scholarly journals Airway Bacteria Quantification Using Polymerase Chain Reaction Combined with Neutrophil and Eosinophil Counts Identifies Distinct COPD Endotypes

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1337
Author(s):  
Augusta Beech ◽  
Simon Lea ◽  
Jian Li ◽  
Natalie Jackson ◽  
Alex Mulvanny ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Bacterial Load ◽  
Bacterial Colonisation ◽  
Chain Reaction ◽  
Cell Counts ◽  
Copd Patients ◽  
Polymerase Chain ◽  
Eosinophil Counts

Background: Chronic obstructive pulmonary disease (COPD) inflammatory endotypes are associated with different airway microbiomes. We used quantitative polymerase chain reaction (qPCR) analysis of sputum samples to establish the bacterial load upper limit in healthy controls; these values determined the bacterial colonisation prevalence in a longitudinal COPD cohort. Bacteriology combined with sputum inflammatory cells counts were used to investigate COPD endotypes. Methods: Sixty COPD patients and 15 healthy non-smoking controls were recruited. Sputum was analysed by qPCR (for Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae and Psuedomonas aeruginosa) and sputum differential cell counts at baseline and 6 months. Results: At baseline and 6 months, 23.1% and 25.6% of COPD patients were colonised with H. influenzae, while colonisation with other bacterial species was less common, e.g., S. pneumoniae—1.9% and 5.1%, respectively. H. influenzae + ve patients had higher neutrophil counts at baseline (90.1% vs. 67.3%, p < 0.01), with similar results at 6 months. COPD patients with sputum eosinophil counts ≥3% at ≥1 visit rarely showed bacterial colonisation. Conclusions: The prevalence of H. influenzae colonisation was approximately 25%, with low colonisation for other bacterial species. H. influenzae colonisation was associated with sputum neutrophilia, while eosinophilic inflammation and H. influenzae colonisation rarely coexisted.

Rapid estimation of spoilage bacterial load in aerobically stored meat by a quantitative polymerase chain reaction

1997 ◽  
Vol 82 (3) ◽  
pp. 359-364 ◽  
Author(s):  
K.S. VENKITANARAYANAN ◽  
C. FAUSTMAN ◽  
J.F. CRIVELLO ◽  
M.I. KHAN ◽  
T.A. HOAGLAND ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Bacterial Load ◽  
Chain Reaction ◽  
Rapid Estimation ◽  
Polymerase Chain

scholarly journals Detection of novel strains genetically related to Anaplasma platys in Tunisian one-humped camels (Camelus dromedarius)

2015 ◽  
Vol 9 (10) ◽  
pp. 1117-1125 ◽  
Author(s):  
Hanène Belkahia ◽  
Mourad Ben Said ◽  
Lotfi Sayahi ◽  
Alberto Alberti ◽  
Lilia Messadi
Keyword(s):  
Polymerase Chain Reaction ◽  
16S Rrna ◽  
Quantitative Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Chain Reaction ◽  
Rrna Sequence ◽  
16S Rrna Sequence ◽  
Nested Polymerase Chain Reaction ◽  
16S Rrna Sequence Analysis ◽  
Polymerase Chain

Introduction: Little information is currently available regarding the presence of Anaplasma species in North African dromedaries. To fill this gap in knowledge, the prevalence, risk factors, and genetic diversity of Anaplasma species were investigated in Tunisian dromedary camels. Methodology: A total of 226 camels from three different bioclimatic areas were sampled and tested for the presence of Anaplasma species by quantitative polymerase chain reaction (qPCR) and nested polymerase chain reaction (nPCR) assays. Detected Anaplasma strains were characterized by 16S rRNA sequence analysis. Results: Overall infection rate of Anaplasma spp. was 17.7%, and was significantly higher in females. Notably, A. marginale, A. centrale, A. bovis, and A. phagocytophilum were not detected. Animals were severely infested by three tick species belonging to the genus Hyalomma (H. dromedarii, H. impeltatum, and H. excavatum). Alignment, similarity comparison, and phylogenetic analysis of the 16S rRNA sequence variants obtained in this study suggest that Tunisian dromedaries are infected by more than one novel Anaplasma strain genetically related to A. platys. Conclusions: This study reports the presence of novel Anaplasma sp. strains genetically related to A. platys in dromedaries from various bioclimatic areas of Tunisia. Findings raise new concerns about the specificity of the direct and indirect diagnostic tests routinely used to detect different Anaplasma species in ruminants and provide useful molecular information to elucidate the evolutionary history of bacterial species related to A. platys.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Development of a Genoserotyping Method for Salmonella Infantis Detection on the Basis of Pangenome Analysis

2020 ◽  
Vol 9 (1) ◽  
pp. 67
Author(s):  
Seung-Min Yang ◽  
Jiwon Baek ◽  
Eiseul Kim ◽  
Hyeon-Be Kim ◽  
Seyoung Ko ◽  
...  
Keyword(s):  
Public Health ◽  
Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Cost Effective ◽  
The Other ◽  
Gene Marker ◽  
Diagnostic Assay ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Substantial Economic Burden

In recent years, Salmonella Infantis has become a predominant serovariant in clinical and poultry isolates, thereby imposing a substantial economic burden on both public health and the livestock industry. With the aim of coping with the steep increase in serovar Infantis prevalence, a polymerase chain reaction (PCR)-based rapid and accurate diagnostic assay was developed in this study through pangenome profiling of 60 Salmonella serovars. A gene marker, SIN_02055, was identified, which is present in the S. Infantis genome but not in the pangenome of the other serovars. Primers specific to SIN_02055 were used to accurately detect serovar Infantis, and to successfully differentiate Infantis from the other 59 serovars in real-time PCR with a R2 of 0.999 and an efficiency of 95.76%. The developed method was applied to 54 Salmonella strains belonging to eight dominant serovars, and distinguished Infantis from the other seven serovars with an accuracy of 100%. The diagnostic primer set also did not show false positive amplification with 32 strains from eight non-Salmonella bacterial species. This cost-effective and rapid method can be considered an alternative to the classic serotyping using antisera.

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