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Published in final edited form as: Parasitol Int. 2011 Aug 16;61(1):178–182. doi: 10.1016/j.parint.2011.08.009

Rapid detection of Opisthorchis viverrini copro-DNA using loop-mediated isothermal amplification (LAMP)

Yuji Arimatsu a, Sasithorn Kaewkes b,c, Thewarach Laha b, Sung-Jong Hong d, Banchob Sripa a,c
PMCID: PMC4444511  NIHMSID: NIHMS319666  PMID: 21871581

Abstract

Opisthorchis viverrini and other foodborne trematode infections are major health problem in Thailand, the Lao People’s Democratic Republic, Vietnam and Cambodia. Differential diagnosis of O. viverrini based on the microscopic observation of parasite eggs is difficult in areas where Clonorchis sinensis and minute intestinal flukes coexist. We therefore established a rapid, sensitive and specific method for detecting O. viverrini infection from the stool samples using the loop-mediated isothermal amplification (LAMP) method. A total of five primers from seven regions were designed to target the internal transcribed spacer 1 (ITS1) in ribosomal DNA for specific amplification. Hydroxy naphthol blue (HNB) was more effective to detect the LAMP product compared to the Real-time LAMP and turbidity assay for its simple and distinct detection. The LAMP assay specifically amplified O. viverrini ITS1 but not C. sinensis and minute intestinal flukes with the limit of detection around 10−3 ng DNA/μL. The sensitivity of the LAMP was 100% compare to egg positive samples. While all microscopically positive samples were positive by LAMP, additionally 5 of 13 (38.5%) microscopically negative samples were also LAMP positive. The technique has great potential for differential diagnosis in endemic areas with mixed O. viverrini and intestinal fluke infections. As is is an easy and simple method, the LAMP is potentially applicable for point-of-care diagnosis.

Keywords: Loop-Mediated Isothermal Amplification, LAMP, Opisthorchis viverrini, Stool examination, Internal transcribed spacer 1, Internal transcribed spacer 2

1. Introduction

Liver fluke infection caused by Opisthorchis viverrini is a major public health problem in Thailand, the Lao People’s Democratic Republic, Cambodia and southern Vietnam with an estimate of over 8-10 million people are infected [1]. Recent surveys have revealed a high prevalence of liver fluke infection with up to 70% in certain villages along the Lawa Lake, Khon Kaen Province, Thailand [2]. Moreover, the infection is also prevalent in cats and dogs, the reservoir hosts (35-36.4% and 0.3-3.8%, respectively) [3, 4] and Cyprinid fish intermediate hosts (up to 95%) [5]. Diagnosis of O. viverrini infection in humans is usually done by microscopic observation of parasite eggs collected from feces. However, the eggs of O. viverrini are morphologically similar to those of Clonorchis sinensis and minute intestinal flukes, such as, Centrocestus caninus and Haplorchis taichui. The diagnoses of O. viverrini and C. sinensis are particularly important because both parasite infections are risk factors for developing cholangiocarcinoma [10, 11]. A rapid, accurate and specific stool analysis is required, since these liver and intestinal flukes coexist in Southeast Asian countries [7-9].

Diagnosis of O. viverrini infection using PCR has been reported for specific diagnosis of opisthorchiasis [6]. Internal transcribed spacers (ITS1 and ITS2) have frequently been used as a molecular marker to differentiate closely related species because a high level of interspecific sequence divergence is common. ITS1 is specific for the discrimination of parasite species, but is less sensitive for diagnosis (i.e. 76.2%), whereas ITS2 is less specific but highly sensitive (i.e. 95.2%) [12]. A decade ago, a loop-mediated isothermal amplification (LAMP) method which amplifies DNA with high specificity, efficiency and rapidity was developed [13]. The LAMP assay does not require a thermocycler and allows visual detection of DNA amplification by turbidity. It has been applied successfully to detect pathogenic microorganisms, including parasites, such as, C. sinensis [14], Schistosoma japonicum [15], Paragonimus westermani [16], Fasciola hepatica and Fasciola gigantica [17]. The objective of the present study was to establish a LAMP-based approach for the sensitive and rapid diagnosis of O. viverrini in stool samples.

2. Materials and methods

2.1. Parasites and stool specimens

Opisthorchis viverrini metacercariae were obtained from naturally infected Cyprinid fish captured from a freshwater reservoir in Khon Kaen Province, Thailand. The fish were digested by pepsin-HCl. After several washings with normal saline, the metacercariae were collected and identified under a dissecting microscope. Viable metacercariae were used to infect hamsters (Mesocricetus auratas), which were maintained at the animal facility of the Khon Kaen University Faculty of Medicine. After 2–3 months of infection, adult O. viverrini flukes were recovered from their bile ducts. Stool specimens were collected from school children who live near the Lawa Lake in Khon Kaen Province, Thailand. The specimens were processed by standard formalin-ethyl acetate concentration technique and examined for parasites’ eggs under a microscope. The number of O. viverrini eggs observed was given as eggs per gram (EPG) of feces [18]. Clonorchis sinensis adult worms were prepared in Korea using similar procedures as those for O. viverrini. Cercariae of O. viverrini, Centrocestus caninus and Haplorchis taichui were obtained from Bithynia snails by standard shedding techniques.

2.2. DNA extraction

Genomic DNA was extracted from adult O. viverrini worms using the PureGene Core kit A (QIAGEN) and dissolved in a 50 μl elution buffer. The DNA was used for analyzing the specificity and sensitivity of the LAMP assay. Stool samples microscopically positive for O. viverrini eggs were subjected to DNA extraction (n= 37). In addition, 13 stool samples negative for O. viverrini eggs by microscopy were also randomly selected and subjected to DNA extraction. Briefly, the stool samples (200 mg) were suspended in 1.4 ml ATL tissue lysis buffer and subjected to 5 cycles of freezing in liquid nitrogen followed by thawing them at 98–100 °C. DNA was then isolated from the supernatant using the QIAamp DNA Mini Stool Kit according to manufacturer’s instruction with slight modifications. Final elution of DNA was made in 50 μl of elution buffer instead of 200 μl as recommended by the manufacturer. Control DNA from C. sinensis, C. caninus and H. taichui was extracted by the same method as described for O. viverrini adults.

2.3. LAMP assay

As shown in Fig. 1A, LAMP primers for the amplification of six distinct regions of the target DNA were designed from the O. viverrini ITS1 region (GenBank accession no. EU038151) using PrimerExplorer V4 software (http://primerexplorer.jp/e). The reaction mixture comprised of Tris-HCl (20 mM, pH 8.8), KCl (10 mM), MgSO4 (8 mM), (NH4)2SO4 (10 mM), Tween 20 (0.1%), Betaine (1.6 M), deoxynucleotide triphosphates (1.4 mM each), primers FIP and BIP (1.6 μM each), primers F3 and B3 (0.2 μM each), primer LB (0.8μM) (Fig. 1B), Bst DNA polymerase (8 U; New England BioLabs), and template DNA (2 μL). Double-distilled water was finally used to fill the system to 25 μL. No template DNA was added in the negative control reaction. The mixture was incubated at 65 °C in a heat block for 60 min and then heated at 80 °C for 5 min to terminate the reaction.

Fig. 1.

Fig. 1

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Opisthorchis viverrini ITS1 sequence and primer sequence (A) Name and location of each target sequence as a primer in the O. viverrini ITS1 gene. (B) Names and sequences of LAMP and PCR primers used in this study.

2.4. Detection of LAMP product

Three different reaction mixtures were prepared for detection of LAMP product. The first LAMP assay contained 1:3000 diluted SYBR green I (Invitrogen) and detected by real time monitoring using Mx3005P QPCR System (STRATAGENE, USA) [19]. The second reaction contained 120 μM hydroxy naphthol blue (HNB), which changes the color of solution from violet to sky blue when the DNA is amplified [20]. The third assay was inspected by turbidity of magnesium pyrophosphate, a by-product of the amplification reaction. For distinct detection of the formed magnesium pyrophosphate, the tubes were centrifuged for 10 seconds to conform a pellet at the bottom of the tube. The amplified product was also visualized using 10 μL of 1:100 diluted SYBR green I (Invitrogen) and adding 1 μL of each LAMP product. The reaction tubes were placed on a light box and an UV box irradiated at 365 nm and images were photographed.

2.5. PCR assay

Conventional PCR was performed with the outer primers F3 and B3 which were used for amplification of O. viverrini ITS1 region in the LAMP assay. The PCR reaction was carried out in a 25 μL system with 10× PCR buffer (2.5 μL), 0.2 mM of each dNTPs, 0.4 μM of each B3 and F3 primers, and 1.25 U of i-Taq DNA polymerase (Intron biotechnology), and 2 μL of DNA sample in a thermocycler (Applied Biosystems) under the following conditions: after an initial denaturation at 95 °C for 7 min, then 35 cycles of denaturation (30 s at 95 °C), annealing (30 s at 60 °C), and extension (30 s at 72 °C), and a final extension for 7 min at 72 °C. Five microliters of PCR products (213 bp) were examined on a 1% agarose gel and stained with ethidium bromide (EtBr) and photographed.

2.6. PCR-RFLP assay

PCR was also performed by using ITS2-specific primers [7] shown in Fig. 1B. The estimated size of the amplified products is 375 bp, 381 bp and 526 bp for O. viverrini, C. sinensis and H. taichui, respectively. The PCR conditions were the same as described above. To differentiate O. viverrini from C. sinensis, amplified ITS2 products were digested with AcuI. The PCR amplicon of O. viverrini does not possess a restriction site for AcuI and remains uncut (375 bp), whereas that of C. sinensis has a single AcuI site and gives rise to two bands at 286 bp and 95 bp. Two μL of PCR product were digested with 2.5 units of the restriction endonuclease AcuI (New England Biolabs) at 37 °C for 3 hours in a volume of 10 μL.

3. Results

3.1. Sensitivity of LAMP

The concentration of the genomic DNA of O. viverrini adult worm was measured by spectrophotometer and diluted with 10 mM Tris HCl (pH 8.8) to a final concentration of 10 ng/μL. Subsequently, a 10-fold serial dilution was prepared ranging from 1×101 to 1×10−5 ng/μL and each of the diluted DNAs was used as a template for LAMP and conventional PCR. After amplifications, the products were visually detected on a 1% agarose gel and the sensitivity of the two assays was compared. The results showed the detection limit of the LAMP assay was 10−3 ng/μL. Similar sensitivity was observed by conventional PCR, using both LAMP outer primers (F3, B3), and ITS2 primers (RTFlukeFa, RTFlukeRa). However the intensity of the amplified product was clearly different between the two methods, indicating that LAMP assay gives distinct results more than the conventional PCR (Suppl. 1).

The Real-time LAMP assay using SYBR green I as an intercalator was successful to detect the LAMP products by Mx3005P QPCR System (Fig. 2). The fluorescence intensity rose during the LAMP reaction using DNA sample ranging from 1×101 to 1×10−3 ng/μL within 40 minutes. The same result was obtained using the HNB as a metal ion indicator, which changes the color of solution after DNA amplification. The LAMP product detecting by turbidity, spin down pellet and SYBR green I as a DNA dye solution (Suppl. 2), also showed similar results.

Fig. 2.

Fig. 2

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Real-time monitoring of LAMP reaction. The generated fluorescence intensity of DNA binding SYBR Green I was monitored at every 1 minute. Template DNAs were prepared from 101 to 10-5 ng/μL. Distilled water (D.W.) was used as a negative control.

3.2. Specificity of LAMP

Specificity of the LAMP was done by using genomic DNA of O. viverrini (adult worms, metacercariae and cercariae), C. sinensis adult worms and minute intestinal flukes (MIF), namely, C. caninus and H. taichui. The concentration of the genomic DNA of each sample was measured by spectrophotometer and diluted with 10 mM Tris–HCl (pH 8.8) to a final concentration of 1.0 ng/μL and applied to the LAMP and PCR assays. The DNA samples were confirmed by PCR-RFLP assay. The results showed that the LAMP could differentiate O. viverrini from C. sinensis, H. taichui and C. caninus, indicating the reaction is specific (Fig. 3).

Fig. 3.

Fig. 3

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Specificity of LAMP and PCR-RFLP assays for Opisthorchis viverrini and minute intestinal flukes. Sample numbers are as follows: 1, negative control; 2, O. viverrini adults; 3, O viverrini metacercariae; 4, O. viverrini cercariae; 5, C. sinensis adults; 6, C. caninus metacercariae; 7, H. taichui adults. (A) Visual examination of LAMP products by HNB. +, positive reaction; -, negative reaction. (B) PCR-RFLP assay. +AcuI, digestion with AcuI endonuclease; -AcuI, without digestion.

3.3. Stool sample analysis

Conventional PCR and LAMP analysis were compared by using stool samples (Table 1). With the PCR method, the results showed 9 positive (18.0%) and 41 negative (82.0%) out of 50 specimens. With reference to microscopic examination, the sensitivity of the PCR was 9 in 37 egg-positive stool specimens (24.3%). Using the same DNA samples, 37 out of the 37 samples (100.0%) showed positive results with the LAMP system. Overall, the percentage of O. viverrini positive by LAMP was higher than that of microscopic examination in the 50 stool samples examined. Of the samples negative for O. viverrini eggs (n=13), 5 (38.5%) were positive by LAMP. Using the microscopic examination as a gold standard, the diagnostic sensitivity and specificity of LAMP assay were calculated at 100% and 61.5%, respectively. The positive rates of LAMP assay were consistently higher than those of conventional PCR within different O. viverrini intensity groups (Table 2).

Table 1.

Diagnostic abilities of PCR and LAMP to detect Opisthorchis viverrini copro-DNA from stool samples.

Microscopic stool examination a
Egg-positive samples (N=37) Egg-negative samples (N=13) Total (N=50)
PCR No. positive 9 (24.3) b 0 (0) 9 (18.0)
No. negative 28 (75.7) 13 (100) 41 (82.0)
LAMP No. positive 37 (100) 5 (38.5) 42 (84.0)
No. negative 0 (0) 8 (61.5) 8 (16.0)
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a

Stool samples confirmed by microscopic examination were used as standard to evaluate the diagnostic abilities of PCR and LAMP.

b

Percentages are shown in parentheses.

Table 2.

Diagnosis of Opisthorchis viverrini infection by PCR and LAMP in different intensity groups based on microscopic analyses.

Intensity (egg/g) No. of samples PCR positive (%) LAMP Positive (%)
0 13 0 (0) 5 (38)
1-49 12 0 (0) 12 (100)
50-99 8 2 (25) 8 (100)
100-499 14 5 (36) 14 (100)
>500 3 2 (67) 3 (100)
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4. Discussion

Our established LAMP is a rapid method, which can obtain results within 40 min, using a heat box or a water bath to maintain the temperature at 65 °C. The performances of LAMP assays using Real-time LAMP, HNB, and turbidity were compared. All methods show clear differences between the negative control and positive samples. Regarding the sensitivities of the three different LAMP methods, similar results were obtained among them. The LAMP products detected by HNB were used throughout because of simplicity, distinct results, and since other detection methods may cause cross contamination through opening the caps of the tubes with amplified DNA. The limit of detection of the LAMP method was 10−3 ng DNA/μL.

The PCR technique has been used to detect O. viverrini infection in fish, snails and experimentally infected hamsters [21, 22]. However, few of these studies have been employed for the detection of O. viverrini DNA in human stool specimens and they have shown varying sensitivity [23, 24]. Therefore, the sensitivity of detection needs to be improved when stool samples are used in the test. The sensitivity of PCR detection depends largely on the quality of extracted DNA, since several inhibitors in stools are known to inhibit Taq DNA polymerase [25]. It is also known that stools may contain high concentrations of polyphenols, tannins, polysaccharides, terpens, and resins which derive from food that limit DNA extraction success [26]. Modified QIAamp DNA Mini Stool Kit DNA extraction procedure using hexadecyltrimethylammonium bromide (CTAB) is useful to remove these compounds and successfully extract DNA from stool of primates [27] and also O. viverrini DNA from eggs in stool samples [28]. However, this study used only the QIAamp stool kit, therefore contaminants which may inhibit the polymerase reaction seems to remain in our stool DNA samples. Interestingly, our LAMP assay showed higher sensitivity than conventional PCR when used the same DNA samples of egg-positive cases. However, LAMP assay showed low sensitivity in stool sample without repeated freeze-thaw step during DNA extraction (data not shown). The results indicate that LAMP amplification is resistant to the contaminants included in the extracted DNA from the stool sample. However, radical physical crush of the O. viverrini eggs is necessary to expose miracidium to reagents during DNA extraction. The Bst DNA polymerase used in LAMP amplification is more resistant to inhibitors than other DNA polymerases [29], and also this DNA polymerase is active at relatively high temperatures (65 °C), which helps the reduction of nonspecific priming. Therefore, LAMP technique is effective for amplifying DNA from the stool samples, which possibly contain inhibitors of DNA polymerase.

In this study 5 false positive reactions occurred in the LAMP assay to detect copro-DNA. There is a possibility that microscopists may miss parasite eggs in stool samples due to light infections. The second possibility may be due to the quantity of stool sample examined. For microscopic analyses, 2 drops of egg sediments are generally examined while DNA is extracted from 100 mg of feces for LAMP assay. The other possibility may be the cross reaction between our LAMP primers with DNA from other organisms that are not included in our study. We did not use the stool samples of healthy people in nonendemic areas as negative control, but our established LAMP had no cross reaction with common minute intestinal flukes (C. caninus and H. taichui) and C. sinensis. The LAMP method is simple having only one step amplification, unlike other PCR techniques that require restriction endonuclease for differential diagnosis of these parasites [7,12]. Therefore, it is feasible to use for differential diagnosis of O. viverrini in areas with high prevalence of minute intestinal flukes in Thailand [8,9], or even in C. sinensis endemic areas in Vietnam [1].

In conclusion, LAMP assay has inherent characteristics that make it advantageous as a diagnostic test which can assist in bringing point-of-care diagnosis to patients. Since DNA amplification and reading of results require minimum equipment, the technique has great potential for use in the endemic areas of mixed O. viverrini and other foodborne trematode infections. Future studies will be aimed at the application of this LAMP procedure for routine diagnosis of opisthorchiasis with further validation of DNA extraction method and improvement of sensitivity.

Supplementary Material

01. Supl. 1.

Sensitivity of the LAMP assay for Opisthorchis viverrini and comparison with conventional PCR by agarose gel electrophoresis. Numbers above the Lanes represent O. viverrini DNA concentration of 101–10−5 ng/μL. Neg. represents no-DNA control. (A) Sensitivity analysis of the LAMP assay. (B) Sensitivity analysis of conventional PCR using LAMP outer primers (F3, B3). (C) Sensitivity analysis of conventional PCR using ITS2 primers (RTFlukeFa, RTFlukeRa).

02. Suppl. 2.

Various visual detection of LAMP products. (A) Detection by hydroxy naphthol blue (HNB). The numbers on the tubes indicate the amount of O. viverrini DNA used in the reaction. (B) Detection by SYBR green I under light box. (C) Detection by SYBR green I under UV light. (D) Detection by turbidity. The tubes were observed just after LAMP amplification. (E) Detection by turbidity. The tubes were spun down to form magnesium pyrophosphate pellets.

Acknowledgments

This work was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Health Cluster (SHeP-GMS), Khon Kaen University, the Lawa Project, Khon Kaen University, Thailand and NIH-NIAID, award number AI065871. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIAID or the NIH or the funders.

Footnotes

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References

  • 1.Sripa B, Kaewkes S, Intapan PM, Maleewong W, Brindley PJ. Food-borne trematodiases in Southeast Asia: epidemiology, pathology, clinical manifestation and control. Adv Parasitol. 2010;72:305–50. doi: 10.1016/S0065-308X(10)72011-X. [DOI] [PubMed] [Google Scholar]
  • 2.Sripa B. Concerted action is needed to tackle liver fluke infections in Asia. PLoS Negl Trop Dis. 2008;2:e232. doi: 10.1371/journal.pntd.0000232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Enes JE, Wages AJ, Malone JB, Tesana S. Prevalence of Opisthorchis viverrini infection in the canine and feline hosts in three villages, Khon Kaen Province, northeastern Thailand. Southeast Asian J Trop Med Public Health. 2010;41:36–42. [PMC free article] [PubMed] [Google Scholar]
  • 4.Aunpromma S, Tangkawattana P, Papirom P, Kanjampa P, Tesana S, Sripa B, et al. High prevalence of Opisthorchis viverrini infection in reservoir hosts in the opisthorchiasis endemic area of Thailand. Parasitol Int. doi: 10.1016/j.parint.2011.08.004. this issue. [DOI] [PubMed] [Google Scholar]
  • 5.Sithithaworn P, Haswell-Elkins M. Epidemiology of Opisthorchis viverrini. Acta Trop. 2003;88:187–94. doi: 10.1016/j.actatropica.2003.02.001. [DOI] [PubMed] [Google Scholar]
  • 6.Johansen MV, Sithithaworn P, Bergquist R, Utzinger J. Towards improved diagnosis of zoonotic trematode infections in Southeast Asia. Adv Parasitol. 2010;73:171–95. doi: 10.1016/S0065-308X(10)73007-4. [DOI] [PubMed] [Google Scholar]
  • 7.Traub RJ, Macaranas J, Mungthin M, Leelayoova S, Cribb T, Murrell KD, et al. A new PCR-based approach indicates the range of Clonorchis sinensis now extends to Central Thailand. PLoS Negl Trop Dis. 2009;3:e367. doi: 10.1371/journal.pntd.0000367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Waikagul J, Wongsaroj T, Radomyos P, Meesomboon V, Praewanich R, Jongsuksuntikul P. Human infection of Centrocestus caninus in Thailand. Southeast Asian J Trop Med Public Health. 1997;28:831–5. [PubMed] [Google Scholar]
  • 9.Sukontason KL, Sukontason K, Piangjai S, Pungpak S, Radomyos P. Prevalence of Opisthorchis viverrini infection among villagers harboring Opisthorchis-like eggs. Southeast Asian J Trop Med Public Health. 2001;32:23–6. [PubMed] [Google Scholar]
  • 10.Sripa B, Kaewkes S, Sithithaworn P, Mairiang E, Laha T, Smout M, et al. Liver fluke induces cholangiocarcinoma. PLoS Med. 2007;4:e201. doi: 10.1371/journal.pmed.0040201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Bouvard V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F WHO International Agency for Research on Cancer Monograph Working Group. A review of human carcinogens--Part B: biological agents. Lancet Oncol. 2009;10:321–2. doi: 10.1016/s1470-2045(09)70096-8. [DOI] [PubMed] [Google Scholar]
  • 12.Sato M, Thaenkham U, Dekumyoy P, Waikagul J. Discrimination of O. viverrini, C. sinensis, H. pumilio and H. taichui using nuclear DNA-based PCR targeting ribosomal DNA ITS regions. Acta Trop. 2009;109:81–3. doi: 10.1016/j.actatropica.2008.09.015. [DOI] [PubMed] [Google Scholar]
  • 13.Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28:E63. doi: 10.1093/nar/28.12.e63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Cai XQ, Xu MJ, Wang YH, Qiu DY, Liu GX, Lin A, et al. Sensitive and rapid detection of Clonorchis sinensis infection in fish by loop-mediated isothermal amplification (LAMP) Parasitol Res. 2010;106:1379–83. doi: 10.1007/s00436-010-1812-3. [DOI] [PubMed] [Google Scholar]
  • 15.Xu J, Rong R, Zhang HQ, Shi CJ, Zhu XQ, Xia CM. Sensitive and rapid detection of Schistosoma japonicum DNA by loop-mediated isothermal amplification (LAMP) Int J Parasitol. 2010;40:327–31. doi: 10.1016/j.ijpara.2009.08.010. [DOI] [PubMed] [Google Scholar]
  • 16.Chen MX, Ai L, Zhang RL, Xia JJ, Wang K, Chen SH, et al. Sensitive and rapid detection of Paragonimus westermani infection in humans and animals by loop-mediated isothermal amplification (LAMP) Parasitol Res. 2011;108:1193–8. doi: 10.1007/s00436-010-2162-x. [DOI] [PubMed] [Google Scholar]
  • 17.Ai L, Li C, Elsheikha HM, Hong SJ, Chen JX, Chen SH, et al. Rapid identification and differentiation of Fasciola hepatica and Fasciola gigantica by a loop-mediated isothermal amplification (LAMP) assay. Vet Parasitol. 2010;174:228–33. doi: 10.1016/j.vetpar.2010.09.005. [DOI] [PubMed] [Google Scholar]
  • 18.Sithithaworn P, Nuchjungreed C, Srisawangwong T, Ando K, Petney TN, Chilton NB, et al. Genetic variation in Opisthorchis viverrini (Trematoda: Opisthorchiidae) from northeast Thailand and Laos PDR based on random amplified polymorphic DNA analyses. Parasitol Res. 2007;100:613–7. doi: 10.1007/s00436-006-0304-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Maeda H, Kokeguchi S, Fujimoto C, Tanimoto I, Yoshizumi W, Nishimura F, et al. Detection of periodontal pathogen Porphyromonas gingivalis by loop-mediated isothermal amplification method. FEMS Immunol Med Microbiol. 2005;43:233–9. doi: 10.1016/j.femsim.2004.08.005. [DOI] [PubMed] [Google Scholar]
  • 20.Goto M, Honda E, Ogura A, Nomoto A, Hanaki K. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques. 2009;46:167–72. doi: 10.2144/000113072. [DOI] [PubMed] [Google Scholar]
  • 21.Wongratanacheewin S, Pumidonming W, Sermswan RW, Maleewong W. Development of a PCR-based method for the detection of Opisthorchis viverrini in experimentally infected hamsters. Parasitology. 2001;122:175–80. doi: 10.1017/s0031182001007235. [DOI] [PubMed] [Google Scholar]
  • 22.Maleewong W, Intapan PM, Wongkham C, Wongsaroj T, Kowsuwan T, Pumidonming W, et al. Detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes by a PCR-based method. Parasitology. 2003;126:63–7. doi: 10.1017/s0031182002002573. [DOI] [PubMed] [Google Scholar]
  • 23.Wongratanacheewin S, Pumidonming W, Sermswan RW, Pipitgool V, Maleewong W. Detection of Opisthorchis viverrini in human stool specimens by PCR. J Clin Microbiol. 2002;40:3879–80. doi: 10.1128/JCM.40.10.3879-3880.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Umesha KR, Kumar S, Parvathi A, Duenngai K, Sithithaworn P, Karunasagar I, et al. Opisthorchis viverrini: detection by polymerase chain reaction (PCR) in human stool samples. Exp Parasitol. 2008;120:353–6. doi: 10.1016/j.exppara.2008.09.004. [DOI] [PubMed] [Google Scholar]
  • 25.Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol. 1997;63:3741–51. doi: 10.1128/aem.63.10.3741-3751.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Doulis AG, Harfouche AL, Aravanopoulos FA. Rapid, high quality DNA isolation from cypress (Cupressus sempervirens L.) needles and optimization of the RAPD marker technique. Plant Mol Biol. 2000;17:1–14. [Google Scholar]
  • 27.Vallet D, Petit EJ, Gatti S, Levréro F, Ménard N. A new 2CTAB/PCI method improves DNA amplification success from faeces of Mediterranean (Barbary macaques) and tropical (lowland gorillas) primates. Conservation Genetics. 2008;9:677–80. [Google Scholar]
  • 28.Duenngai K, Sithithaworn P, Rudrappa UK, Iddya K, Laha T, Stensvold CR, et al. Improvement of PCR for detection of Opisthorchis viverrini DNA in human stool samples. J Clin Microbiol. 2008;46:366–8. doi: 10.1128/JCM.01323-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, Abeyewickreme W, et al. Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin Chem. 2006;52:303–6. doi: 10.1373/clinchem.2005.057901. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

01. Supl. 1.

Sensitivity of the LAMP assay for Opisthorchis viverrini and comparison with conventional PCR by agarose gel electrophoresis. Numbers above the Lanes represent O. viverrini DNA concentration of 101–10−5 ng/μL. Neg. represents no-DNA control. (A) Sensitivity analysis of the LAMP assay. (B) Sensitivity analysis of conventional PCR using LAMP outer primers (F3, B3). (C) Sensitivity analysis of conventional PCR using ITS2 primers (RTFlukeFa, RTFlukeRa).

NIHMS319666-supplement-01.zip (149.9KB, zip)
02. Suppl. 2.

Various visual detection of LAMP products. (A) Detection by hydroxy naphthol blue (HNB). The numbers on the tubes indicate the amount of O. viverrini DNA used in the reaction. (B) Detection by SYBR green I under light box. (C) Detection by SYBR green I under UV light. (D) Detection by turbidity. The tubes were observed just after LAMP amplification. (E) Detection by turbidity. The tubes were spun down to form magnesium pyrophosphate pellets.

NIHMS319666-supplement-02.jpg (367.6KB, jpg)

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