Study On Detection Of Trichinella Spiralis DNA By Loop-mediated Isothermal Amplification (LAMP)

Trichinellosis is a foodborne zoonosis caused by the parasitic nematode Trichinella, which is characterized by an extremely wide host range and geographical distribution. It is a tissue-dwelling nematode acquired by the ingestion of raw or inadequately cooked meat-products containing encapsulated larvae. Outbreaks of trichinellosis have been regularly reported during the past two centuries and there is evidence suggesting that this parasitic disease may be emerging or re-emerging in some parts of the world. Evaluating the worldwide prevalence of the disease is difficult because the definitive localization of the larvae in the muscles precludes simple parasitological surveys(parasite identification requires muscle biopsy).Extensive surveys have been based on the examination of diaphragms from cadavers but there are no recent data from such studies. Serological surveys are possible but have several drawbacks:they are expensive, antibody titers fall quite rapidly though patients still harbor the parasite and cross-reaction can occur, requiring the use of expensive western blots. Application of the detection technique of nucleic acid molecules are more extensive, more standardized and automated, with lower costs.Early diagnosis, on site rapid detection and epidemiological survey and other aspects by Loop-mediated isothermal amplification(LAMP) will be used more and more in Trichinellosis. There are many advantages of the LAMP assay.The strict spatial separation of reagent preparations and test procedures is necessary to avoid contamination. However, the colour visualisation detection method greatly increases the scope of use relative to the real time PCR assay, because it does not require fluorescent quantitative PCR instrument. This means that detection of T. spiralis is no longer limited by space and equipment, and only requires a constant temperature water bath.The experiment successfully developed a LAMP diagnostic kit for rapid detection of Trichinella DNA, dedicated to the research trichinosis diagnostic techniques. Establishment of a low, medium and high degree Trichinella spiralis infection dose of mouse model, acollecting muscle, blood, stool sample of early infection stages. The LAMP diagnostic kit can detect one Trichinella spiralis larva in samples, with high sensitivity and specificity, especially for larval muscle is not formed capsule package, larval density is very low, the nucleic acid moleculedetection technique also highlighted its important diagnostic value. Previous studies have shown that PCR was used to detect Trichinella has a certain value of early diagnosis, this experiment proved LAMP method is more sensitive than PCR, rapid,simple, and can be used in the early infection diagnosis, meat inspection and field epidemiology research.Materials and methods1 Parasites, mice and DNA extractionThe Trichinella isolates used in the study were T. spiralis(ISS534), obtained from a domestic pig in Nanyang city of Henan Province, China, and Trichinella nativa(ISS10), Trichinella pseudospiralis(ISS13), Trichinella nelsoni(ISS29), all of which were obtained from the Trichinella Reference Center(TRC, Rome, Italy).Isolates of Ascaris lumbricoides, Enterobius vermicularis, Trichuris trichiura,Ancylostoma duodenale, Clonorchis sinensis, Fasciolopsis buski, Schistosoma japonicum, Taenia solium, Taenia saginata and Spirometra mansoni were obtained from the Department of Parasitology, Zhengzhou University, China. All of these Trichinella isolates were cultured in Kunming mice by serial passages in a laboratory at the Experimental Animal Center, Academy of Military Medical Sciences, Beijing,China. The mice were produced and maintained at the Experimental Animal Center in accordance with the guidelines of the American Association for Accreditation of Laboratory Animal Care. The muscle larvae of these Trichinella isolates were released from the infected mouse muscles by the digestion of carcasses with 1% pepsin(1:3,000 preparation, Beijing Solarbio Science & Technology Co.,Ltd., China) and1% hydrochloric acid. Genomic DNA of all samples and parasites were extracted using the DNeasy Blood & Tissue Kit(Tiangen Biotech Beijing Co., Ltd., China)according to the manufacturer’s instructions.2 Design of Trichinella-specific LAMP assay primers and evaluation of primers for the rapid detection of T. spiralis LAMP primers were designed that targeted a 1.6 kb repetitive element of the T.spiralis genome. The sequence of this repetitive element(accession number: X06625)was downloaded from the NCBI Gen Bank. The sequence was further analysed by Primer Explorer V4 software(http://primerexplorer.jp/lamp), and16 sets of primers were designed. Under the same reaction conditions, using the real time LAMP assay,evaluation of primers for the rapid detection of T. spiralis.3 Specificity of the LAMP assay on detection of T. spiralis DNA was extracted from the muscle larvae of T. spiralis(ISS534), T. nativa(ISS10), T. pseudospiralis(ISS13) and T. nelsoni(ISS29) as the positive controls.The total DNA of several heterologous parasites were extracted, including A.lumbricoides, E. vermicularis, T. trichiura, A. duodenale, C. sinensis, Fasciolopsis,S. japonicum, T. solium,T. saginata and S. mansoni, in addition to double distilled water as a negative control, to assess the specificity of the LAMP assay and two different detection methods.4 Determination of the LAMP detection limit using pure T. spiralis larval DNA DNA was extracted from thousands of T. spiralis larvae and pure T. spiralis DNA templates were prepared by a serial 10 fold dilution with concentrations that ranged from 3,620 ng/μl to 3.62 fg/μl. The results were also monitored with the direct visual method by the addition of 1μl of fluorescence detection reagent to 25μl of the reaction mixture, prior to the LAMP reaction. For the purposes of comparison, PCRs using the TS2F3 and TS2B3 primer pair with the same amount of T. spiralis DNA were also performed.5 Determination of the LAMP detection limit using pure T. spiralis larvae To verify the sensitivity of the LAMP reaction, DNA was extracted from 10 T.spiralis larvae in 1 ml of PBS. Pure T. spiralis DNA templates were prepared by a serial 10 fold dilution with concentrations that ranged from 10 to 0.00001 larvae/ml PBS. Two microliters of the template were put into every LAMP reaction.6 The comparison of different T.spiralis samples DNA extraction method Evaluation the DNA extraction Kit and Chelex method extraction of T.spiralis different samples. Respectively to add 1 T.spiralis muscle larva into 50μl healthy mice blood, 200 mg healthy mice feces, 250 mg healthy muscle tissue, genomic DNA was extracted by Tiangen company’s blood/cell/tissue genomic DNA extraction kit and LAMP Chelex cracking liquid. Evaluation the LAMP amplification efficiency of two kinds method.7 Impact Assessment sealent on LAMP reaction and calcein chromogenic effectDetect whether sealent affect the LAMP reaction and calcein color effect, the melting point of 42 ℃ solid paraffin wax after melting, made of the same diameter cylindrical with reaction tube, which was added before the experiment as solid.During the reaction, the liquid can be well prevented formation of aerosols. After completion of the reaction becomes solid, the amplified product is fully enclosed in the tube end. Design experiment comparing six groups with or without add paraffin and calcein into LAMP reaction.8 LAMP detection of T. spiralis in mouse muscle infected with T.spiralis larvae( 1) To determine the sensitivity of this technique in muscle tissue, 1 g of muscle sample from uninfected mice together with 10 T. spiralis larvae were homogenised with a glass tissue grinder and DNA was extracted. The spiked muscle sample DNA templates were prepared by the addition of a serial 10 fold dilution of larvae with concentrations that ranged from 10 to 0.00001 larvae into 1 g of muscle.( 2) One gram of mince muscle tissue(including the diaphragm, pectoral,gastrocnemius, deltoid and tongue muscles) was assayed from each mouse. A total of18 muscle sample DNA templates from mice, either uninfected or with an infecting dose of 10 or 50 T. spiralis larvae, were prepared for LAMP assays. Similar results were observed for both LAMP with colour visualization and PCR assays.9 LAMP detection of T. spiralis in mouse blood infected with T.spiralis larvae6w healthy male age Kunming mice, 15 mice were randomly divided into three groups, each 5 infected with T. spiralis by orally. Each group of mice were infected with 10, 100, 300 T. spiralis muscle larvae, establish low, medium and high degree of infection mouse model. During 1d ~ 20 d after infection tail venous blood were collected daily from each mouse, 50μl blood samples extracted genomic DNA for LAMP assay.10 LAMP detection of T. spiralis in mouse faeces infected with T.spiralis larvae6w healthy male age Kunming mice, 15 mice were randomly divided into three groups, each 5 infected with T. spiralis by orally. Each group of mice were infected with 10, 100, 300 T. spiralis muscle larvae, establish low, medium and high degree of infection mouse model. During 1d~20d after infection, 800 mg feces collected from each group of mice daily, extraction of genomic DNA for LAMP assay.11 Statistical analysisAll statistical analyses of data were done with SPSS for Windows version 17.0Chisquare test and Mean comparison, single factor variance analysis and Correlation analysis were used, and the level of significance used was 5%(P<0.05).Results1 evaluation of primers for the rapid detection of T. spiralisThe LAMP primers target was the 1.6 kb repetitive sequence of T. spiralis.Sixteen sets of primers were designed for the detection of T. spiralis larvae. Under the same reaction conditions, using the real time LAMP assay, we observed that 15 curves occurred after the 43 min reaction, which demonstrated that 15 out of 16 primer sets amplified the target sequence. The TS1 and TS2 sets that could amplify the target gene in the shortest time were the fastest and the optimal reaction primers.It was observed that the increased turbidity curve only appeared when T.spiralis, T. nativa, T. pseudospiralis and T. nelsoni were used as templates, but not with the negative control(double-distilled water) or the other 10 heterologous parasites.2 Determination of the LAMP detection limit using pure T. spiralis larval DNAIt was observed that the detection limit of the real time LAMP assay for T.spiralis larval DNA was 362 fg/μl. For the purposes of comparison, PCRs using the TS2F3 and TS2B3 primerpair with the same amount of T. spiralis DNA were also performed. We observed that the detection limit for the PCR was 3.62 pg/μl. The sensitivity of LAMP with the detection limit was >10 times higher than that for PCR.3 Determination of the LAMP detection limit using pure T. spiralis larvaeTo verify the sensitivity of the LAMP reaction using pure T. spiralis larvae,it can be observed that the LAMP detection limit was 0.001 larvae/ml. It demonstrates that the PCR detection limit was 0.01 larvae/ml. The sensitivity of LAMP with the detection limit 0.001 larvae/ml >10 times higher than that for PCR.4 The comparison of T. spiralis samples different DNA extraction methodsTo verify the sensitivity of the LAMP reaction using T. spiralis different samples.Genomic DNA was extracted by Tiangen company’s blood/cell/tissue genomic DNA extraction kit and LAMP Chelex cracking liquid. Two methods to extract the DNA template for LAMP amplification efficiency roughly the same. This put an end to the formation of volatile aerosols and the formation of false positive and can be a good solution to false positives caused by amplified products.5 Impact Assessment sealent on LAMP reaction and calcein chromogenic effectFrom the experimental results it can be seen the same time adding a blocking agent group and the group without the blocking agent to react, indicating that the blocking agent does not inhibit the LAMP reaction; added calcein for group blocking agent is added or not has no effect on the time of the LAMP reaction, showed sealers added LAMP reaction does not inhibit calcein.6 LAMP detection of T. spiralis in mouse muscle infected with T. spiralis larvae(1)To determine the sensitivity of this technique in muscle tissue,it was observed that the LAMP detection limit was 0.01 larvae/g of muscle tissue,whereas it shows that the PCR detection limit was 0.1 larvae/g,The sensitivity of LAMP>10 times higher than that for PCR.(2)A total of 18 muscle sample DNA templates from mice, either uninfected or with an infecting dose of 10 or 50 T. spiralis larvae, were prepared for LAMP assays. As figers shown that curves occurred only in the samples from mice infected by T.spiralis larvae,showing that mouse muscle infected with 50 and 10 T. spiralis larvae could be detected by real time LAMP. The total detection rate was 100%(6/6).Similar results were observed for both LAMP with colour visualization and PCR assays.7 LAMP detection of T. spiralis in mouse blood infected with T. spiralis larvae(1)The LAMP was used to detect diifferent doses of T. spiralis infection in mice during 1~ 20 dpi blood samples, low infection(10 muscle larvae) group. There are 10 blood samples were positive result. The total detection rate was10%(10/100).The T. spiralis DNA was detected on 9d、10d、13d、18d~20dpi,detection rates were 20%(1/5). On the 15d、17dpi detection rates were 40%(2/5).(2)The LAMP was used to detect diifferent doses of T. spiralis infection in mice during 1~ 20 dpi blood samples, mild infection(100 muscle larvae) group. There are 17 blood samples were positive result. The total detection rate was17%(17/100).The T. spiralis DNA was detected on 1d、3d、11d、12d、15dpi, detection rates were 20%(1/5). On the 7d、8d、10d、17d、19d、20dpi detection rates were 40%(2/5).(3)The LAMP was used to detect diifferent doses of T. spiralis infection in mice during 1~ 20 dpi blood samples, high infection(300 muscle larvae) group. There are 26 blood samples were positive result. The total detection rate was26%(26/100).The T. spiralis DNA was detected on 4d、5d、9d、13d、14d、16d、20dpi, detection rates were 20%(1/5),On the 3dpi detection rates were 40%(2/5), On the 8d、10d、11d、19dpi detection rates were 60%(3/5), On the 15 dpi detection rates were 80%(4/5).(4)LAMP positive rate difference between the three groups of mice infected with different degrees was not statistically significant(c(17)=4.899, P=0.086).LAMP positive rate was no significant difference in mice infected with a different number of days between mild(c(17)(28)20.000, P=0.395). No significant difference of the positive rate of LAMP in moderate infection of mice between different days(c(17)=23.459,P=0.218).The positive rate of LAMP infection in mice with severe differences between different stages had statistical significance(c(17)=31.393, P=0.037).8 LAMP detection of T. spiralis in mouse faeces infected with T. spiralis larvae(1)The LAMP was used to detect diifferent doses of T. spiralis infection in mice during 1~ 20 dpi faeces samples, low infection(10 muscle larvae) group. There are 17 faeces samples were positive result. The total detection rate was21%(17/80).The T. spiralis DNA was detected on 6~10d、13~15d、17dpi, detection rates were 25%(1/4). On the 11d、18d、19dpi detection rates were 50%(2/4).(2)The LAMP was used to detect diifferent doses of T. spiralis infection in mice during 1~ 20 dpi faeces samples, mild infection(100 muscle larvae) group. There are 33 faeces samples were positive result. The total detection rate was41%(33/80).The T. spiralis DNA was detected on 2d、4d、9d、11d、19d、20dpi,detection rates were 25%(1/4). On the10d、14d、 18 dpi detection rates were 50%(2/4). On the 5d、7d、12d、13d、15~17dpi detection rates were 75%(3/4).(3)The LAMP was used to detect diifferent doses of T. spiralis infection in mice during 1~ 20 dpi faeces samples, high infection(300 muscle larvae) group. There are 33 faeces samples were positive result. The total detection rate was59%(47/80).The T. spiralis DNA was detected on 2d、3d、6d、9d、18dpi, detection rates were 25%(1/4). On the 5d、14d、19dpi detection rates were 50%(2/4). On the4d、7d、8d、10d、13d、15d、16d、20dpi detection rates were 75%(3/4). On the 11d、12d、17dpi detection rates were100%(4/4).( 4) Statistically significant differences between the 3 groups were LAMP positive rate(c(17)=14.823, P=0.001). There is a correlation between the positive rate and the infection dose(rs=0.500, P<0.001), the positive rate increased with the increase of infection dose increased(F=20.492, P<0.001). No statistically significant difference between mild infection group positive rate among different days(c(17)=15.761, P=0.673); the difference was not statistically significant and moderate infection positive rate between the groups of different days(c(17)=27.339, P=0.096);severe infection group differences between the positive rate of different days of no statistical significance(c(17)=27.389, P=0.096).Conclusions1 The LAMP primers target was the 1.6 kb repetitive sequence of T. spiralis.Sixteen sets of primers were designed for the detection of T. spiralis larvae. Under the same reaction conditions, using the real time LAMP assay, The TS2 primer set was chosen as the final set of LAMP primers for T. spiralis detection. it was observed that the increased turbidity curve only appeared when T. spiralis, T. nativa, T.pseudospiralis and T. nelsoni were used as templates, but not with the negative control(double-distilled water) or the other 10 heterologous parasites. These results suggest that the TS2 primer set was specific for the detection of T. spiralis.2 LAMP amplification process can be carried out under constant temperature conditions, a stable heat source equipment(such as a water bath or in a metal bath)will be able to meet requirements, testing costs are significantly reduced. Since the advent of LAMP technology, it has been widely used in on-site diagnosis of T.spiralis DNA.3 To determine the sensitivity of this technique in mouse muscle tissue、mouse blood、mouse stool. The specific DNA was detected early in muscle of mice with low infection and on 2 dpi in blood 、 faeces sample of mice. This experiment proved LAMP method is more sensitive than PCR, rapid, simple, and can be used in the early infection diagnosis, meat inspection and field epidemiology research.