| Part 1 Stability of silica gel membrane spin column for nucleic acid extraction from rabbit model infected with Schistosoma japonicumObjective:To verify the stability of silica gel membrane spin column for extraction DNA from sera of rabbits infected with S.japonicum,which lay a foundation for the development of a rapid diagnostic kit for S.Japonicum.Methods:Nine rabbits were randomly divided into three groups with 3 rabbits in each group.Each rabbit was infected with S.japonicum cercariae via abdomen according to routine steps.The rabbits of group Ⅰ and group Ⅱ were infected with 500 cercariae and 200 cercariae(half male and half female),respectively.At 7 and 8 weeks after infection,praziquantel was given at 200 mg/kg dosage for insecticidal treatment.The rabbits of group Ⅲ was infected with 200 cercariae of S.japonicum without treatment.Blood samples from middle ear artery were collected once a week before and 1-17 weeks after infection in each rabbit.The sera of each rabbit were collected and extracted twice by silica column method under the same conditions.Nested PCR based on SjCHGCS19 target was used to repeat detection three times.Results:1.The sera of rabbits with different infection degrees was extracted twice by silica column method.Nested-PCR results showed that the positive detection rates of group I(500 cercariae praziquantel treated group)after infection of ld-4w fluctuated in a low level(11%-56%).The positive detection rate of 5w-7w for the first fluctuated between 78%-89%,which was different from that of the second positive detection rate(89%-100%).After praziquantel treatment,the positive detection rate declined sharply at 2w(9w after infection),and fluctuated at a low level after 9w-17w.There was no significant difference in the positive detection rate of two extraction tests with silica column method in different stages(early stage,acute stage and post-treatment)(P>0.05).The consistency of the results of two extractions of sera DNA from infected rabbits by silica column method was good(P<0.001,Kappa＝0.712>0.4),and there was no statistical difference in consistency P>0.05 by McNemar test).2.The positive detection rate of the first for group Ⅱ(200 cercariae praziquantel treated group)fluctuated between 11%and 33%at 1d-4w after infection,which was different from the second positive detection rate(11%-44%).The positive detection rate of 5w-7w all fluctuated between 78%and 100%.After praziquantel treatment,the positive detection rate declined sharply at 2w(9w after infection),and fluctuated at a low level from 9w to17w.There was no significant difference in the positive detection rate of two extraction tests with silica column method in different stages(early stage,acute stage and post-treatment)(P>0.05).The consistency of the results of two extractions of sera DNA from infected rabbits by silica column method was good(P<0.001,Kappa=0.576>0.4),but there was statistical difference in consistency(P<0.05 by McNemar test).3.The second positive detection rate of group Ⅲ(200 cercariae infected untreated group)at 4w after infection was 22%higher than that of the first(11%).The positive detection rate reached 100%for the second at 6 weeks after infection,which was higher than 78%for the first.The positive detection rate of 8w-11w after infection fluctuated from 67%to 89%for the first and the the second positive detection rate fluctuated from 33%to 67%.The positive detection rate of 12w-17w after infection fluctuated at a higher level.There was no significant difference in the positive detection rate of two extraction tests with silica column method in different stages(early stage,acute stage,chronic stage and late stage)(P>0.05).The consistency of the results of two extractions of sera DNA from infected rabbits by silica column method was good(P<0.001,Kappa=0.489>0.4),and there was no statistical difference in consistency(P>0.05 by McNemar test).Conclusion:There was no significant difference in the positive detection rate of sera DNA of rabbits with different infection degrees extracted twice by silica column method,and the consistency of the two detection results was good.The results show that the silica column method has the advantages of rapid,simple and good stability,and it is hopeful to further construct a serum DNA detection kit for schistosomiasis in epidemic areas,which has potential application value.Part 2 Evaluation the efficacy of LAMP assay based on silica gel membrane spin column for detection of Schistosoma japonicum infection in host seraObjective:The extraction efficiency silica column method and phenol chloroform method for sera DNA from infected rabbits was compared,and the detection effect of LAMP assay based on silica gel membrane spin column for host sera DNA in S.japonicum infection rabbit was evaluated.Methods:Six rabbits were randomly divided into two groups with 3 rabbits in each group.Each rabbit was infected with S.japonicum cercariae via abdomen according to routine steps.The rabbits of Group Ⅰ were infected with 200 cercariae and was treated with praziquantel at 200 mg/kg dosage at 6th and 7th week after infection.The rabbits of Group Ⅱ were infected with 200 cercariae of S.japonicum without treatment.Blood samples were collected once a week from the middle ear artery of each rabbit before and 1-20 weeks after infection.Blood samples were collected from the middle ear artery every two weeks from 20w to 28w.Serum DNA was extracted by silica column method and phenol chloroform method respectively.Nested PCR and LAMP based on SjCHGCS19 target were used for repeated detection three times.Results:1.The positive detection rate of the two methods for sera DNA extraction in the treatment group(group I)was all at a low level after infection of 1-3 weeks,but the positive detection rate of phenol chloroform method for serum DNA at 2 week after infection(67%)was significantly higher than that of silica column method(11%).At 4-6 weeks after infection,the positive detection rate of the two methods was higher than 50%.After praziquantel treatment,the positive detection rate of phenol chloroform method(89%)for 2nd wee k(8th week after infection)was higher than that of silica column method(44%).From the 3th week after treatment(9th week after infection),the positive detection rates of the two extraction methods fluctuated at a lower level.There was no significant difference in the positive detection rate between the two extraction methods in different stages(early stage,acute stage and post-treatment)(P>0.05);the consistency of the two extraction methods was good P<0.001,Kappa=0.475>0.4),and there was no statistical difference in consistency(P>0.05 by McNemar test).2.The positive detection rates of the two methods of sera DNA extraction in the untreated group(group Ⅱ)were lower after infection of 1-3 weeks,and the positive detection rate of phenol chloroform method at 2w after infection(44%)was higher than that of silica column method(0).The positive detection rate of 4-7w after infection increased significantly after,which were all higher than 50%.The positive detection rate of the two methods maintained in a high level after infection of 8-22 weeks and fluctuated slightly.There was no significant difference in the positive detection rate between the two extraction methods in different stages(early stage,acute stage and chronic stage and late stage)(P>0.05);the consistency of the two extraction methods was good P<0.001,Kappa=0.630>0.4),and there was no statistical difference in consistency(P>0.05 by McNemar test).3.The positive detection rate of LAMP based on two extraction methods for sera DNA of rabbits in the treatment group(group Ⅰ)was similar at 1d-4w after infection.The positive detection rate of LAMP based on two extraction methods was higher than 50%at 5w-6w after infection.When the rabbits were treated with praziquantel,the positive detection rate of serum DNA after infection of 7w-28w extracted by phenol chloroform fluctuated between 22%and 89%,and that by silica column fluctuated from 11%to 78%.There was no significant difference in the positive detection rate of LAMP based on two extraction methods in different stages(early stage,acute stage and post-treatment)of rabbits in the treatment group(P>0.05);the consistency of the two extraction methods was good(P<0.001,Kappa=0.416>0.4),and there was no statistical difference in consistency(P>0.05 by McNemar test).4.The positive detection rate of LAMP based on two extraction methods was higher in untreated group(group Ⅱ)at 1d after infection.The positive detection rate of phenol chloroform method(67%)was slightly higher than that of silicon column method(56%).The positive detection rate of two methods was higher than 50%at 4w-7w after infection,and there was no significant difference.From 8w to 11w after infection,the positive detection rate of the two extraction methods fluctuated greatly,phenol chloroform method fluctuated between 33%and 89%,and silica column method fluctuated from 33%to 78%.The positive detection rate of phenol chloroform and silica column methods fluctuated between 33%and 100%at 12w-28w after infection.There was no significant difference in the positive detection rate of LAMP based on two extraction methods in different stages(early stage,acute stage and chronic stage and late stage)of rabbits in the treatment group(P>0.05);the consistency of the two extraction methods was good(P<0.001,Kappa=0.416>0.4),and there was no statistical difference in consistency(P>0.05 by McNemar test).5.The extraction step of silica column method is 5 steps,less than that of phenol chloroform method(6 steps).It takes 1.5 hours to extract 20 samples,significantly less than that of phenol chloroform method(4 hours).It takes only 3 steps to complete LAMP detection less than that of nested PCR method(5 steps).The detection time of 40 samples(2 hours)is significantly less than that of nested PCR method(4.5 hours).LAMP based on silica column method can significantly shorten the detection time of serum DNA.Conclusion:This study showed that the method of nucleic acid extraction with silica gel membrane spin column could replace the method of phenol chloroform combined with LAMP for rapid detection of DNA in sera of rabbits infected with S.japonicum and this extraction and detection method has the advantages of simplicity and rapidity that phenol chloroform and nested PCR method can not match.It is hopeful to develop LAMP kit based on silica column method for DNA detection in schistosomiasis field,which has potential application value.Part 3 Establishment LAMP assay based on nucleic acid colorimetry with AuNP probe for detection of Schistosoma japonicum infectionObjective:To establish a DNA detection system for S.japonicum based on nucleic acid colorimetry with gold nanoprobe with SjR2 sequence,so as to make up for the deficiency of fluorescent dye method in detecting amplified products of LAMP,and further improve the sensitivity and specificity of LAMP method for DNA detection of S.japonicum.Methods:The oligonucleotide probe was designed to connect with gold nanoparticles,and the AuNP probe was determine the success of the connection by UV-Vis absorption spectroscopy.DNA template of adult S.japonicum and negative serum DNA template were used for LAMP amplification,and the detection conditions of LAMP amplification products were optimized by using AuNP probe under the condition that negative and positive reactions could be clearly distinguished by naked eyes.The sensitivity and specificity of the detection system were analyzed,and compared with the fluorescence dye detection method.Nucleic acid gel electrophoresis was used to verify the amplified products of LAMP.Results:1.The results of two connections showed that the absorbance of the washing solution at 260 nm was-0.006 and 0.005 Abs,and that of the AuNP probe solution at 260 nm was 0.586 and 0.532 Abs,respectively,and also absorbance at 526 nm was 0.505 and 0.485 Abs.2.The optimum salt concentration(MgSO4)was 150 mM(final concentration was 50 mM)in the DNA detection system of adult S.japonicum by LAMP based on nucleic acid colorimetry with gold nanoprobe and the optimum ratio of AuNP probe to LAMP amplification product was 5:5.3.The detection sensitivity of the first and second AuNP probe was 1:105 and 1:106 respectively,which was consistent with detection sensitivity of the fluorescence dye method and the nucleic acid gel electrophoresis.In addition to the positive results of S.japonicam and S.mansoni,all the other results were negative,consistent with the results of fluorescent dye and nucleic acid gel electrophoresis.Conclusion:The oligonucleotide probes can be successfully linked to gold nanoparticles,and LAMP based on SjR2 target for nucleic acid colorimetric analysis had good detection sensitivity and specificity for adult DNA of S.japonicum.It is expected to construct a nucleic acid diagnostic kit for schistosomiasis japonica by combining it with the nucleic acid extraction of silica gel membrane spin column,which has potential field application value. |
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