Standardization of an LNA-based TaqMan assay qPCR analysis for Aspiculuris tetraptera DNA in mouse faeces

Abstract

Background: Aspiculuris tetraptera, as a parasitic pinworm, is most frequently detected in laboratory mice, and transmission is mediated by the eggs contained in the faeces of infected mice. A highly sensitive and quantitative faeces-based diagnostic tool would be useful for the early detection of A. tetraptera to inhibit the expansion of infection. In this study, we developed a quantitative assay that exhibits high sensitivity in detecting A. tetraptera in faeces using PCR techniques.

Results: Endpoint PCR demonstrated the detection of A. tetraptera DNA in 0.5 ng genomic DNA extracted from the faeces of infected mice. To quantitatively detect the small amount of A. tetraptera DNA, locked nucleic acid (LNA)-based primers and LNA-based TaqMan probes were used for the quantitative PCR assay (qPCR). The combination of LNA-based DNA increased detection sensitivity by more than 100-fold compared to using normal oligo DNAs. The copy number of the A. tetraptera DNA detected was positively related to the infected faeces-derived genomic DNA with a simple linearity regression in the range of 20 pg to 15 ng of the genomic DNA. To more conveniently detect infection using faeces, the LNA-based TaqMan assay was applied to the crude fraction of the faeces without DNA purification. An assay using ethanol precipitation of the faeces yielded results consistent with those of direct microscopic observation.

Conclusion: The LNA-TaqMan assay developed in this study quantitatively detects A. tetraptera infection in mouse faeces.

Keywords: A. tetraptera; Faeces; Locked nucleic acid; Mice; PCR; TaqMan assay.

Fig. 1

Optimizing PCR experimental conditions to detect A. tetraptera DNA. a-c Synthesized A. tetraptera DNA was amplified with Taq polymerase, as described in the “Materials and methods” section, at the indicated annealing temperature (a) and primer concentrations (b) using the indicated cycle numbers (c), and the products were subsequently observed by agarose gel electrophoresis using ethidium bromide staining. d The bands of the PCR products were quantified using the Metamorph imaging software. The signal intensity was expressed by division of total gray value by number of pixels, and corrected by background intensities. The correlation coefficient indicates the relationship between the band intensity and the copy number of synthesized A. tetraptera DNA in the range from 10 to 1000 copies. Data represent the mean obtained from three independent experiments. The genomic DNA were purified from the faeces of A. tetraptera-positive mice and amplified using the indicated amounts of genomic DNA under the optimal PCR experimental conditions. The PCR products were visualized by agarose gel electrophoresis using ethidium bromide staining (e), and the bands were quantified using the Metamorph imaging software (f). The data represent the mean ± SE obtained from three independent experiments. PC shows the data of the synthesized A. tetraptera DNA in the absence of genomic DNA

Fig. 2

qPCR analysis of A. tetraptera DNA using LNA based-primers and LNA-TaqMan probe. a Schematic of the qPCR method using LNA-based primers and LNA-based TaqMan probe is shown. qPCR was conducted with the combination of the LNA-based primers and LNA-based TaqMan probe, which was modified with FAM, a fluorescent reporter at the 5′-end— and IBFQ, a fluorescence quencher at the 3′-end. The fluorescence of FAM within the probe disappeared by IBFQ during hybridization. The probe was cut using Taq polymerase during the extension reaction, which resulted in the emission of the reporter’s fluorescence. b qPCR was conducted using 100 copies of synthesized A. tetraptera DNA as template DNA in the presence of the indicated fluorescence reagents and primers. The detected RFU values were plotted and the representative data obtained from three independent experiments are shown. c qPCR was conducted using 3-fold serial dilutions of synthesized A. tetraptera DNA from 3000 to 1.37 copies as template DNA in the presence of the indicated fluorescence reagents and primers. The data of the copy numbers were converted to logarithm. The correlation coefficient indicates the relationship between the Ct value and the copy number of the template DNA. The data represent the mean ± SE obtained from 3 independent experiments. d qPCR was conducted using genomic DNA extracted from the faeces of A. tetraptera-infected mice in the presence of LNA-based TaqMan probes and LNA-based primers. The genomic DNA were used with a 3-fold serial dilution that ranges from 15,000 pg to 20.6 pg. The data of the copy numbers were converted to logarithm. The correlation coefficient indicates the relationship between the detected copy number of A. tetraptera DNA and the input genomic DNA. The dashed lines show the 95% confidence interval. The data represent the mean ± SE obtained from three independent experiments

Fig. 3

Development of direct PCR using faeces preparations in mice. a The indicated amounts of healthy mouse faeces were diluted with TE buffer, and the final 1 ng/μl concentration of synthesized A. tetraptera DNA was added to the dilutions. The DNA preparations were amplified using the optimal PCR experimental conditions, and the PCR products were observed, as described in the “Materials and methods” section. b The DNA preparations were treated with heat at 95 °C for 5 min after dilution, as shown in (a), and the PCR was performed, as described in the legend of (a). c One % BSA, 0.1% TritonX-100, or 0.1% Tween-20 was added to the DNA preparations after the dilution, as shown in (a), and then the PCR was performed, as described in the legend of (a). d Ethanol precipitation was conducted after the dilution as shown in (a), and then the PCR was performed as described in the legend of (a). e DNA preparations after ethanol precipitation were quantified using qPCR. qPCR was conducted using the LNA-based TaqMan probes and LNA-based primers as described in the “Materials and methods” section. The data represent the mean ± SE obtained from three independent experiments. f Four micrograms of faeces from healthy mice were added to the 3-fold serial dilutions of genomic DNA extracted from the faeces of A. tetraptera-infected mice (gDNA) from 13.3 ng to 0.5 ng, and subsequently precipitated by ethanol. qPCR was conducted using the LNA-based TaqMan probe and LNA-based primers as described in the “Materials and methods” section. The data of the copy numbers were converted to logarithm. The correlation coefficient indicates the relationship between the input amounts of gDNA and the detected copy numbers. The dashed lines show the 95% confidence interval. The data represent the mean ± SE obtained from three independent experiments

Fig. 4

Screening of A. tetraptera infection in faeces of the breeding mice using the LNA-TaqMan assay. a The indicated A. tetraptera eggs were added into fresh faeces collected from healthy mice and subsequently precipitated by ethanol after the dilution with TE buffer. qPCR was conducted in triplicates using the LNA-based TaqMan probe and LNA-based primers as described in the “Materials and methods” section (left panel). The region of dashed line in the left panel was enlarged (right panel) b The faeces in the cages of the breeding mice were collected and diluted with TE buffer. The dilutions were precipitated by ethanol, and then qPCR was conducted using LNA-based TaqMan probes and LNA-based primers as described in the “Materials and methods” section. Representative data of the qPCR in 3 independent experiments are shown. c The copy number of A.tetraptera DNA in faeces collected from each cage was determined using Ct values as observed in (b). The data represent the mean ± SE obtained from 3 independent experiments. *P < 0.05 (one-way ANOVA with post-hocTukey’s multiple comparison test). d Photographs are representative data of the adult pinworm (1) in the mouse colon and the egg (2) in the faeces that were detected in the cages. Scale bars, 500 μm (1), 50 μm (2)