In this study, a simple fluorescence aptasensor was developed for rapid and sensitive detection of Listeria monocytogenes in food. The aptasensor was comprised of two parts. Aptamer-functionalized ...upconversion nanoparticles (UCNPs) recognized and adhered to the L. monocytogenes cells and provided strong fluorescent signals. Aptamer-functionalized magnetic nanoparticles (MNPs), which also recognized and adhered to the cells, were used to concentrate the aptamer-UCNP/L. monocytogenes/aptamer-MNP complex. The fluorescence intensity was measured and calibrated to a concentration series of L. monocytogenes. A low limit of detection of 8 cfu mL−1 was estimated from the range of 68 to 68 × 106 cfu mL−1. Compared with other methods currently available, the proposed method had wider detection range and higher sensitivity. Furthermore, the selectivity and functionality when used to detect L. monocytogenes in real food samples suggest it may be useful for practical applications.
•A fluorescence aptasensor was successfully developed.•Rapid and sensitive detection of L. monocytogenes was achieved.•Application of the fluorescence aptasensor in food produced satisfactory results.•Fluorescence aptasensors could be created for detecting other foodborne pathogens by changing the aptamer.
Diagnostics is crucial for a prompt identification of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infected patients, their isolation and treatment. Real‐time PCR is the reference ...method for the diagnosis of SARS‐CoV‐2 infection; however, the unprecedented increase in the number of infections worldwide calls for faster and easy methods that do not require skilled personnel and special equipment. Rapid antigen tests have been developed and used as first line screening. Here, we assessed the performance of a rapid antigen test in comparison to a real‐time qualitative PCR as gold standard. Fifty nasopharyngeal swabs from suspected cases of SARS‐CoV‐2 infection have been tested by Coris coronavirus disease 2019 Ag Respi‐Strip test and Allplex 2019n‐CoV assay. Of the 50 nasopharyngeal swabs tested, 11 were negative by both tests, 27 were negative by Ag test but positive by real‐time PCR, and 12 were positive by both methods. PCR detected the 39 positive samples at a median cycle threshold (Ct) value of 22.78 (mean: 24.51; range: 13.59–39.6). In the 12 concordant samples, the median Ct value was 17.37. The sensitivity of the Ag test was 30.77% (95% confidence interval CI: 17.02%–47.57%), specificity 100% (95% CI: 71.51%–100.00%), positive predictive value 100%, negative predictive value 85.25% (95% CI: 82.42%–87.69%), and accuracy 86.15% (95% CI: 73.45%–94.28%). The level of agreement between the two tests was poor, k = 0.164. The Ag test performs well in the presence of high viral loads, whereas lower levels are missed. Considering the poor sensitivity of the method, real‐time PCR remains the gold standard as front line screening for SARS‐CoV‐2 infection.
The effects of silicon (Si) and salicylic acid (SA) applications on proline content and expression of Δ
1
-pyrrolin-5-carboxylate synthetase (P5CS) were examined under different drought levels and ...different drought exposure times. Two wheat cultivars, a drought tolerant and a drought sensitive were used. The experiment was a factorial based on completely randomized design with three replicates. Expression analysis by the quantitative real time PCR showed that the tolerant cultivar had significantly higher P5CS expressions compared to the sensitive one under drought stress. In sampling time points, the maximum level of mRNA was observed at 48 h after stress was applied. At 48 h after stress induction, the expression of P5CS was almost 3.1 fold higher in the tolerant cultivar compared to the sensitive one. In both cultivars, gene expression decreased from 48 to 72 h. The stressed plants treated with Si + SA showed a higher expression. Proline content started to increase by Si and SA treatments and the maximum proline content was obtained at simultaneous application of Si + SA. Drought stress significantly reduced chlorophyll content, relative water content and leaf water potential of both cultivars, while increased electrolyte leakage (EL) of the leaves. In contrast, foliar-applied Si and SA significantly increased these parameters and reduced EL, and the effect of simultaneous application of Si and SA was greater. The results suggest that the P5CS is a stress inducible gene. This gene has the potential to be used for improvement of drought stress tolerance in wheat. Network analysis highlighted positive interaction of osmotic stress, drought and cold stress on P5CS1 and the regulatory role of MYB2, ERF-1, and EIN3 transcription factors. In conclusion, alleviation of drought stress by application of Si and SA was associated partially with enhanced expression of P5CS gene and following proline accumulation.
Pitfalls in SARS‐CoV‐2 PCR diagnostics Wernike, Kerstin; Keller, Markus; Conraths, Franz J. ...
Transboundary and emerging diseases,
March 2021, Letnik:
68, Številka:
2
Journal Article
Recenzirano
Odprti dostop
To combat the COVID‐19 pandemic, millions of PCR tests are performed worldwide. Any deviation of the diagnostic sensitivity and specificity will reduce the predictive values of the test. Here, we ...report the occurrence of contaminations of commercial primers/probe sets with the SARS‐CoV‐2 target sequence of the RT‐qPCR as an example for pitfalls during PCR diagnostics affecting diagnostic specificity. In several purchased in‐house primers/probe sets, quantification cycle values as low as 17 were measured for negative control samples. However, there were also primers/probe sets that displayed very low‐level contaminations, which were detected only during thorough internal validation. Hence, it appears imperative to pre‐test each batch of reagents extensively before use in routine diagnosis, to avoid false‐positive results and low positive predictive value in low‐prevalence situations. As such, contaminations may have happened more widely, and COVID‐19 diagnostic results should be re‐assessed retrospectively to validate the epidemiological basis for control measures.
Quantitative real‐time PCR (qPCR) has been widely used in quantifying bacterial and fungal populations in various ecosystems, as well as the fungi to bacteria ratio (F:B ratio). Recently, researchers ...have begun to apply droplet digital PCR (ddPCR) to this area; however, no study has systematically compared qPCR and ddPCR for quantitating both bacteria and fungi in environmental samples at the same time. Here, we designed probe‐primer pair combinations targeting the 16S rRNA gene and internal transcribed spacer (ITS) for the detection of bacteria and fungi, respectively, and tested both SYBR Green and TaqMan approaches in qPCR and ddPCR methods for mock communities and in real environmental samples. In mock communities, the quantification results of ddPCR were significantly closer to expected values (p < .05), and had smaller coefficients of variations (p < .05) than qPCR, suggesting ddPCR was more accurate and repeatable. In environmental samples, ddPCR consistently quantified ITS and 16S rRNA gene concentrations in all four habitats without abnormal overestimation or underestimation, and the F:B ratio obtained by ddPCR was consistent with phospholipid fatty acid analysis. Our results indicated that ddPCR had better precision, repeatability, sensitivity, and stability in bacterial and fungal quantitation than qPCR. Although ddPCR has high cost, complicated processes and restricted detection range, it shows insensitivity to PCR inhibitors and the potential of quantifying long target fragments. We expect that ddPCR, which is complementary to qPCR, will contribute to microbial quantification in environmental monitoring and evaluation.
Background: MicroRNAs (miRNAs) are an emerging field of interest in many diseases. Some of the miRNAs have been reported to be expressed differentially in diseased states of pregnancy. The current ...study was designed to measure and compare the levels of microRNA 182-3-p, 519-d-5p, and 378-3p and it was hypothesized that the microRNA 182-3-p, 519-d-5p, and 378-3p can be used as a non-invasive predictor of preeclampsia. Methods: Expression level of the miRNAs 182-3-p, 519-d-5p, and 378-3p was measured in the serum of preeclamptic and normal pregnancies by real-time PCR. Data was entered and analysed by Statistical Package for the Social Sciences 22 (SPSS). Results: Significantly high expression levels of MiRNA 182-3p, 519-d-5p and low levels of miR-378-3p were associated with preeclampsia (PE). Conclusion: The results revealed that miR-182-3p is a powerful predictor of PE with an Odds Ratio of 5.9 and can be used as a noninvasive, reliable predictor of PE to screen these patients at an early stage. Screening at early gestation with follow-up studies can emphasize the results
•Alpha-1 antitrypsin deficiency (AATD) remains underdiagnosed.•AATD is mainly caused by PI*Z and PI*S alleles of the SERPINA1 gene.•A novel genotyping test for these variants is described.•This ...method combines allele-specific tailed primers with SYBR-Green.•Genotyping costs are greatly reduced, compared with other available approaches.
Alpha-1 antitrypsin deficiency is an underdiagnosed genetic condition that predisposes to pulmonary complications and is mainly caused by rs28929474 (PI*Z allele) and rs17580 (PI*S allele) mutations in the SERPINA1 gene.
Development of a homogeneous genotyping test for detection of PI*S and PI*Z alleles based on the principles of allele-specific PCR and amplicon melting analysis with a fluorescent dye.
Sixty individuals, which included all possible genotypes that result from combinations of rs28929474 and rs17580 single nucleotide variants, were assayed with tailed allele-specific primers and SYBR Green dye in a real-time PCR machine.
A clear discrimination of mutant and wild-type variants was achieved in the genetic loci that define PI*S and PI*Z alleles. Specific amplicons showed a difference of 2.0 °C in melting temperature for non-S and S variants and of 2.9 °C for non-Z and Z variants.
The developed genotyping method is robust, fast, and easily scalable on a standard real-time PCR platform. While it overcomes the handicaps of non-homogeneous approaches, it greatly reduces genotyping costs compared with other homogeneous approaches.
Nanotechnology covers many disciplines, including the biological sciences. In this study, selenium nanoparticles (Se‐NPs) were synthesized using Artemisia annua extract and investigated against ...clinical strains of klebsiella pneumoniae (K. pneumoniae) for their anti‐biofilm effects. In this experimental study, from May 1998 to September 1998, 50 clinical samples of blood, urine, and sputum were collected, and K. pneumoniae strains were isolated using microbiological methods. Subsequently, the antibacterial effects of Se‐NPs at concentrations of 12‐25‐50‐100/5‐6/3‐25/125 μg/mL were studied. Finally, biofilm‐producing strains were isolated, and the expression of mrkA biofilm gene was studied in real‐time strains treated with Se‐NPs using real‐time polymerase chain reaction (PCR). Out of 50 clinical samples, 20 strains of K. pneumoniae were isolated. Minimum inhibitory concentration (MIC) results of Se‐NPs showed that Se‐NPs were capable of significant cell killing. Real‐time PCR results also showed that mrkA gene expression was significantly reduced in strains treated with Se‐NPs. According to this study, Se‐NPs could reduce bacterial growth and biofilm formation, therefore, could be considered a candidate drug in the medical application for infections caused by K. pneumoniae.
Objectives/Hypothesis
Acute rhinosinusitis is a frequent common cold‐related complication in children. Despite the need for appropriate treatment, its underlying microbiology remains unclear. This ...study aimed to investigate the microbiology of acute rhinosinusitis in children.
Study Design
Prospective non controlled study.
Methods
Thirty‐one pediatric acute maxillary sinusitis patients with severe symptoms were assessed. The subjects were 17 males and 14 females aged 5 to 14 years (mean age, 9.1 years). Maxillary sinus aspirates were collected and cultured, with subsequent viral and bacterial polymerase chain reaction (PCR) analysis. Bacteria were analyzed using culturing and PCR, and viruses were analyzed using PCR. The PCR kits used identify 18 types of respiratory viruses and 13 types of bacteria.
Results
At least one pathogen was detected in 30 of 31 aspirates (97%) using PCR, and none of the aspirates contained respiratory viruses alone. Ten aspirates (32%) contained both viruses and bacteria. The most common viruses detected were rhinovirus (13%) and influenza virus (10%). The most common bacteria were Haemophilus influenzae (45%), Streptococcus pneumoniae (32%), Moraxella catarrhalis (16%), and Chlamydophila pneumoniae (13%). Bacteria were found in 21 of 31 cases (68%) via bacterial culturing. Culturing revealed that H influenzae was the most common pathogen (42%).
Conclusions
In pediatric acute maxillary sinusitis, respiratory bacteria were detected in 65% of the sinus aspirates and both bacteria and viruses in 32%. The most common viruses were rhinovirus and influenza virus, and the most common bacteria were H influenzae and S pneumoniae. Viral and bacterial PCR is useful for accurately investigating the microbiology in pediatric sinusitis.
Level of Evidence
3 Laryngoscope, 131:E2705–E2711, 2021
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