Establishment Of Loop-mediated Isothermal Amplification For Detection Of Canine Leishmanial Infection In China Using Canine Conjunctival Swabs Samples

Background and objectiveLeishmaniasis is a zoonotic parasitic disease caused by infection of Leishmania amastigotes in macrophages of mammals, including human beings. In China, four of the six current visceral leishmaniasis endemic provinces (regions) are zoonotic and dogs are infectious source. Canine management is the key to control this type of visceral leishmaniasis. Thus it is important to develop the methods, which are sensitive, specific, rapid, simple and suitable for use in field, for detection of canine leishmanial infection. Leishmania contains a kinetoplast DNA (kDNA) which consists of tens of maxicircle molecules and thousands of minicircle molecules. Minicircles present highly heterogeneity among genus, species, even strains. Thus, minicircles are thought to be sensitive and specific molecular target for detection of leishmanial infection. The Loop-mediated isothermal amplification technology (LAMP) developed in recent year is a sensitive, simple and efficient method. The aim of this study is establishment of a LAMP method based on minicircles of kDNA for sensitive, specific detection of canine Leishmanial infection using conjunctival swabs samples.MethodsMinicircles of kDNA of Leishmania isolated from Wenchuan, Sichuan, China were amplified using a set of primers from conserved region and sequenced. Then these sequences were analyzed. Several sets of primers for LAMP were designed based on the determined sequence using online software. Each set of primers was evaluted using a established initial reaction system and conditions. Eventually a set of primers, which prime a sensitive and specific amplification, was selected for establishing the LAMP method. Then initial reaction system was optimized by selection of proper concentration of magnesium ions and deoxy-ribonucleoside triphosphates which are critical to LAMP reaction. Three reading methods of reaction result, i.e, turbidity method, S YBR Green method and calcein method, were compared. The detection limit and specificity of the LAMP method were determined using a series of dilution of Leishmanial kDNA of Wenchuan strain and using kDNA from different Leishmania species and/or strains as template, respectively. Conjunctival swabs of dogs from endemic area and non-endemic area in Gansu province were collected and boiled as templates for LAMP. Similarly canine bone marrow and whole blood samples were collected, and examed or detected by Microscopy or PCR, respectively. The results from the three methods were compared.ResultsA single segment, about850bp of PCR product was obtained using kDNA of Wenchuan strain of Leishmania as template and KP1/kp2as primers. Sequence analysis of20recombinant minicircle clones showed that the sequences can be divided into two classes.75%of the clones fell into a predominant sequence class of minicircles (CQ18, the sequence length is858bp) and25%of the clones contained the other sequence class of minicircles(CQ07, the sequence length is854bp). There exsits82.7%nucleotide identity between CQ18and CQ07. Phylogenetic analysis showed the two sequence classes have a highly level of nucleic acid identity with sequences from L. donovani complex. According to the sequence CQ18, five sets of primers were obtained designed online. Only the second set of primers can lead to specific, sensitive LAMP reaction. The optimized concentration of magnesium ions is8mM while that of deoxy-ribonucleoside triphosphates is1.4mM. Comprison of three methods for result observation showed that calcein can significantly distinguish positive LAMP reaction from negative reaction. The detection limit of the LAMP method was up to10"7parasite per milliliter, and the detection limit was10"2and10-3parasite per milliliter by conventional PCR using RV1/RV2and K13A/K13B primers, respectively. Three strains of Leishmania (JS5, Ganquan and801) isolated from China showed positive results in LAMP reation. The microscopy, PCR and LAMP method were used for detecting samples from dogs in endemic areas and the positive rate were7.62%(8/105),48.1%(53/110) and59.46%(66/111), respectively. There was no statistically significant difference between PCR and LAMP in endemic area(X2=2.83, P>0.05). Thirty-three dogs in non-endemic areas were detected by PCR and LAMP and1(1/33) and3(3/32) false-positive were observed, respectively. There was no statistically significant difference between PCR and LAMP in non-endemic area(X2=0.0007, P>0.05).ConclusionThe LAMP method is successfully established using counjuctival swabs for detection of canine Leishmanial infection in this study. Compared with conventional PCR, the specificity is no difference and sensitivity of LAMP is higher than that of-, PCR. The established LAMP method shows simplicity of sampling, rapidity of reaction and suitable use in field. The results of two molecular methods showed that infection rate was quite high in zoonotic endemic areas and management of dog should be strengthened to control spread of the disease.