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

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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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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Quantification of Yeast and Bacterial Gene Transcripts in Retail Cheeses by Reverse Transcription-Quantitative PCR

Applied and Environmental Microbiology ◽

10.1128/aem.02360-12 ◽

2012 ◽

Vol 79 (2) ◽

pp. 469-477 ◽

Cited By ~ 9

Author(s):

Christophe Monnet ◽

Cécile Straub ◽

Jessie Castellote ◽

Djamila Onesime ◽

Pascal Bonnarme ◽

...

Keyword(s):

Quantitative Pcr ◽

Reverse Transcription ◽

Debaryomyces Hansenii ◽

Gene Expression Measurement ◽

Gene Transcript ◽

Bacterial Gene ◽

Content Type ◽

Total Rna ◽

Reverse Transcription Quantitative Pcr ◽

Gene Transcripts

ABSTRACTThe cheese microbiota contributes to a large extent to the development of the typical color, flavor, and texture of the final product. Its composition is not well defined in most cases and varies from one cheese to another. The aim of the present study was to establish procedures for gene transcript quantification in cheeses by reverse transcription-quantitative PCR. Total RNA was extracted from five smear-ripened cheeses purchased on the retail market, using a method that does not involve prior separation of microbial cells. 16S rRNA and malate:quinone oxidoreductase gene transcripts ofCorynebacterium casei,Brevibacterium aurantiacum, andArthrobacter arilaitensisand 26S rRNA and beta tubulin gene transcripts ofGeotrichum candidumandDebaryomyces hanseniicould be detected and quantified in most of the samples. Three types of normalization were applied: against total RNA, against the amount of cheese, and against a reference gene. For the first two types of normalization, differences of reverse transcription efficiencies from one sample to another were taken into account by analysis of exogenous control mRNA. No good correlation was found between the abundances of target mRNA or rRNA transcripts and the viable cell concentration of the corresponding species. However, in most cases, no mRNA transcripts were detected for species that did not belong to the dominant species. The applications of gene expression measurement in cheeses containing an undefined microbiota, as well as issues concerning the strategy of normalization and the assessment of amplification specificity, are discussed.

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