Digital Microfluidic qPCR Cartridge for SARS-CoV-2 Detection.

Point-of-care (POC) tests capable of individual health monitoring, transmission reduction, and contact tracing are especially important in a pandemic such as the coronavirus disease 2019 (COVID-19). We develop a disposable POC cartridge that can be mass produced to detect the SARS-CoV-2 N gene through real-time quantitative polymerase chain reaction (qPCR) based on digital microfluidics (DMF). Several critical parameters are studied and improved, including droplet volume consistency, temperature uniformity, and fluorescence intensity linearity on the designed DMF cartridge. The qPCR results showed high accuracy and efficiency for two primer-probe sets of N1 and N2 target regions of the SARS-CoV-2 N gene on the DMF cartridge. Having multiple droplet tracks for qPCR, the presented DMF cartridge can perform multiple tests and controls at once.

[Detection and genotyping of rotavirus in diarrhea infant feces in Chengdu].

OBJECTIVE: To study the genotype of rotavirus and the genetic variations of the major neutralization antigen VP4 of group A rotavirus in fecal samples from infants with diarrhea in Chengdu, Sichuan province, China. METHODS: The fecal specimens were collected from infant patients with diarrhea in the spring of 2010 at West China Second University Hospital. Reverse transcription polymerase chain reaction (RT-PCR) was performed to identify rotavirus G serotypes and P genotypes. VP4 gene fragments of the virus were amplified from two strains drawn randomly from the prevailing genotype and cloned into a T-A clone vector to generate the recombinants for sequencing. RESULTS: A group rotaviruses were detected in 13 of 75 specimens (17.3%). Serotype G1 was the predominant type (7/13) and two were serotype G3, four strains' serotypes were unidentified. Analysis of P gene demonstrated that genotype P [8] was the predominant type (6/13), whereas only two P[4] genotype were detected and genotypes for two strains were not determined. G1P [8] was the predominant type of G/P dominance combination (5/13). Sequencing results of the VP4 gene for the analyzed two strains implied that they were genotype P[8] with a 97% homology in sequence. Compared with the standard strain, homologies were also more than 90%. CONCLUSION: Rotavirus is one of the major etiological agents of viral diarrhea among infants in Chengdu. G1 was the dominant type G in Chengdu. G1P[8] was the predominant type of G/P dominance combination.

Metabolic and genetic assessments interpret unexplained aggressive pulmonary hypertension induced by methylmalonic acidemia: A case report.

BACKGROUND: Pulmonary hypertension (PH) causes significant morbidity and mortality in diverse childhood diseases. However, limited information has been reported to obtain a good understanding of pediatric PH. Gaps exist between genome sequencing and metabolic assessments and lead to misinterpretations of the complicated symptoms of PH. Here, we report a rare case of a patient who presented with severe PH as the first manifestation without significant cardiovascular malformation and was finally diagnosed with methylmalonic aciduria (MMA) after metabolic and genomic assessments. CASE SUMMARY: An 11-year-old female presented with an aggressive reduction in activity capability and shortness of breath for only 4 mo and suffered from unexplained PH. A series of examinations was performed to evaluate any possible malformations or abnormalities of the cardiovascular system and lungs, but negative results were obtained. The blood tests were normal except for manifestations of microcytic anemia and elevated total homocysteine. Computed tomography and magnetic resonance imaging failed to identify any pulmonary diseases. Cardiac catheterization examination identified a small right coronary artery to pulmonary artery shunt and severe PH. During the follow-up, PH progressed rapidly. Then, genome sequencing and metabolic disorder screening were performed, which confirmed a diagnosis of MMA with MMACHC c.80A > G/c and 609G > A mutations. Vitamin B12, betaine and bosentan were then administered as the main treatments. During the 6-mo follow-up, the pulmonary artery pressure dropped to 45 mmHg, while the right ventricle structure recovered. The patient's heart function recovered to NYHA class II. Metabolic disorder analysis failed to identify significant abnormalities. CONCLUSION: As emerging types of metabolic dysfunction have been shown to present as the first manifestation of PH, and taking advantage of next generation sequencing technology, genome sequencing and metabolic disorder screening are recommended to have a more superior role when attempting to understand unclear or aggressive PH.

An Investigation on the Molecular Characteristics and Intracellular Growth Ability among Environmental and Clinical Isolates of Legionella pneumophila in Sichuan Province, China.

OBJECTIVE: To investigate the molecular characteristics and intracellular growth ability of Legionella pneumophila (L. pneumophila) strains from 1989 to 2016 in Sichuan Province, China. METHODS: Seventy-nine isolates of L. pneumophila were collected from environmental and clinical sources, including cooling towers, hot springs, bath water, fountains, and patients, and identified with 16S rRNA gene analysis and serum agglutination assay. The isolates were then typed by Sequence-Based Typing (SBT), and Genotyping of forty-two LP1 strains were analyzed by means of multiple-locus VNTR analysis with 8 loci (MLVA-8). All strains were further analyzed for two virulence genes: Legionella vir homologue (lvh) and repeats in structural toxin (rtxA). The intracellular growth ability of 33 selected isolates was determined by examining their interaction with J774 cells. RESULTS: All isolates were identified to L. pneumophila including 11 serogroups, among which the main serogroup were LP1, accounting for 54.43%. Thirty-three different sequence types (STs) from five main clonal groups and five singletons were identified, along with 8 different MLVA patterns. Both the lvh and rtxA loci were found in all 79 strains. Thirty isolates showed high intracellular growth ability in J774 cells. CONCLUSION: L. pneumophila is a potential threat to public health, and effective control and prevention strategies are urgently needed.

Effects of Ligustrum robustum on gut microbes and obesity in rats.

AIM: To investigate the anti-obesity and antibacterial effects of Ligustrum robustum (L. robustum) in vivo and in vitro and its possible mechanisms. METHODS: The effects of L. robustum aqueous extract (LR) on various gut bacteria in vitro were evaluated. The effects of LR on high-fat diet-fed (HFD) rats in vivo were also assessed. Culture methods, quantitative polymerase chain reaction, and terminal-restriction fragment length polymorphism were used to analyze the effects of LR on gut bacteria. Biochemical tests were also performed to detect the changes in obesity-related indicators after LR treatment. RESULTS: LR treatment lowered adipose weight and decreased Lee's index, blood glucose, total cholesterol, and lipid in the tested groups relative to control (P < 0.05). To determine the reasons for these changes, we assessed the potential bacteriostatic and bactericidal effects of LR on specific bacterial species in vitro. LR affected the richness, diversity, and evenness of gut bacteria, increased fecal Lactobacillus, and decreased Enterococci in HFD rats (P < 0.05). CONCLUSION: L. robustum may be a safe and effective food for weight loss and obesity control, and the effects of L. robustum might be mediated by the regulation of gut bacteria.