In this study, loop-mediated isothermal amplification (LAMP) assay in conjunction with calcein for visualized detection of Mycobacterium tuberculosis (MTB) was established. Firstly, four LAMP primers were designed according to the region of 16S rDNA sequences of MTB. Secondly, clinical sputum samples were collected, decontaminated and their DNA was extracted. Thirdly, standard MTB strains were used to evaluate the specificity and sensitivity of LAMP. At the same time, electrophoresis was used for products detection and calcein was used for visualized verification. At last, Chi-squared test function in SPSS 17.0 software was used for consistency evaluation of LAMP assay as compared with the gold standard (culture method). Results showed that there was no nonspecific amplification appeared in the specificity assay and the detection limit was 10 copies/tube in the sensitivity assay. In addition, visualized method by calcein had a comparable sensitivity with that of electrophoresis method. After evaluation of clinical practicability, the sensitivity of LAMP was calculated as 94.74% and the specificity was 90%, respectively. And Chi-squared test showed that LAMP and culture method had no statistic difference, and the two methods were in good consistency (P>0.05). In conclusion, LAMP assay introduced in our study has the characteristics of high efficiency and visualized detection so that this technique has great application prospects in the resource-limited environment, such as work field and primary care hospitals.
Objective To investigate the effect of cholecystolithiasis with cholecystitis and cholecystectomy on intestinal flora in patients with colorectal cancer. Methods A total of 168 patients with colorectal cancer who admitted to the Department of Anorectal Surgery in Gansu Provincial Hospital from June 2020 to March 2021 were selected, and 29 patients with colorectal cancer who met the criteria were selected as the research objects, including 10 colorectal cancer patients with gallstones and cholecystitis (cholecystolithiasis with cholecystitis+colorectal cancer group), 10 colorectal cancer patients after cholecystectomy (cholecystectomy+colorectal cancer group), and 9 colorectal cancer patients with normal gallbladder (normal gallbladder+colorectal cancer group). Clinical data of the patients in three groups were collected and compared. The fresh fecal samples of the patients included in the study were collected, and the 16S rDNA high-throughput sequencing method was used to determine and analyze the composition and distribution of the intestinal flora in the obtained samples. Results The interleukin-6 level in the cholecystolithiasis with cholecystitis+colorectal cancer group was statistically higher than that in the normal gallbladder+colorectal cancer group and the cholecystectomy+colorectal cancer group (P<0.05). At the phylum level of the fecal flora in three groups patients: ① In the samples of three groups, the relative abundances of Bacteroidetes, Firmicutes, Proteobacteria, Fusobacteria and Verrucomicrobia phylums were all high, accounting for almost more than 95% of the total intestinal bacteria. ② The relative abundance of Fusobacteria phylum in the cholecystolithiasis with cholecystitis+colorectal cancer group was statistically higher than that in the normal gallbladder+colorectal cancer group (P<0.05). ③ The relative abundance of Verrucomicrobia phylum in the normal gallbladder+colorectal cancer group was statistically higher than that in the cholecystolithiasis with cholecystitis+colorectal cancer group and the cholecystectomy+colorectal cancer group (P<0.05). ④ The relative abundance of Synergistetes phylum in the cholecystectomy+colorectal cancer group was statistically higher than that in the cholecystolithiasis with cholecystitis+colorectal cancer group and the normal gallbladder+colorectal cancer group (P<0.05). At the genus level: ① The relative abundances of Bacteroidetes and Roseburia genus were lower in the gallstone with cholecystitis+colorectal cancer group than those in the cholecystectomy+colorectal cancer group and the normal gallbladder+colorectal cancer group (P<0.05). ② The relative abundance of Shigella genus in the cholecystectomy+colorectal cancer group was higher than that in the cholecystolithiasis with cholecystitis+colorectal cancer group (P<0.05). ③ The relative abundance of the Lachnospira genus in the cholecystolithiasis with cholecystitis+colorectal cancer group was lower than that in the normal gallbladder+colorectal cancer group (P<0.05). ④ The relative abundances of Prevotella and Fusobacteria genus were higher in the cholecystolithiasis with cholecystitis+colorectal cancer group than that in the cholecystectomy+colorectal cancer group and the normal gallbladder+colorectal cancer group (P<0.05). ⑤ The relative abundances of Clostridium and Akkermansia genus were lower in the cholecystolithiasis with cholecystitis+colorectal cancer group and the cholecystectomy+colorectal cancer group than that in the normal gallbladder+colorectal cancer group (P<0.05). ⑥ The relative abundance of Enterococcus genus was higher in the normal gallbladder+colorectal cancer group than that in the cholecystectomy+colorectal cancer group (P<0.05).Conclusions ① Long-term occurrence of cholecystolithiasis with cholecystitis can cause obvious decrease in the abundances of Bacteroides, Roseburia, Lachnospira, etc. ② Cholecystectomy can cause changes in the relative abundances of Clostridium, Enterococcus, Verrucomicrobia, Synergistetes, etc. ③ The relative abundance of Fusobacterium is obviously increased in colorectal cancer patients with gallstones and cholecystitis, then promotes the release of inflammatory cytokines and causes intestinal inflammation, which is conducive to the growth of opportunistic pathogens, thus may affect the occurrence and development of colorectal cancer.
ObjectiveTo explore the composition of intestinal microbiota between patients with fixed airflow obstruction asthma, reversible airflow obstruction asthma, and healthy control, and analyze the correlation between key differential bacterial distribution and clinical characteristics. MethodsFifteen patients with fixed airflow obstruction asthma (FAO) and 13 patients with reversible airflow obstruction asthma (RAO) were included, along with 11 matched healthy control subjects. Clinical data were collected, and lung function tests and induced sputum examination were performed. Blood and stool samples were tested to compare the gut microbiota status among the groups, and analyze the relationship between gut microbiota abundance and patients' blood routine, IgE levels, lung function, and induced sputum. Results The dominant bacterial compositions were similar in the three groups, but there were differences in the abundance of some species. Compared to the RAO group, the FAO group showed a significant increase in the genera of Bacteroides and Escherichia coli, while Pseudomonas was significantly decreased. The phylum Firmicutes was negatively correlated with the course of asthma, while the phylum Bacteroidetes and genus Bacteroides were positively correlated with the asthma course. Bacteroidetes was negatively correlated with Pre-BD FEV1/FVC, Pseudomonas was positively correlated with Pre-BD FEV1, Escherichia coli was negatively correlated with Post-BD FEV1/FVC, and Bacteroides was negatively correlated with Post-BD MMEF. The class Actinobacteria and the order Actinomycetales were negatively correlated with peripheral blood EOS%, while the order Enterobacteriales and the family Enterobacteriaceae were positively correlated with peripheral blood IgE levels. Furthermore, Actinobacteria and Actinomycetales were negatively correlated with induced sputum EOS%. Conclusions There are differences in the gut microbiota among patients with fixed airflow obstruction asthma, reversible airflow obstruction asthma, and healthy individuals. Bacteroides and Escherichia coli are enriched in the fixed airflow obstruction asthma group, while the Firmicutes are increased in the reversible airflow obstruction asthma group. These three microbiota may act together on Th2 cell-mediated inflammatory responses, influencing the process of airway remodeling, and thereby interfering with the occurrence of fixed airflow obstruction in asthma.