Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes

Cai, Xiaoming; Yu, Hai-Qiong; Bai, Jian-Shan; Tang, Jing; Hu, Xuchu; Chen, Dinghu; Zhang, Renli; Chen, Mu-Xin; Ai, Lin; Zhu, Xingquan

doi:10.1016/j.parint.2011.06.010

Cited by 30 publications

(23 citation statements)

References 17 publications

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“…The detection limit of our method was as little as one C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample, equivalent to 10 and 20 EPG, respectively. This result is similar with the egg detection limits in fecal samples of previous reports of 5-10 EPG by singleplex Taqman probe-based real-time PCR assay for C. sinensis eggs (Cai et al 2012;Rahman et al 2011) or of 10 EPG by singleplex FRET-probe-based real-time PCR assay for O. viverrini eggs (Intapan et al 2009a). Variations in the detection limits noted in various reports were possibly due to the analytical sensitivity examined the parasite DNA spiked in distilled water or fecal samples.…”

Section: Discussionsupporting

confidence: 89%

“…The real-time FRET PCR was sensitive enough to detect four copies of each artificial positive template controls. The detection limit for C. sinensis and O. viverrini genomic DNA was both 0.2 pg, which is quite similar to the detection ranges of 0.1-1 pg of C. sinensis genomic DNA (Cai et al 2012;Rahman et al 2011) and far smaller than that of O. viverrini genomic DNA in a real-time PCR method, which was 30 pg (Intapan et al 2009a). The detection limit of our method was as little as one C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample, equivalent to 10 and 20 EPG, respectively.…”

Section: Discussionsupporting

confidence: 74%

“…Some of the examples are multiplex PCR approaches (Le et al 2006), PCR and sequencing of nuclear ribosomal and mitochondrial DNA (Park 2007) and nuclear marker sequences (PM-int9) (Shekhovtsov et al 2009), PCR-RFLP analysis of the 18S-ITS1-5.8S nuclear ribosomal DNA region (Kang et al 2008), and PCR targeting ribosomal DNA ITS regions (Sato et al 2009). A rapid, high-throughput, specific, and sensitive realtime PCR has been applied for the detection of C. sinensis (Kim et al 2009;Cai et al 2012;Rahman et al 2011) and O. viverrini (Intapan et al 2008a, 2008b, 2009aSuksumek et al 2008;Sri-Aroon et al 2011;Janwan et al 2011) DNA. However, those methods have been applied for detecting both parasites in separate assays and are useful in endemic areas of either C. sinensis or O. viverrini alone but may be problematic where C. sinensis and O. viverrini are co-endemic.…”

Section: Introductionmentioning

confidence: 99%

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Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis

et al. 2012

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We developed a single step duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis for the fast detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples. Two species of mitochondrial NADH dehydrogenase subunit 2 (nad2) DNA elements, the 165-bp nad2 product of C. sinensis and the 209-bp nad2 product of O. viverrini, were amplified by species-specific primers, and the fluorescence melting curve analyses were generated from hybrid of amplicons and two pairs of species-specific fluorophore-labeled probes. By their different fluorescence channels and melting temperatures, both C. sinensis and O. viverrini eggs in infected human fecal samples were detected and differentiated with high (100%) sensitivity and specificity. Detection limit was as little as a single C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasitosis, as well as from the well-defined genomic DNA of human leukocytes and other parasites. It can reduce labor time of microscopic examination and is not prone to carry over contamination of agarose electrophoresis. Our duplex real-time FRET PCR method would be useful to determine the accurate range of endemic areas and/or to discover the co-endemic areas of two liver flukes, C. sinensis and O. viverrini, in Asia. This method also would be helpful for the differential diagnosis of the suspected cases of liver fluke infections among travelers who had visited the endemic countries of those parasites.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionsupporting

confidence: 74%

Section: Introductionmentioning

confidence: 99%

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Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis

et al. 2012

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“…The C T values were strongly correlated with the intensity of infections, as determined by egg counts in Kato-Katz smears (369). A similar approach with primers and a hydrolysis probe designed from the C. sinensis ITS1 sequence showed specific (with the exception of O. viverrini) and sensitive detection of C. sinensis in a small number of fecal samples and fish tissue samples (370) Primer sets for the detection of O. viverrini DNA in stool samples using LAMP were designed from the mitochondrial nad1 sequence and from the ribosomal ITS1 sequence of O. viverrini (373,374). The ITS1-based LAMP assay showed a 100% sensitivity compared to microscopy when tested on stool samples (n ϭ 50) from schoolchildren from an area of endemicity in Thailand, compared to a remarkably low sensitivity of only 24% using conventional PCR on the same target (373).…”

Section: Food-borne Trematodesmentioning

confidence: 94%

Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

2014

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SUMMARY Over the past few decades, nucleic acid-based methods have been developed for the diagnosis of intestinal parasitic infections. Advantages of nucleic acid-based methods are numerous; typically, these include increased sensitivity and specificity and simpler standardization of diagnostic procedures. DNA samples can also be stored and used for genetic characterization and molecular typing, providing a valuable tool for surveys and surveillance studies. A variety of technologies have been applied, and some specific and general pitfalls and limitations have been identified. This review provides an overview of the multitude of methods that have been reported for the detection of intestinal parasites and offers some guidance in applying these methods in the clinical laboratory and in epidemiological studies.

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“…Both conventional PCR and LAMP have high risk of contamination. Moreover, even with expensive probe labeled real-time PCR, the two liver flukes are difficult to be distinguished [13]. …”

Section: Introductionmentioning

confidence: 99%

Rapid Detection and Differentiation ofClonorchis sinensisandOpisthorchis viverriniUsing Real-Time PCR and High Resolution Melting Analysis

Cai¹,

et al. 2014

The Scientific World Journal

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Clonorchis sinensis and Opisthorchis viverrini are both important fish-borne pathogens, causing serious public health problem in Asia. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the specific detection and rapid identification of C. sinensis and O. viverrini. Primers targeting COX1 gene were highly specific for these liver flukes, as evidenced by the negative amplification of closely related trematodes. Assays using genomic DNA extracted from the two flukes yielded specific amplification and their identity was confirmed by sequencing, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria of C. sinensis. Moreover, C. sinensis and O. viverrini were able to be differentiated by their HRM profiles. The method can reduce labor of microscopic examination and the contamination of agarose electrophoresis. Moreover, it can differentiate these two flukes which are difficult to be distinguished using other methods. The established method provides an alternative tool for rapid, simple, and duplex detection of C. sinensis and O. viverrini.

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Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes

Cited by 30 publications

References 17 publications

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis

Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

Rapid Detection and Differentiation ofClonorchis sinensisandOpisthorchis viverriniUsing Real-Time PCR and High Resolution Melting Analysis

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