Establishment of an Analytical System for the Human Fecal Microbiota, Based on Reverse Transcription-Quantitative PCR Targeting of Multicopy rRNA Molecules

ABSTRACT An analytical system based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) was established for the precise evaluation of human intestinal microbiota. Group- and species-specific primer sets for Clostridium perfringens, Lactobacillus spp. (six subgroups and three species), Enterococcus spp., and Staphylococcus spp. targeting 16S rRNA gene sequences were newly developed for the quantitative analysis of such subdominant populations in human intestines. They were used together with previously reported group-specific primer sets for Enterobacteriaceae, Pseudomonas spp., and six predominant bacterial groups (the Clostridium coccoides group, the Clostridium leptum subgroup, the Bacteroides fragilis group, Bifidobacterium spp., the Atopobium cluster, and Prevotella spp.) for the examination of fecal samples from 40 healthy adults by RT-qPCR with lower detection limits of 102 to 104 cells per g of feces. The RT-qPCR method gave data equivalent to those yielded by qPCR for predominant populations of more than 108 cells per g of feces and could quantify bacterial populations that were not detectable (Staphylococcus and Pseudomonas) or those only detected at lower incidences (Prevotella, C. perfringens, Lactobacillus, and Enterococcus) by qPCR or the culture method. The RT-qPCR analysis of Lactobacillus spp. at the subgroup level revealed that a subject has a mean of 4.6 subgroups, with an average count of log10(6.3 ± 1.5) cells per g of feces. These results suggest that RT-qPCR is effective for the accurate enumeration of human intestinal microbiota, especially the entire analysis of both predominant and subdominant populations.

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Sensitive Quantification of Clostridium difficile Cells by Reverse Transcription-Quantitative PCR Targeting rRNA Molecules

Applied and Environmental Microbiology ◽

10.1128/aem.07990-11 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5111-5118 ◽

Cited By ~ 31

Author(s):

Kazunori Matsuda ◽

Hirokazu Tsuji ◽

Takashi Asahara ◽

Takuya Takahashi ◽

Hiroyuki Kubota ◽

...

Keyword(s):

Clostridium Difficile ◽

Quantitative Pcr ◽

Reverse Transcription ◽

Care Facility ◽

23S Rrna ◽

Rrna Gene ◽

Carriage Rate ◽

Content Type ◽

Reverse Transcription Quantitative Pcr ◽

Pcr Targeting

ABSTRACTWe established a sensitive and accurate quantification system forClostridium difficilein human intestines, based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR). We newly developed a species-specific primer set forC. difficiletargeting 23S rRNA gene sequences. Both the vegetative cells and the spores ofC. difficilein human feces were quantified by RT-qPCR, with a lower detection limit of 102.4cells/g of feces. In an analysis of the feces of residents (n= 83; age, 85 ± 8 years) and staff (n= 19; age, 36 ± 10 years) at a care facility for the elderly,C. difficilewas detected by RT-qPCR in 43% of the residents (average count, log104.0 ± 2.0 cells/g of feces) and 16% of the staff (average count, log102.2 ± 0.1 cells/g of feces); these rates were far higher than those detected by qPCR (residents, 19%; staff, 0%) or selective cultivation (residents, 18%; staff, 5%). Another analysis of healthy adults (n= 63; age, 41 ± 11 years) also revealed the significant carriage rate ofC. difficilein the intestines (detection rate, 13%; average count, log104.9 ± 1.2 cells/g of feces). From these results, it was suggested that rRNA-targeted RT-qPCR should be an effective tool for analyzing population levels ofC. difficilein the human intestine.

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Development of a rapid and sensitive analytical system for Pseudomonas aeruginosa based on reverse transcription quantitative PCR targeting of rRNA molecules

Emerging Microbes & Infections ◽

10.1080/22221751.2021.1906164 ◽

2021 ◽

pp. 1-43

Author(s):

Mai Niikura ◽

Satomi Atobe ◽

Akira Takahashi ◽

Yukiko Kado ◽

Takuya Sugimoto ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quantitative Pcr ◽

Reverse Transcription ◽

Reverse Transcription Quantitative Pcr ◽

Analytical System ◽

Pcr Targeting

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Measurement of 2-methylthio Modifications in Mitochondrial Transfer RNAs by Reverse-transcription Quantitative PCR

BIO-PROTOCOL ◽

10.21769/bioprotoc.1695 ◽

2016 ◽

Vol 6 (1) ◽

Author(s):

Fan-Yan Wei ◽

Kazuhito Tomizawa

Keyword(s):

Quantitative Pcr ◽

Reverse Transcription ◽

Transfer Rnas ◽

Mitochondrial Transfer ◽

Reverse Transcription Quantitative Pcr

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Establishment and Application of Rapid Diagnosis for Reverse Transcription-Quantitative PCR of Newly Emerging Goose-Origin Nephrotic Astrovirus in China

mSphere ◽

10.1128/msphere.00380-18 ◽

2018 ◽

Vol 3 (6) ◽

Cited By ~ 4

Author(s):

Xiaoyuan Yuan ◽

Kai Meng ◽

Yuxia Zhang ◽

Lihong Qi ◽

Wu Ai ◽

...

Keyword(s):

Quantitative Pcr ◽

Clinical Application ◽

Reverse Transcription ◽

Test Sample ◽

Sensitivity Testing ◽

Content Type ◽

Reverse Transcription Quantitative Pcr ◽

Emerging Virus ◽

New Type ◽

And Control

ABSTRACT In 2017, a new type of goose-origin astrovirus (GoAstV) that is completely different from previously identified avian astroviruses (which have only 30.0% to 50.5% homology with GoAstV) has been isolated from diseased geese in China. This disease can cause joint swelling in sick geese, and the anatomy shows a clear precipitation of urate in the kidney. The rate of death and culling can reach more than 30%, revealing the disease’s severe pathogenicity. To quickly and accurately diagnose the newly emerging disease, we established a highly specific reverse transcription-quantitative PCR (RT-qPCR) method of detecting GoAstV. Sensitivity testing showed that the minimum amount of test sample for this method is 52.5 copies/μl. Clinical application confirmed that this method can quickly and effectively detect GoAstV, providing a diagnostic platform for the prevention and control of goose disease. IMPORTANCE Goose-origin astrovirus (GoAstV), as a newly emerging virus in 2017, is different from previously known astroviruses in the genus Avastrovirus. So far, few studies have focused on the novel virus. Considering the infectious development of astrovirus (AstV), we established a reverse transcription-quantitative PCR (RT-qPCR) assay with a strong specificity to quickly and accurately diagnose GoAstV. Confirmed by clinical application, this method can quickly and accurately detect prevalent GoAstV. The assay is thus convenient for clinical operation and is applicable to the monitoring of GoAstV disease.

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