Detection of Eight Respiratory Bacterial Pathogens Based on Multiplex Real-Time PCR with Fluorescence Melting Curve Analysis

Hu, Liuyang; Han, Bing; Tong, Qin; Xiao, Hui; Cao, Deliang

doi:10.1155/2020/2697230

Cited by 15 publications

(13 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…pneumoniae, H. influenzae, E. coli, Staph. aureus and Pseudomonas aeruginosa are the main bacterial species that have been frequently reported to cause respiratory infections associated with morbidity and mortality in developing countries (12).…”

Section: Introductionmentioning

confidence: 99%

Isolation and antimicrobial resistance of Staphylococcus spp., enteric bacteria and Pseudomonas spp. associated with respiratory tract infections of sheep

Aziz¹,

Lafta²

2021

IJVS

View full text Add to dashboard Cite

Sheep are considered as an important part of livestock in the worldwide, particularly in Iraq, as they provide meat, milk, leather, wool, and manure. The present study aim is isolation and identification of staphylococci, enteric bacteria and Pseudomonas spp. Totally, 115 samples were collected from sheep (100 samples were collected from the nasal cavity of local sheep suffering from respiratory infections, and 15 samples were collected from apparently healthy local sheep). All the samples were collected from seven flocks located in Abu Ghraib and Al-Radwaniyah, Baghdad governorate, Iraq. The samples were taken during the period from October 2020 to February 2021. Staphylococcus spp., Pseudomonas spp., and enteric bacteria were detected firstly by using classical diagnostic methods, and secondly they were identified to the species level by using the corresponding analytical profile index (API 20 staph, API 20NE and API 20E) and via the Vitek2 system. Importantly, from nasal swabs, 79% bacterial isolates were obtained, including Enterobacter cloacae, Escherichia coli, Raoutltella planticola, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus among others. The antimicrobial susceptibility test showed multi-drug resistant S. aureus, P. aeruginosa and K. pneumoniae. To conclude, several aerobic bacteria were isolated from the nasal cavity of diseased sheep. An extensive study is needed to determine the economic and public health impacts of these bacteria.

show abstract

Section: Introductionmentioning

confidence: 99%

Isolation and antimicrobial resistance of Staphylococcus spp., enteric bacteria and Pseudomonas spp. associated with respiratory tract infections of sheep

Aziz¹,

Lafta²

2021

IJVS

View full text Add to dashboard Cite

show abstract

“…This allows us to overcome the use of high doses of nanomaterials, otherwise low safe doses in conjugation with natural benefit materials as the combination of ZnONPs with Curcumin and enable to inhibit the viability of virulent genes expression as detected here by PCR. Similarly, the PCR enables the rapid detection of pathogens' genes as recommended by Hu et al (2020), Hassan et al (2021Hassan et al ( , 2022 who found that the use of nanocomposites with natural plant materials caused significant changes in aflatoxin regulatory genes. Other studies illustrated that the traditional antifungals and herbs against C. albicans increased the activity of secreted proteinase (Sap) and SAP genes, which can be observed by PCR (Copping et al 2005;Saleh 2011).…”

Section: Resultsmentioning

confidence: 99%

Evaluation of Zinc Oxide Nano particles and Curcumin effect on some virulence genes of Staphylococcus aureus and Candida albicans causing subclinical mastitis in buffalo

El-Hamaky

Mohamed²,

El-Khalek³

et al. 2023

Egyptian Journal of Agricultural Research

View full text Add to dashboard Cite

Nowadays, there are progressive advancements in the biomedical employment of nanotechnology in human and animal health. Today, the dangers of infectious animal diseases and the continuous application of traditional antibiotics resulted in the elevation of the activities of the drugs resistant genes and the failure of their control. Hence, it is necessary to prevent fungal and bacterial pathogens pollution and their toxicity in dairy animal products. Sixty samples were collected from dairy buffalo farms suffering from subclinical mastitis (20 Samples of each from subclinical mastitis milk, animal feeds, and bedding). The species of Staphylococcus is considered the most predominant species, and Staphylococcus aureus was the most prevalent bacterial isolate. While, yeast, especially Candida albicans, was the most dominant fungal isolate from different samples of dairy buffalo farms. Our antibiogram of S. aureus and Candida albicans showed a high resistance rate against several traditional antimicrobial agents. Therefore, searching for effective drug materials to overcome drug resistance is needed. Herein, ZnONPs and Curcumin are evaluated singly and, in a composite, to inhibit the growth of C. albicans and S. aureus. The inhibitory dose of ZnONPs for C. albicans and S. aureus were (600 and 600 μg /ml) and in Curcumin were (2% and 3%), respectively. However, the hybrid nanocomposite of ZnONPs (100 μg/ml) with Curcumin (0.25% and 1%) caused suppression of the growth of these pathogens. The nanocomposite of low levels of ZnONPs (100 μg /ml) with (0.25% curcumin) significantly increased the inhibitory effects against C. albicans and S. aureus. The molecular study to detect the efficiency of the treatment doses indicated that the exposure of C. albicans and S. aureus to a low dose of ZnONPs (100 µg/ml) did not affect the DNA expression of virulent genes. While the high dose (600 µg/ml) prevented the DNA expression. However, in the Curcumin treatments of isolates with low doses (0.25), the genes' DNA expression was not affected. While, the high dose (1%), removed the DNA band. All inhibition and removal of virulent genes' DNA of pathogens can be obtained by the combination of low levels of ZnONPs and Curcumin. Hence, it is concluded that the nanocomposites of ZnONPs and Curcumin caused significant effects against pathogens.

show abstract

“…We have also demonstrated that the sensitivity of this assay is considerably higher than that of standard qPCR (LoD of approximately 64 CFU/mL). (11,12) It is a limitation of this study that the Droplet Reader only has two channels, which makes it di cult to do a probe-based multiplexing with more than two targets. However, a recent study by Nyaruaba et al 2021 has demonstrated that an amplitude multiplexing is possible for different SARS-CoV-2 applications by varying the primer-probe concentrations for each target.…”

Section: Discussionmentioning

confidence: 99%

Development of Duplex Droplet Digital PCR for Rapid Detection of Blood Stream Infections Caused by S. aureus, E. coli, S. pneumoniae and P. aeruginosa

Badran

Andersen

Chen

et al. 2022

Preprint

View full text Add to dashboard Cite

Purpose: The aim of this study was to develop highly specific primer-probes pairs for the detection of species-specific genes, and establish a multiplex ddPCR assay for the detection of low copies of genomic DNA, of common bacteria causing bloodstream infections in our institution, i.e. Staphylococcus aureus, Streptococcus pneumonia, Escherichia coli, and Pseudomonas aerugnosa..Methods: Pubmed and GenBank databases were used to select well-characterized candidate genes for each microorganism. The newly designed primer-probe pairs were designed using AllelID 7.85 software. The designed primer-probe pairs were tested and validated using Droplet Digital PCR QX200 by using quantitative ATCC genomic DNA from S. aureus (ATCC® 29213DQ™), S. pneumoniae (ATCC® 700669DQ™), E. coli (ATCC® 25922DQ™), and P. aeruginosa (ATCC® 27853DQ™).Results: All primer-probe pairs demonstrated an optimal annealing temperature at 56°C. The ddPCR detected 162 copies for S. aureus and 152 copies S. pneumonia in singleplex reactions as compared to 152 copies and 132 copies in duplex reactions, respectively, and 324 copies for P. aeruginosa, 30 copies for E. coli in singleplex reactions as compared to 356 copies and 34 copies, respectively, in duplex reactions. The results of the limit of detection of the ddPCR assay was as low as 1 copy/reaction for S. aureus, E. coli, and P. aeruginosa and 2 copies/reaction for S. pneumonia. Conclusion: In this study, we have designed highly specific PP pairs for use in a ddPCR assay for the accurate and reliable detection and quantification of species-specific genes for four common blood culture pathogens in our institution.

show abstract

Detection of Eight Respiratory Bacterial Pathogens Based on Multiplex Real-Time PCR with Fluorescence Melting Curve Analysis

Cited by 15 publications

References 38 publications

Isolation and antimicrobial resistance of Staphylococcus spp., enteric bacteria and Pseudomonas spp. associated with respiratory tract infections of sheep

Isolation and antimicrobial resistance of Staphylococcus spp., enteric bacteria and Pseudomonas spp. associated with respiratory tract infections of sheep

Evaluation of Zinc Oxide Nano particles and Curcumin effect on some virulence genes of Staphylococcus aureus and Candida albicans causing subclinical mastitis in buffalo

Development of Duplex Droplet Digital PCR for Rapid Detection of Blood Stream Infections Caused by S. aureus, E. coli, S. pneumoniae and P. aeruginosa

Contact Info

Product

Resources

About