manilensis (Orthoptera: Acrididae) DNA and discriminate it from other species, by using material from different stages of development. A pair of specific primers and crRNA were designed based on the Gene DEAD‐box DDX10 mRNA sequence. For visual readout, a FAM‐RNA‐biotin reporter was used for detection. The CRISPR‐Cas13a‐based detection method utilizes the visual identification of L. migratoria manilensis at different developmental stages or with the use of incomplete samples. The test results could be distinguished within 2 h. The sensitivity of the PCR‐Cas13a‐LFD detection system reached 0.1 ng/μL, a 10‐fold increase compared with that of the PCR with electrophoresis. The LFD coupled with CRISPR‐Cas13a (Cas13a‐LFD) is promising for the portable, onsite and fast identification of insect pests. This study provides a reference for the development of a new method for insect identification.

Detection of ATXN2 Expansions in an Exome Dataset: An Underdiagnosed Cause of Parkinsonism.

CAG-repeat expansions in Ataxin 2 (ATXN2) are known to cause spinocerebellar ataxia type 2 (SCA2), but CAA interrupted expansions may also result in autosomal dominant Parkinson's disease (AD PD). However, because of technical limitations, such expansions are not explored in whole exome sequencing (WES) data. To identify ATXN2 expansions using WES data from PD cases. We explored WES data from a cohort of 477 index cases with PD using ExpansionHunter (Illumina DRAGEN Bio-IT Platform, San Diego, CA). Putative expansions were confirmed by combining polymerase chain reaction and fragment length analysis followed by sub-cloning and sequencing methods. Using ExpansionHunter, we identified three patients from two families with AD PD carrying either ATXN2 22/39 or 22/37 repeats, both interrupted by four CAA repeats. These findings demonstrate the usefulness of WES to detect pathogenic CAG repeat expansions, which were found in 1.7% of AD PD in the ATXN2 gene in our exome dataset.

Detection of Tobacco Bacterial Wilt Caused by Ralstonia solanacearum by Combining Polymerase Chain Reaction with an α-Hemolysin Nanopore.

Tobacco bacterial wilt is a serious disease caused by the soil-borne bacterium Ralstonia solanacearum (R. solanacearum). Herein, a rapid and purification-free α-hemolysin (α-HL) nanopore-sensing strategy based on polymerase chain reaction (PCR) and lambda exonuclease digestion was established to detect R. solanacearum. A 198-nucleotide-long single-stranded DNA was obtained via asymmetric PCR or the lambda exonuclease-mediated digestion of the PCR product. The DNA fragment produced unique long-lived, current-blocking signals when it passed through the α-HL nanopore. This sensing approach can allow for the determination of R. solanacearum in tobacco samples and can be conveniently extended to other DNA monitoring because of the extremely wide range of PCR applications.

Molecular typification of Escherichia coli from community-acquired urinary tract infections in Mexico

One hundred and five uropathogenic Escherichia coli (UPEC) strains from patients with community-acquired urinary tract infections were characterized according to phylogenetic group, virulence factors, serogroup, antibiotic resistance, and genotype. The pathogenic phylogenetic groups (B2, D, and F) were found in 71.4% of the tested strains. Among them, the main uropathogenic serogroups were O8, O25, and O75, in which 97.1% of the strains had a multidrug-resistant profile. Sixteen virulence genes were analysed using a combination of polymerase chain reaction (PCR) assays, with the fimH, irp-2, iutA, aer, iucC, PAI, sat, iroN, usp, and cnf1 genes being mainly found in pathogenic phylogroups. The E. coli O25b-ST131 clone was identified in 32% of the strains assigned to the pathogenic phylogroup B2. These findings demonstrate that virulence genes encoding adhesin components, iron-acquisition systems, toxins, and pathogenicity-associated islands were highly prevalent among the pathogenic phylogroup of UPEC strains.

A Longitudinal Nine-Year Study of the Molecular Epidemiology of Carbapenemase-Producing Enterobacterales Isolated From a Regional Hospital in Taiwan: Predominance of Carbapenemase KPC-2 and OXA-48.

Enterobacterales clinical isolates are now being resistant to clinically achievable concentrations of most commonly used antibiotics that makes treatment of hospitalized patients very challenging. We hereby determine the molecular characteristics of carbapenemase genes in carbapenem-resistant Enterobacterales (CRE) isolates in Taiwan. A total of 455 CRE isolates were identified between August 2011 to July 2020. Minimum inhibitory concentrations for selected carbapenems were tested using Vitek 2, and carbapenemase genes were determined using polymerase chain reaction in combination with sequencing. Phenotypic detection of carbapenemase was determined by modified carbapenem inactivation method (mCIM) and EDTA-modified carbapenem inactivation method (eCIM) to validate our PCR screening results. Pulsed-field gel electrophoresis (PFGE) was used to determine the clonality of carbapenemase-producing Enterobacterales (CPE) isolates, and the transferability of carbapenemase-carrying plasmids was determined by conjugation assays. A slight increase in carbapenem-resistant E. coli (CREC) was observed, however, the prevalence of carbapenem-resistant K. pneumoniae (CRKP) was steady, during 2011–2020. The dominant species among our CRE was K. pneumoniae (270/455, 59.3%), followed by E. coli (81/455, 17.8%), Morganella morganii (32/455, 7.0%), and Enterobacter cloacae (25/455, 5.5%). From 2011 to 2020, the total percentage of CPE increased steadily, accounting for 61.0% of CRE in 2020. Moreover, 122 of 455 CRE isolates (26.8%) were CPE. Among the CPE isolates, the dominant carbapenemase gene was blaOXA–48–like (54/122, 44.3%), and the second most common carbapenemase gene was blaKPC–2 (47/122, 38.5%). The sensitivity and specificity for mCIM to detect carbapenemase in the 455 isolates were both 100% in this study. The PFGE results showed that 39 carbapenemase-producing E. coli and 69 carbapenemase-producing K. pneumoniae isolates carrying blaKPC–2 and/or blaNDM–5 could be classified into 5 and 12 clusters, respectively. In conclusion, our results showed an increase in CPE isolates in Taiwan. Moreover, the distribution of carbapenemase and antimicrobial susceptibility in CPE were associated with PFGE typing.

Low-salt diet increases mRNA expression of aldosterone-regulated transporters in the intermediate portion of the endolymphatic sac.

The endolymphatic sac is a small sac-shaped organ at the end of the membranous labyrinth of the inner ear. The endolymphatic sac absorbs the endolymph, in which the ion balance is crucial for inner ear homeostasis. Of the three sections of the endolymphatic sac, the intermediate portion is the center of endolymph absorption, particularly sodium transport, and is thought to be regulated by aldosterone. Disorders of the endolymphatic sac may cause an excess of endolymph (endolymphatic hydrops), a histological observation in Meniere's disease. A low-salt diet is an effective treatment for Meniere's disease, and is based on the assumption that the absorption of endolymph in the endolymphatic sac abates endolymphatic hydrops through a physiological increase in aldosterone level. However, the molecular basis of endolymph absorption in each portion of the endolymphatic sac is largely unknown because of difficulties in gene expression analysis, resulting from its small size and intricate structure. The present study combined reverse transcription-quantitative polymerase chain reaction and laser capture microdissection techniques to analyze the difference of gene expression of the aldosterone-controlled epithelial Na+ channel, thiazide-sensitive Na+-Cl- cotransporter, and Na+, K+-ATPase genes in the three individual portions of the endolymphatic sac in a rat model. A low-salt diet increased the expression of aldosterone-controlled ion transporters, particularly in the intermediate portion of the endolymphatic sac. Our findings will contribute to the understanding of the physiological function of the endolymphatic sac and the pathophysiology of Meniere's disease.

Effect of Sodium on Methanogens in a Two-Stage Anaerobic System

This study evaluated the effects of sodium on anaerobic biomass from the second-stage reactor of a two-stage anaerobic digester. The results indicated that methanogens showed a relatively high sodium tolerance of 2.4 g Na+ L−1. Microbial community analysis showed that viable Methanomicrobiales was the most abundant population by a combined propidium monoazide cross-linking quantitative polymerase chain reaction technique. There was a population shift towards higher abundance of Thermotoga (0.02%), Clostridium (2.50%) and Methanoculleus (13.80%). Biomass activity in relation to increased sodium concentrations was investigated with the adenosine triphosphate test coupled with extracellular polymeric substances measurement. The results showed biomass activity decreased from 33 to 16 µg g−1 volatile suspended solids as sodium concentrations increased from 1.3 to 9.1 g Na+ L−1. Higher EPS production, particularly a greater predominance of carbohydrates, was stimulated by higher sodium concentrations. This study provides insights into the superiority of sodium tolerance of two-stage anaerobic digester in compared with a single-stage anaerobic system.

Prevalence of Campylobacter spp. in marine fishes, crustaceans and molluscs in Kozhikode district, Kerala#

Campylobacteriosis is one of the leading causes of bacterial gastroenteritis world over, with a self-limiting nature. Nevertheless some rare sequelae like Guillain-Barre syndrome, Reiter syndrome and reactive arthritis are evident. With poultry as a major reservoir, the organism is ubiquitous in nature and has been isolated from various sources like animals and the environment. The present study was undertaken to evaluate the occurrence of Campylobacter spp. in marine fishes (n=48), crustaceans (n=35) and molluscs (n=50) from Puthiyappa, a fish catchment area of coastal Kerala (India) using multiplex polymerase chain reaction (mPCR) in combination with conventional plating technique using Blood-free campylobacter broth and modified Charcoal Cefoperazone Deoxycholate (mCCD) agar. Campylobacter spp. was found to be present in marine fishes, crustaceans and molluscs at a level of 6.3, 8.6 and 28.0 per cent, respectively. The predominant species was Campylobacter jejuni in fishes, while Campylobacter coli was more prevalent in molluscs. Of the seasonally available fishes, the organism was found to be present in ribbon fish, pink perch, pony fish, while among molluscs, it was present in mussel, squid and clam. Only 8.3 and 6.0 per cent of Campylobacter spp. were recoverable from fishes and molluscs, respectively, on mCCDA plates. Campylobacter spp. in these marine fishes and shellfishes indicate that these can act as possible sources for the transmission of food-borne campylobacteriosis.

Polymerase chain reaction (PCR) とloop-mediated isothermal amplification (LAMP) 法の コンビネーションによるクジラ型パラコクシジオイデス症の診断法の研究

Polymerase chain reaction (PCR) とloop-mediated isothermal amplification (LAMP) 法の コンビネーションによるクジラ型パラコクシジオイデス症の診断法の研究

Determination of critical cut-off values of PCA3 index in patients with prostate gland pathology

Introduction. Prostate cancer antigen-3 (PCA3) is a genetic biomarker, which got widespread and demonstrated high diagnostic potential. This gene expresses only in prostate gland tissue, furthermore, malignant prostate neoplastic transformation leads to 70 – 100 time overexpression of it. Polyadenylated matrix RNA (mRNA) is a functional product of this gene, which do not translating into protein. Gene contains of four introns and three exon. Product of PCA’s gene detecting in post massage urine using polymerase chain reaction (PCR) in combination with reverse transcription. It could use as prognostic biomarker in patients with first negative biopsy or before it performing. Moreover, index of PCA3 can be useful in detecting tumor aggressiveness and decisions about further treatment options. Taking into account these facts, patients with high initial PSA level can avoid unnecessary biopsies. Objective: to determine critical cut-off value of PCA3 index as prognostic biomarker of PCa development. Materials and methods. Research based on routine and special test’s data of 243 men in Institute of Urology, National Academy of Medical Sciences of Ukraine in period of 2015–2020 year. RNA extraction from postmassage urine samples performed using RNeasy (Qiagen) kit. Statistical analysis performed using SPSS Statistics 19.0 (IBM SPSS Statistics 19.0) та SAS 12.0 (IBM Statistical Analysis System 12.0). Critical cut-off values determined using three methods: calculating of average value and (Xave) and standard deviation (SD) of it, Heiner index, and Youden’s index. Evaluation of optimality of determined critical cut-off values performed by comparative analysis using index of positive prognosis (IPP), index of negative prognosis (INP), diagnostic accuracy of method (DAM), Sp and Se for each of used methods. Results and discussion. First method demonstrated than 97,5 % of all results, which means absent of disease, was below critical cut-off values, due to statistical deviations and accurate demonstrate than in case of asymmetry or multimodal distribution this method was unsatisfied. Wherein IPP was 95 %, INP – 72 %, DAM – 78 %, Sp – 97,5 % and Se – 52 %. Calculation of PCA3 index area under ROC-curve was 0,810 (0,771–0,849). Using Heiner’s method critical cut-off value of PCA3 index was 14,0 с. u., where in IPP was 75 %, INP – 82 %, DAM – 78 %, Sp – 78 % and Se – 78 %. Critical cut-off values of PCA3 index 33,4 c. u. which calculated by Youden’s index demonstrated IPP – 98 %, INP – 73 %, DAM – 80 %, Sp – 99 % and Se – 57 %. Conclusions. The most optimal critical cut-off value for PCA3 index was determined using Youden’s index and was 33,4 c. u. Keywords: prostate cancer, PCA , prostate cancer antigen-3, PCA3, prostate cancer antigen-3 index, PCA3 index, critical cut-off values of PCA3 index.

Snus users: intraoral lesions, biofilm properties, communication and treatment strategies

Objective: The link between snus, periodontal diseases and oral malignancy is still in question in different literature. This study aims to explore the impact of snus on mucosal lesions and oral malignancy along with evaluation of strategies for snus cessation and approaches to communication with patients. Methods: A questionnaire about tobacco consumption habits was made. A heavy snus group, a light snus group and a control group were made. Oral biopsy samples were tested for protein gene product 9.5, tissue inhibitor of matrix metalloproteinase 2, chromogranin A and B, matrix metalloproteinase 2, interleukin-1, interleukin-10 using immunohistochemical techniques. Periodontal pocket biofilms were tested with combined polymerase chain reaction and were subsequently analyzed in order to determine the presence of pathogenic periodontal bacteria, such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola and Prevotella intermedia. Results: Biopsy results showed cellular disorganization, apoptosis, hyperkeratosis and prevalence of keratotic seborrhea in the area of snus sachets. Microbiological examination revealed the presence of periodontal pathogens in the snus users group. High concentration of pathogenic periodontal bacteria Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia was found in groups of both heavy and light snus users, yet they were absent in the samples of the control group. High concentration of Tannerella forsythia and Treponema denticola was also found in the groups of heavy and light snus users, whereas they were present in samples of only two patients of the control group. Conclusions: Snus changes cell function, it can lead to oral malignancy and promote periodontal disease regardless of the frequency and amount of snus used.

A prospective, multicentre, cohort study to assess the incidence of dengue illness in households from selected communities in Brazil (2014–2018)

ObjectivesTo estimate the incidence of dengue infection across geographically distinct areas of Brazil. MethodsThis prospective, household-based, cohort study enrolled participants in five areas and followed them up for up to 4 years (2014–2018). Dengue seroprevalence was assessed at each scheduled visit. Suspected dengue cases were identified through enhanced passive and active surveillance. Acute symptomatic dengue infection was confirmed through reverse-transcriptase quantitative polymerase chain reaction in combination with an antigenic assay (non-structural protein 1) and serology. ResultsAmong 3300 participants enrolled, baseline seroprevalence was 76.2%, although only 23.3% of participants reported a history of dengue. Of 1284 suspected symptomatic dengue cases detected, 50 (3.9%) were laboratory-confirmed. Based on 8166.5 person-years (PY) of follow-up, the incidence of laboratory-confirmed symptomatic infection (primary endpoint) was 6.1 per 1000 PY (95% confidence interval [CI]: 4.5, 8.1). Incidence varied substantially in different years (1.8–7.4 per 1000 PY). The incidence of inapparent primary dengue infection was substantially higher: 41.7 per 1000 PY (95% CI: 31.1, 54.6). ConclusionsOur findings, highlighting that the incidence of dengue infection is underestimated in Brazil, will inform the design and implementation of future dengue vaccine trials. Clinical trial registrationNCT01751139

A universal method for rapid identification of alfalfa and burr medic seeds with an emphasis on discriminating different forage species

AbstractUnlike many crop seeds which are large in size, forage seeds are relatively small. Moreover, some forage seeds from the same genus, or even different genera, share similar morphologies in terms of colour, size, weight and so on. Alfalfa (Medicago sativa L.) and burr medic (Medicago polymorpha L.) also share similar morphology. However, alfalfa is a perennial, cross‐pollinated herbaceous species, whereas burr medic is considered an annual, self‐pollinating plant. Therefore, a universal and rapid method of discriminating burr medic seeds from alfalfa seeds or other similar forage seeds from different plant species is needed. Previous methods of discriminating similar‐looking forage seeds have been shown to be unreliable or time consuming. Here, a universal method that combines polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis techniques was developed to discriminate alfalfa and burr medic. Based on the differences in the lengths of homologous genes caused by insertions and deletions between two species, a pair of primers was designed to discriminate the accessions of 15 alfalfa and 9 burr medic via visualizing the clearly different PCR amplicon sizes. In addition, the melting curve analysis was performed to investigate the specificity and reliability of the primer pair. The results showed two different melting peaks between the two species, further demonstrating this method can accurately and sensitively identify the two species. Thus, we believe that this method has the great potential to be extensively applied for the identification of contaminated seeds and breeding of forages.

Approaches for the sensitive detection of rare base and prime editing events.

Precision chemistry entailing user-directed nucleotide substitutions and template-specified repair can be facilitated by base editing and prime editing, respectively. Recently, the diversification of adenine, cytosine, and prime editor variants obliges a considered, high-throughput evaluation of these tools for optimized, end-point applications. Herein, we outline novel, cost-effective and scalable approaches for the rapid detection of base editing and prime editing outcomes using gel electrophoresis. For base editing, we exploit primer mismatch amplification (SNP genotyping) for the gel-based detection of base editing efficiencies as low as 0.1%. For prime editing, we describe a one-pot reaction combining polymerase chain reaction (PCR) amplification of the target region with restriction digestion (restriction fragment length polymorphism; RFLP). RFLP enables the rapid detection of insertion or deletion events in under 2.5h from genomic DNA extraction. We show that our method of SNP genotyping is amenable to both endogenous target loci as well as transfected, episomal plasmid targets in BHK-21 cells. Next, we validate the incidence of base and prime editing by describing Sanger sequencing and next-generation sequencing (NGS) workflows for the accurate validation and quantification of on-target editing efficiencies. Our workflow details three different methods for the detection of rare base and prime editing events, enabling a tiered approach from low to high resolution that makes use of gel electrophoresis, Sanger sequencing, and NGS.