polymerase chain reactions
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2022 ◽  
Vol 82 ◽  
Author(s):  
M. F. Ribeiro ◽  
V. R. Carvalho ◽  
A. L. Favoreto ◽  
B. R. De Marchi ◽  
C. Jordan ◽  
...  

Abstract Endosymbiont bacteria can affect biological parameters and reduce the effectiveness of natural enemies in controlling the target insect. The objective of this work was to identify endosymbiont bacteria in Anaphes nitens (Girault, 1928) (Hymenoptera: Mymaridae), the main natural enemy used to manage Gonipterus platensis (Marelli, 1926) (Coleoptera: Curculionidae). Genomic DNA from six A. nitens populations was extracted and polymerase chain reactions (PCR) were performed with the primers to detect endosymbiont bacteria in this insect. The PCR products were amplified, sequenced, and compared with sequences deposited in the GenBank for the bacteria identification. All A. nitens populations had the bacterium Yersinia massiliensis (Enterobacteriales: Enterobacteriaceae). This bacterium was originally described as free-living, and it is associated with and composes part of the A. nitens microbiota. This is the first report of Y. massiliensis in an insect host.


Author(s):  
Maral Farzin ◽  
Seyed Ahmad Rasoulinejad ◽  
Mansour Babaei ◽  
Farzin Sadeghi ◽  
Mahmoud Sadeghi-Haddad-Zavareh ◽  
...  

Background and Objectives: This study aimed to detect SARS-CoV-2 in conjunctival samples of COVID-19 patients to investigate the transmission route of COVID-19 and its correlation with laboratory indexes. Materials and Methods: In this cross-sectional study, 44 COVID-19 patients were tested for conjunctival PCR in Ayatollah Rouhani hospital of Babol, Iran, in January and February 2021. The conjunctival samples were collected using a conjunctival swab and suspended in a viral transport medium. After RNA extraction and cDNA synthesis, real-time PCR was performed to investigate the SARS-CoV-2 genome in samples. The ocular manifestations and laboratory indexes were evaluated for all patients. Results: Among 44 COVID-19 patients, 6 samples (13.63%) were positive in terms of conjunctival PCR. The mean ± SD age of conjunctival PCR-positive patients was 76.17 ± 16.61-year-old, while conjunctival PCR-negative COVID-19 pa- tients were aged 57.54 ± 13.61-year-old (p <0.05). D-dimer serum level is significantly higher in conjunctival PCR-positive COVID-19 patients (4001.00 ± 3043.36 µg/ml) compared to normal individuals (496.80 ± 805.92 µg/ml, p <0.01). Conclusion: Our study showed that the conjunctiva and tear contain the SARS-CoV-2 in COVID-19 patients as a possible transmission route.


2021 ◽  
Vol 12 ◽  
Author(s):  
Ruihua Cao ◽  
Tao Sun ◽  
Ruyi Xu ◽  
Jin Zheng ◽  
Hao Wang ◽  
...  

Objective: Low plasma level of high-density lipoprotein cholesterol (HDL-C) associated with poor outcomes in several cardiovascular diseases, including pulmonary arterial hypertension (PAH). Regulation of miR-638 have been proved to be associated with PAH. The aim of this study was to evaluate the expression of miR-638 after Xuezhikang (XZK) therapy in patients with low HDL-C.Methods: Plasma levels of miR-638 were quantified by real-time polymerase chain reactions in 20 patients with PAH and 30 healthy controls. A total of 40 subjects with low HDL-C were assigned to receive an XZK therapy for 6 months. The miR-638 expression profiles were detected in PAH patients, XZK-treated subjects and lovastatin treated pulmonary arterial smooth muscle cells (PA-SMCs).Results: The relative expression level of miR-638 in the plasma was lower in the PAH patients than that in the controls (p &lt; 0.001). An increase of 11.2% from baseline in the HDL-C level was found after XZK therapy (p &lt; 0.001). The relative expression of miR-638 was increased after XZK treatment (p &lt; 0.01). The changes of miR-638 were inversely associated with baseline HDL-C levels. A significantly reduction in miR-638 expression were found in PDGF-BB-treated hPA-SMCs compared to the control cells, and the pre-treatment of the cells with lovastatin significantly re-gain the expression levels in miR-638.Conclusion: In patients with low HDL-C levels, XZK therapy raised the expression of miR-638, suggesting that the potential therapeutic effect of XZK in PAH patients with low serum HDL-C levels deserves further exploration.


2021 ◽  
Vol 12 ◽  
Author(s):  
Jianjun Zhang ◽  
Yan Zeng ◽  
Yuefeng Wang ◽  
Jiaming Fan ◽  
Haijiang Chen ◽  
...  

RHD variants in D¯ Chinese pregnant women arose difficulties in management during pregnancy. Therefore, this study aims to precisely manage D¯ pregnant women by evaluating the spectrum of RHD mutations in D¯ pregnant women and getting insight into the possible rare alleles of RHD. A total of 76 D¯ pregnant women were analyzed by performing polymerase chain reactions with sequence-specific primers (PCR-SSP), the 10 RHD exons Sanger sequencing, RHD zygosity detection, and mRNA sequencing (mRNA-seq). About 40% of alleles are variations of RHD, including RHD 1227A homozygous, RHD-CE(2-9)-D, et al. Therefore, we developed a molecular diagnostic strategy for Chinese women, and most D¯ pregnant women can be diagnosed with this simple decision tree. After RHD genotyping for D¯ pregnancy women, we eliminated at least 15% unnecessary ante- and postpartum injections of Rh immunoglobulin (RhIG). As the first pedigree study and the first functional analysis under physiological conditions, mRNA-seq revealed that c.336-1G&gt;A mutation mainly led to the inclusion of the intron 2, which indirectly explained the D¯ phenotype in this family. We also developed a robust protocol for determining fetal RhD status from maternal plasma. All 31 fetuses were predicted as RhD positive and confirmed the RhD status after birth.


Author(s):  
Sacha Laurent ◽  
Corinne Gehrig ◽  
Thierry Nouspikel ◽  
Sami S Amr ◽  
Andrea Oza ◽  
...  

Bi-allelic loss-of-function variants of OTOA are a well-known cause of moderate-to-severe hearing loss. Whereas non-allelic homologous recombination-mediated deletions of the gene are well known, gene conversions to pseudogene OTOAP1 have been reported in the literature but never fully described nor their pathogenicity assessed. Here, we report two unrelated patients with moderate hearing-loss, who were compound heterozygotes for a converted allele and a deletion of OTOA. The conversions were initially detected through sequencing depths anomalies at the OTOA locus after exome sequencing, then confirmed with long range polymerase chain reactions. Both conversions lead to loss-of-function by introducing a premature stop codon in exon 22 (p.Glu787*). Using genomic alignments and long read nanopore sequencing, we found that the two probands carry stretches of converted DNA of widely different lengths (at least 9 kbp and around 900 bp, respectively).


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Beatriz Carvalho Henriques ◽  
Avery Buchner ◽  
Xiuying Hu ◽  
Yabing Wang ◽  
Vasyl Yavorskyy ◽  
...  

AbstractMany antidepressants, atomoxetine, and several antipsychotics are metabolized by the cytochrome P450 enzymes CYP2D6 and CYP2C19, and guidelines for prescribers based on genetic variants exist. Although some laboratories offer such testing, there is no consensus regarding validated methodology for clinical genotyping of CYP2D6 and CYP2C19. The aim of this paper was to cross-validate multiple technologies for genotyping CYP2D6 and CYP2C19 against each other, and to contribute to feasibility for clinical implementation by providing an enhanced range of assay options, customizable automated translation of data into haplotypes, and a workflow algorithm. AmpliChip CYP450 and some TaqMan single nucleotide variant (SNV) and copy number variant (CNV) data in the Genome-based therapeutic drugs for depression (GENDEP) study were used to select 95 samples (out of 853) to represent as broad a range of CYP2D6 and CYP2C19 genotypes as possible. These 95 included a larger range of CYP2D6 hybrid configurations than have previously been reported using inter-technology data. Genotyping techniques employed were: further TaqMan CNV and SNV assays, xTAGv3 Luminex CYP2D6 and CYP2C19, PharmacoScan, the Ion AmpliSeq Pharmacogenomics Panel, and, for samples with CYP2D6 hybrid configurations, long-range polymerase chain reactions (L-PCRs) with Sanger sequencing and Luminex. Agena MassARRAY was also used for CYP2C19. This study has led to the development of a broader range of TaqMan SNV assays, haplotype phasing methodology with TaqMan adaptable for other technologies, a multiplex genotyping method for efficient identification of some hybrid haplotypes, a customizable automated translation of SNV and CNV data into haplotypes, and a clinical workflow algorithm.


Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3186
Author(s):  
Michał Pac ◽  
Natalia Krata ◽  
Barbara Moszczuk ◽  
Aleksandra Wyczałkowska-Tomasik ◽  
Beata Kaleta ◽  
...  

Glomerular diseases (GNs) are responsible for approximately 20% of chronic kidney diseases. Glucocorticoid receptor gene (NR3C1) single nucleotide polymorphisms (SNPs) are implicated in differences in predisposition to autoimmunity and steroid sensitivity. The aim of this study was to evaluate the frequency of the NR3C1 SNPs—rs6198, rs41423247 and rs17209237—in 72 IgA nephropathy (IgAN) and 38 membranous nephropathy (MN) patients compared to 175 healthy controls and to correlate the effectiveness of treatment in IgAN and MN groups defined as a reduction of proteinuria <1 g/24 h after 12 months of treatment. Real-time polymerase chain reactions and SNP array-based typing were used. We found significant rs41423247 association with MN (p = 0.026); a significant association of rs17209237 with eGFR reduction after follow-up period in all patients with GNs (p = 0.021) and with the degree of proteinuria after 1 year of therapy in all patients with a glomerulopathy (p = 0.013) and IgAN (p = 0.021); and in the same groups treated with steroids (p = 0.021; p = 0.012). We also observed the association between rs41423247 and IgAN histopathologic findings (p = 0.012). In conclusion, our results indicate that NR3C1 polymorphisms may influence treatment susceptibility and clinical outcome in IgAN and MN.


Antibiotics ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1383
Author(s):  
Shiuh-Bin Fang ◽  
Tsai-Ling Yang Lauderdale ◽  
Chih-Hung Huang ◽  
Pei-Ru Chang ◽  
Yuan-Hung Wang ◽  
...  

This study analyzed the genetic diversity of ciprofloxacin (CIP) nonsusceptibility and the relationship between two major mechanisms and minimum inhibitory concentrations (MICs) of CIP in nontyphoidal Salmonella (NTS). Chromosomal mutations in quinolone resistance-determining regions (QRDRs) and plasmid-mediated quinolone resistance (PMQR) genes were searched from ResFinder, ARG-ANNOT, and PubMed for designing the sequencing regions in gyrA, gyrB, parC, and parE, and the 13 polymerase chain reactions for PMQR genes. We found that QRDR mutations were detected in gyrA (82.1%), parC (59.0%), and parE (20.5%) but not in gyrB among the 39 isolates. Five of the 13 PMQR genes were identified, including oqxA (28.2%), oqxB (28.2%), qnrS (18.0%), aac(6′)-Ib-cr (10.3%), and qnrB (5.1%), which correlated with the MICs of CIP within 0.25–2 μg/mL, and it was found that oxqAB contributed more than qnr genes to increase the MICs. All the isolates contained either QRDR mutations (53.8%), PMQR genes (15.4%), or both (30.8%). QRDR mutations (84.6%) were more commonly detected than PMQR genes (46.2%). QRDR mutation numbers were significantly associated with MICs (p < 0.001). Double mutations in gyrA and parC determined high CIP resistance (MICs ≥ 4 μg/mL). PMQR genes contributed to intermediate to low CIP resistance (MICs 0.25–2 μg/mL), thus providing insights into mechanisms underlying CIP resistance.


Animals ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 2966
Author(s):  
Alexandra Hund ◽  
Johannes Reithofer ◽  
Bita Shahi Barogh ◽  
Maria Sophia Unterköfler ◽  
Josef Harl ◽  
...  

Veterinarians reported cases of cutaneous bleeding in cattle in Austria in the spring and summer of 2020. It was our goal to confirm the tentative diagnosis of parafilariosis by identifying Parafilaria bovicola in exudate samples using molecular methods for the first time in Austria. We asked veterinarians in the field to collect exudate from typical lesions on cattle. We performed polymerase chain reactions (PCRs) and sequenced a 674-bp section of the mitochondrial cytochrome oxidase subunit I in all positive samples. Overall, in 57 of 86 samples, P. bovicola was confirmed by PCR in cattle from Lower Austria, Upper Austria, Styria, Salzburg, Carinthia, and Tyrol. Sequencing detected four different haplotypes or genotypes, respectively, indicating multiple routes of introduction. We conclude that parafilariosis has spread in Austria and we expect that the number of reports of clinical signs and losses due to carcass damage will increase in the future.


2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Axel O. G. Hoarau ◽  
Steven M. Goodman ◽  
Dana Al Halabi ◽  
Beza Ramasindrazana ◽  
Erwan Lagadec ◽  
...  

AbstractCo-infections have a key role in virus transmission in wild reservoir hosts. We investigated the simultaneous presence of astroviruses, coronaviruses, and paramyxoviruses in bats from Madagascar, Mayotte, Mozambique, and Reunion Island. A total of 871 samples from 28 bat species representing 8 families were tested by polymerase chain reactions (PCRs) targeting the RNA-dependent RNA-polymerase genes. Overall, 2.4% of bats tested positive for the presence of at least two viruses, only on Madagascar and in Mozambique. Significant variation in the proportion of co-infections was detected among bat species, and some combinations of co-infection were more common than others. Our findings support that co-infections of the three targeted viruses occur in bats in the western Indian Ocean region, although further studies are needed to assess their epidemiological consequences.


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