Touchdown pcr microbiome7/24/2023 ![]() For example, numerous studies of microbial ecology show that WGA using phi29 DNA polymerase enriches environmental template DNAs with the highest amplification efficiency, and the amplified DNA can be used to characterize the microbial community structure in low-biomass environments. Whole genome amplification (WGA) using phi29 DNA polymerase overcomes insufficient sample sizes. However, the low biomass obtained from oropharyngeal swabs impedes the application of the DGGE technique for characterizing microbial populations of the oropharyngeal mucosa. Therefore, it is a preferred technique for studies of the composition, structure, and stability of complex microbial communities. Moreover, polymerase chain reaction (PCR) amplicons are isolated from the DGGE profile, which can be further amplified and sequenced. DGGE facilitates rapid analyses and comparisons of microbial communities. Moreover, samples are acquired with minimal disturbance of the microbiome, and the technique exposes patients to minimal risk.ĭenaturing gradient gel electrophoresis (DGGE) is a DNA fingerprinting technique used to accurately assess the members of a microbial community by generating patterns or profiles of genetic diversity. A better understanding of the significance of variations of the oropharyngeal mucosal microbiome in preclinical conditions and disease may provide insights into selective oropharyngeal decontamination that is used to prevent susceptible patients from contracting pneumonia. The importance of the microbial composition of the oral and respiratory tracts is increasingly considered as a source of biomarkers to facilitate noninvasive detection of disease. The oropharynx serves the respiratory and digestive systems and is colonized by bacterial pathogens that affect healthy or immunocompromised individuals. Studying the microbial variations of patients with pneumonia is challenging, because sampling the lung microbiome requires an invasive surgical procedure that may harm subjects. Although the presence of pathogens is a prerequisite for infections and alterations of the community, which may lead to overgrowth and invasion, it is a key factor leading to infection. These organisms live in a complex, yet balanced relationship, and therefore manipulation of one may affect the other members of the community. However, the upper and lower respiratory tracts harbor a vast range of commensal and potentially pathogenic bacteria that forms an indispensable part of the human microbiome. Current treatment strategies for pneumonia are based on the routine identification of cultured bacteria, including the isolation of clinically significant bacterial species from sputum. Increasing awareness of the role of the microbiome of humans in the progression of liver and lung inflammation raises the importance of assessing the composition of the respiratory microbiome as well as the nature of disease-induced changes caused by these microbiomes during disease progression. Pneumonia is a far more common complication in patients with decompensated cirrhosis. Liver disease and associated complications represent a major healthcare burden in China. Quantitative polymerase chain reaction assays revealed that the populations of Bacteroides, Neisseria, and Actinomycetes increased, while that of Streptococcus decreased in cirrhotic patients with pneumonia versus others ( p < 0.001, versus Healthy controls p < 0.01, versus cirrhotic patients without pneumonia). Moreover, we identified variants of Bacteroides, Eubacterium, Lachnospiraceae, Neisseria, Actinomyces, and Streptococcus through phylogenetic analysis. Further, species richness and the value of Shannon’s diversity and evenness index increased significantly in patients with cirrhosis and pneumonia versus others ( p < 0.001, versus healthy controls p < 0.01, versus cirrhotic patients without pneumonia). The microbial composition of cirrhotic patients with pneumonia differed from those of others and clustered together in subgroup analysis. Whole genome amplification combined with denaturing gradient gel electrophoresis analysis monitored successfully oropharyngeal microbial variations and showed that the composition of each subject’s oropharyngeal microbiome remained relatively stable during the follow-up.
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