ABSTRACT
@#Abstract: Objective To establish the duplex TaqMan RT-PCR method for detection of Entamoeba histolytica and Giardia lamblia in fecal samples. Methods Primer pairs and probes for Entamoeba histolytica and Giardia lamblia were designed and duplex TaqMan RT-PCR amplification system was constructed. PCR products were inserted into the pUC57 plasmid, and the lower limit of detection of the method was determined. Clinical stool samples were tested in order to evaluated the efficacy of the method. Results The detection limits of duplex TaqMan RT-PCR were 31.6 copies/μL for Entamoeba histolytica and 32 copies/μL for Giardia lamblia, respectively. Of the total of 212 clinical stool samples tested, all 3 samples with E. histolytica-positive patients by microscopy were positive by PCR, while 1 from 209 samples with E. histolytica-negative patients by microscopy were positive by PCR, and the remaining samples were negative. For Giardia lamblia, all 8 samples positive by microscopy were positive by PCR, and 1 from 204 sample with a microscopy-negative patient was positive by PCR, and the remaining samples were negative. The amplification product sequencing and blast analysis were used to confirm that the amplified sequence in the specimen of a patient with negative microscopy but positive PCR belongs to the targeted pathogen, supported by clinical symptoms and laboratory test results. PCR results for other diarrhea-causing pathogens were negative, indicating no cross-reactivity. Conclusions The dual TaqMan RT-PCR method developed in this study can not only detect microscopy-positive samples of Entamoeba histolytica and Giardia lamblia but also can detect samples that were missed by microscopy, with higher sensitivity than the microscopy method. Further, this detection method does not cross-react with other diarrhea-causing pathogens, including cross-react with other diarrhea-causing pathogens including Iodamoeba butschlii, Blastocystis hominis, Plesiomonas, Aeromonas, Salmonella, Shigella, Sphaerozoum fuscum, and Entamoeba hartmani, thus has a good specificity.
ABSTRACT
The Flanders virus (FLAV) is a number of family Rhabdoviridae ,contains a single‐stranded ,negative‐sense vi‐ral RNA .Here we describe a molecular detection method developed for fast measurement of FLAV based on Taqman RT‐PCR method .In this study ,FLAV specific primers and probe were designed based on the FLAV L gene sequences published in GeneBank .Quantitative standard curve of FLAV TaqMan PCR was also successfully established .The specificity and stability test showed that the system is specific and the coefficient variables were all less than 1 .7% .Quantitative standard curve based on the genomic copy was drawn ,and the lowest detectable limit (LOD) of system was 100 copies/PCR ,with higher sensitivity and stability than that of the conventional RT‐PCR assay targeting the same gene .
ABSTRACT
Ebola virus (EBOV) and Marburg virus (MARV) are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates and there is currently no licensed vaccine or therapeutics.To date,there is no specific laboratory diagnostic test in China,while there is a national need to provide differential diagnosis during outbreaks and for instituting acceptable quarantine procedures.In this study,the TaqMan RT-PCR assays targeting the nucleoprotein genes of the Zaire Ebolavirus (ZEBOV) and MARV were developed and their sensitivities and specificities were investigated.Our results indicated that the assays were able to make reliable diagnosis over a wide range of virus copies from 103 to 109,corresponding to the threshold of a standard RNA transcript.The results showed that there were about 1010 RNA copies per milliliter of virus culture supernatant,equivalent to 10,000 RNA molecules per infectious virion,suggesting the presence of many non-infectious particles.These data indicated that the TaqMan RT-PCR assays developed in this study will be suitable for future surveillance and specific diagnosis of ZEBOV and MARV in China.