Method for detecting schistosoma japonicum katsurada and host oncomelania thereof through double-probe fluorescent quantitative PCR

Abstract

The invention discloses a method for detecting schistosoma japonicum katsurada and host oncomelania of the schistosoma japonicum katsurada through fluorescent quantitative PCR (polymerase chain reaction) by using a double probe method. The target gene of the schistosoma japonicum katsurada is one of 16S rRNA (ribosomal ribonucleic acid), 18S small subunit rRNA gene, non-LTR (long-term transposon) retrotransposon SjCHGCS19 and non-LTR retrotransposon SjCHGCS20; the host oncomelania quality control gene is one of a COX1 gene, a 18S rRNA (ribosomal Ribonucleic Acid) gene and a 28S rRNA (ribosomal Ribonucleic Acid) gene. Preferably, the target gene of the schistosoma japonicum katsurada is the 18S small subunit rRNA gene, and the host oncomelania quality control gene is the 28S rRNA gene. The method can be used for rapidly and accurately detecting the schistosoma japonicum katsurada and the host oncomelania of the schistosoma japonicum katsurada, so that whether a detection sample is infected by the schistosoma japonicum katsurada or not can be rapidly judged, the schistosoma japonicum katsurada can also be used for evaluating the schistosoma japonicum katsurada spreading risk, and reference is provided for

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a method for detecting schistosoma japonicum and its host Oncomelania snail using a double probe method of fluorescent quantitative PCR. Background Art

[0002] Oncomelania snails are the only intermediate host of Schistosoma japonicum. They spread with water and play an important role in the spread of schistosomiasis. As the prevention and control process progresses, my country has achieved remarkable results in the prevention and control of schistosomiasis, and the disease rates of humans and animals have dropped to the lowest level in history. However, the snail breeding area in the country has remained at 3.6 billion m for many years. 2 , in some areas, new snail breeding environments are still discovered or reappeared every year. The snail breeding area is large and the density is high. At the same time, the transplantation of seedlings and wetland construction have led to the improvement of the ecological restoration of the natural environment, which is suitable for the breeding of snails. The presence of wild mammals and the re-breeding and open release of oxen have created a major potential risk of schistosomiasis transmission. Therefore, strengthening the monitoring of infectious snails is a key task in the prevention and control of schistosomiasis.

[0003] Traditional methods for detecting infected snails are mainly crushing microscopy and larvae detection. Under low-level epidemic conditions, these two etiological detection methods may miss detection due to low sensitivity. In addition, since it takes about 2 months for schistosoma miracidia to develop into cercariae after drilling into the snail, etiological methods for detecting cercariae often cannot identify snails that are still in the early stage of infection. Molecular biological detection technology can effectively solve this problem.

[0004] At present, the molecular biological detection method for schistosomiasis-infected snails used in the national schistosomiasis risk monitoring points is a loop-mediated isothermal amplification (LAMP) kit designed and developed by the Institute of Parasitic Diseases Prevention and Control of the Chinese Center for Disease Control and Prevention. However, after years of field application, laboratory testing technicians in grassroots schistosomiasis prevention agencies often report that the performance of the kit is not stable enough, and due to the lack of snail quality control genes, it is impossible to determine the false negatives caused by the low quality of the extracted snail DNA. Therefore, providing a method for detecting Japanese Schistosoma and its host snails with higher accuracy and stability is a problem that technicians in the field urgently need to solve. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting Japanese schistosoma and its host Oncomelania snail by dual probe fluorescence quantitative PCR. The method can realize rapid and accurate detection of Japanese schistosoma and its host Oncomelania snail, thereby being used to quickly determine whether the test sample is infected with schistosomiasis, and can also be used to evaluate the risk of schistosomiasis transmission, providing a reference for schistosomiasis prevention and control.

[0006] To achieve the above-mentioned purpose, the present invention provides a method for detecting Schistosoma japonicum and its host Oncomelania hupensis by double probe fluorescence quantitative PCR.

[0007] The target gene of Schistosoma japonicum is one of 16S rRNA, 18S small subunit rRNA gene, non-LTR retrotransposon SjCHGCS19, and non-LTR retrotransposon SjCHGCS20;

[0008] When the target gene of Schistosoma japonicum is 16S rRNA, the forward primer of the gene is 5′-GGGTTTGTATTATAAGTTTATGA-3′, the rear primer is 5′-ACAGCACTAATCAATCTATAC-3′, and the fluorescent probe is 5′-TCCAAGAACCTCAACCTAACATCAACA-3′;

[0009] When the target gene of Schistosoma japonicum is the 18S small subunit rRNA gene, the forward primer of the gene is 5'-GCTACTACCGATTGAATG-3', the rear primer is 5'-TCGTCTTCTCGATATCAG-3', and the fluorescent probe is 5'-CCACTACAACGACACCAATCCAAC-3';

[0010] When the target gene of Schistosoma japonicum is the non-LTR retrotransposon SjCHGCS19, the forward primer of the gene is 5′-CAGAGTGTTGTTGAATAGAA-3′, the rear primer is 5′-CCTTCTCATAGTCGATGA-3′, and the fluorescent probe is 5′-TACAGTGACGAGTTCCATTCCATTGA-3′;

[0011] When the target gene of Schistosoma japonicum is the non-LTR retrotransposon SjCHGCS20, the forward primer of the gene is 5′-GGTGAAGACACTGATAAGA-3′, the rear primer is 5′-CCAGTCCTGAAGTAACAA-3′, and the fluorescent probe is 5′-CTTCTGGTCGCACTGAGCAAC-3′;

[0012] The host snail quality control gene is one of the COX1 gene, 18S rRNA gene, and 28S rRNA gene;

[0013] When the host snail quality control gene is COX1 gene, the forward primer of the gene is 5'-CCACTTGCTGGAAATCTA-3', and the rear primer is 5'-CCAGCCAGATGTAAAGAA-3';

[0014] When the host snail quality control gene is the 18S rRNA gene, the forward primer of the gene is 5'-GCATCTTTCAAATGTCTGCCCTATC-3', and the rear primer is 5'-TGTTATTTTTCGTCACTACCTCCCC-3';

[0015] When the host snail quality control gene is the 28S rRNA gene, the forward primer of the gene is 5'-CGGACTCCCCTCTTGCTTC-3', the rear primer is 5'-ACCTTGCTCACCGCCTTCT-3', and the fluorescent probe is 5'-TTGACGTCGAGCAACCG-3'.

[0016] Preferably, the target gene of Schistosoma japonicum is the 18S small subunit rRNA gene; and the host snail quality control gene is the 28S rRNA gene.

[0017] Preferably, in the reaction system used in this method, the primer concentration of the schistosome target gene is 400 nM, and the probe concentration is 200 nM; the primer concentration of the snail quality control gene is 200-800 nM, and the probe concentration is 100-400 nM, and the presence of the snail quality control gene primers and probes in the system does not affect the quantification of the schistosome target gene.

[0018] Preferably, in the reaction system used in this method, the primer concentration of the schistosome target gene is 400 nM, and the probe concentration is 200 nM; the primer concentration of the snail quality control gene is 200 nM, and the probe concentration is 100 nM, and the presence of the snail quality control gene primers and probes in the system does not affect the quantification of the schistosome target gene.

[0019] Preferably, the total volume of the reaction system used in the method is 20 μl, including 10 μl of 2×TaqMan PCR master Mix, 1 μl of DNA template, and double distilled water to make up to 20 μl.

[0020] Preferably, the reaction conditions used in this method are pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s, annealing / extension at 62°C for 30 s, for a total of 40 cycles.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The dual probe qPCR method of the present invention is used to detect the DNA of infected snails and negative snails, and its sensitivity, specificity, positive predictive value, negative predictive value and correct index are all 100%; when one cercariae is mixed with 50 negative snails to extract DNA, the positive detection rate is 26.67%; and when one infected snail is mixed with 200 negative snails to extract DNA, the positive detection rate is 100%, indicating that the dual probe qPCR method established by the present invention is significantly better than the existing LAMP technology in the detection of Schistosoma japonicum infected snails, and has broad prospects for field application; in the study of the mixed detection ratio of infected snails, the detection results of samples with a mixed ratio of 1:100 (number of infected snails: total number of snails) show that the detection performance of the dual probe qPCR method is equivalent to that of the RAA method (recombinase-mediated nucleic acid isothermal amplification fluorescence method), and when the mixed detection ratio is larger, it is found that at high mixing ratios of 1:150 and 1:200, the positive detection rate of this method is still 100%.

[0023] (2) The present invention uses the 18S small subunit rRNA, a specific gene of Schistosoma japonicum, as the target gene and the 28S rRNA, a gene of Oncomelania hupensis, as the quality control gene, to establish a dual TaqMan probe fluorescent quantitative PCR technology to detect Schistosoma japonicum-infected Oncomelania snails. The sensitivity and specificity are both as high as 100%, and it can effectively solve the problem that the current LAMP technology in schistosomiasis-endemic areas in my country cannot eliminate the false negative problem caused by the low quality of Oncomelania snail DNA, greatly improving the level of accurate detection of Oncomelania schistosome infectivity.

[0024] (3) The present invention can realize rapid and accurate detection of Schistosoma japonicum and its host Oncomelania japonica, thereby being used to quickly determine whether the test sample is infected with Schistosoma japonicum. It can also be used to evaluate the risk of schistosomiasis transmission and provide a reference for schistosomiasis prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the sensitivity results of the single-probe qPCR method for detecting different candidate target genes of Schistosoma japonicum in the embodiment, wherein A is 16S rRNA; B is 18S small subunit rRNA; C is SjCHGCS19; D is SjCHGCS20;

[0026] Figure 2 1 is a schematic diagram of the specific results of the single-probe qPCR method for detecting different candidate target genes of Schistosoma japonicum in the embodiment, wherein A is 16S rRNA; B is 18S small subunit rRNA; C is SjCHGCS19; D is SjCHGCS20;

[0027] Figure 31 is a peak diagram of double probe qPCR detection with different primer and probe concentration ratios in the embodiment, wherein SJ represents Schistosoma japonicum; OH represents Oncomelania hupensis;

[0028] Figure 4 : is a graph of the performance evaluation of the dual qPCR method in the embodiment; wherein SJ represents Schistosoma japonicum; OH represents Oncomelania hupensis; A is a Schistosoma japonicum infected Oncomelania snail sample; B is a negative Oncomelania snail sample;

[0029] Figure 5 The double qPCR method in the embodiment detects infected snail samples with different mixed ratios; wherein, SJ represents Schistosoma japonicum; OH represents Oncomelania hupensis; A is a 1:100 mixed sample; B is a 1:150 mixed sample; C is a 1:200 mixed sample. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] The raw materials and reagents used in the following examples, unless otherwise specified, are all commercially available.

[0032] Example

[0033] 1 Experimental animals

[0034] The snails infected with Schistosoma japonicum were provided by the Parasitic Disease Vector Control Office of Anhui Schistosomiasis Control and Prevention Institute, and the snails infected with Oncomelania japonica were provided by Dangtu County Center for Disease Control and Prevention, Ma'anshan City.

[0035] 2 Main reagents and instruments

[0036] 2.1 Reagents

[0037] Hieff Taqman multiplex qPCR master mix(11208ES)、 qPCR SYBRGreen Master mix (11201ES) was purchased from Shanghai Yisheng Biotechnology Co., Ltd.; blood / cell / tissue genomic DNA extraction kit (centrifugal column type, DP304) was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0038] Primers and probes were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0039] 2.2 Instruments

[0040] Electric constant temperature water bath (DK-S28, Shanghai Senxin Experimental Instrument Co., Ltd.), desktop centrifuge (Micro 17, Thermo Fisher Scientific), real-time fluorescence quantitative PCR instrument (Q3, Thermo Fisher Scientific), micro-spectrophotometer (nanophotometer, Implen).

[0041] 3 Methods

[0042] 3.1 Screening of Schistosoma japonicum-specific target genes

[0043] 3.1.1 Target gene selection

[0044] The mitochondrial 16S rRNA gene (Accession No.: JQ781206.1), 18S small subunit rRNA gene (Accession No.: AY157226.1), non-LTR retrotransposon SjCHGCS19 (Accession No.: FN356221.1) and SjCHGCS20 (Accession No.: FN356222.1) of Schistosoma japonicum were selected as candidate Schistosoma japonicum-specific target genes, and primers and TaqMan probes for fluorescence quantitative PCR (qPCR) amplification of the candidate target genes were designed using Beacon Designer 8 software (Table 1).

[0045] Table 1 qPCR primers and probes for candidate target genes of Schistosoma japonicum

[0046]

[0047] 3.1.2 Comparison of sensitivity of different candidate target genes

[0048] Ten infected snails were taken, and genomic DNA was extracted from the meat tissue of a single snail. Singleplex qPCR was performed using the primer and probe systems of the above four groups of candidate target genes of Schistosoma japonicum, respectively, and Schistosoma japonicum adult DNA and nuclease-free water were used as templates as positive and blank controls, respectively. The reaction system was 20 μl, including 400 nM of forward and reverse primers, 200 nM of TaqMan probe, 10 μl of 2×TaqMan PCR master Mix, 1 μl of DNA template, and double distilled water to 20 μl. The reaction parameters were 95°C pre-denaturation for 1 min; 95°C denaturation for 10 s, 62°C annealing / extension for 30 s, and 40 cycles. Ct values ​​≤ 35 and an S-shaped amplification curve were judged as positive, and no Ct value and no S-shaped amplification curve were judged as negative. Statistical analysis was performed to compare the Ct values ​​of the four reaction systems. The lower the average Ct value, the higher the reaction sensitivity, and the candidate target genes with high sensitivity were preferred.

[0049] Ten infected snails were collected, and 10 positive DNA samples were obtained by extracting the meat tissue of a single snail. The mitochondrial 16S rRNA gene, 18S small subunit rRNA gene, and non-LTR retrotransposons SjCHGCS19 and SjCHGCS20 of Schistosoma japonicum were used as candidate target genes, and primers and probes were designed. The single-plex qPCR method was used to detect 10 DNA samples of Schistosoma japonicum infected snails. The results showed that effective Ct values ​​were obtained for the detection of the above four genes, and the average Ct values ​​were 33.62±0.94, 14.23±0.42, 24.82±1.45 and 24.75±0.99, respectively. Among them, the average Ct of the 18S small subunit rRNA gene was the lowest (F=25.32, P<0.0001), indicating that its detection sensitivity was the highest. In addition, the four candidate target genes in the positive control of Schistosoma japonicum adult DNA all obtained effective Ct values, while no peaks were observed in the blank control (see Figure 1 ).

[0050] 3.1.3 Comparison of specificity of different candidate target genes

[0051] Take 15 negative snails, extract genomic DNA from a single snail meat tissue as a negative control, and use nuclease-free water instead of template as a blank control. Use the above four groups of primers and probe systems of Schistosoma japonicum candidate target genes to perform single-plex qPCR reactions. The reaction system and reaction conditions are the same as 3.1.2. A Ct value ≤ 35 and an S-shaped amplification curve are judged as positive, and no Ct value and no S-shaped amplification curve are judged as negative. Compare the false positive rates of the four reaction systems, and select candidate target genes with high specificity.

[0052] Fifteen negative snails were taken, and 15 negative DNA samples were obtained by extracting the meat tissue of a single snail. At the same time, the DNA of adult Schistosoma japonicum was used as a positive control, and enzyme-free water was used as a blank control. The four candidate target genes of Schistosoma japonicum in the above samples, namely 16S rRNA, 18S small subunit rRNA, SjCHGCS19 and SjCHGCS20, were detected by single-probe qPCR method. The results showed that there was no amplification curve in the 15 negative DNA samples and the blank control, while the positive control had a good amplification curve, indicating that the detection of the four candidate target genes had good specificity (see Figure 2 ).

[0053] 3.1.4 Comparison of minimum detection limits of different candidate target genes

[0054] Extract DNA from adult Schistosoma japonicum, and determine the total DNA concentration using a micro-spectrophotometer. Dilute the total DNA in a 10-fold concentration gradient, i.e., 1:10, 1:100, 1:1000, 1:10000, 1:100000, 1:1000000, and 1:1000000, to prepare DNA templates of different concentrations, and use nuclease-free water instead of the template as a blank control. Use the primer and probe systems of the above four groups of Schistosoma japonicum candidate target genes to perform single-plex qPCR reactions. The reaction system and reaction conditions are the same as 3.1.2. A Ct value of ≤35 and an S-shaped amplification curve are judged as positive, and a Ct value and an S-shaped amplification curve are judged as negative. Compare the minimum detection concentrations of the four reaction systems, and select the candidate target gene with the lowest candidate detection concentration.

[0055] The adult DNA of Schistosoma japonicum was diluted in a 10-fold concentration gradient to prepare DNA templates of different concentrations. The single-plex qPCR method was used to detect the four candidate target genes of Schistosoma japonicum in adult DNA of different dilutions, and the minimum detection limits of different candidate target genes were compared. The results showed that when the adult DNA concentration was as low as 10 -5 ng, effective Ct values ​​could still be obtained by detecting the 18S small subunit rRNA gene (Table 2).

[0056] Table 2 Minimum detection limits of different candidate target genes of Schistosoma japonicum by single-plex probe qPCR

[0057]

[0058] Note: “-” indicates no valid Ct value was measured

[0059] 3.2 Quality control gene screening of Oncomelania hupensis in Hubei

[0060] 3.2.1 Quality control gene selection

[0061] The genes of Oncomelania hupensis cytochrome oxidase subunit 1 (COX1) (Accession No.: MW025080.1), 18S rRNA (Accession No.: AF367667.1) and 28S rRNA (Accession No.: AY207042.1) were selected as candidate quality control genes of Oncomelania hupensis, and primers and TaqMan probes for qPCR amplification of candidate target genes were designed using Beacon Designer 8 software (Table 3).

[0062] Table 3 qPCR primers and probes for candidate quality control genes of Oncomelania hupensis

[0063]

[0064] 3.2.2 Comparison of sensitivity of different candidate quality control genes

[0065] 15 snails were taken, DNA was extracted from single snail meat tissue, nuclease-free water was used instead of template as blank control, and single-plex qPCR reaction was performed on three sets of candidate quality control gene primers of Hubei snails using SYBR Green method. The reaction system was 20μl, including 400nM of forward and reverse primers, 10μl of SYBR Green master mix, 1μl of DNA template, and double distilled water to 20μl. The reaction conditions were 95℃ pre-denaturation for 1min; 95℃ denaturation for 10s, 62℃ annealing / extension for 30s, and 40 cycles in total. Ct value ≤ 35 and S-shaped amplification curve were judged as positive, and no Ct value and no S-shaped amplification curve were judged as negative. Statistical analysis and comparison of the Ct values ​​of the three reaction systems showed that the lower the average Ct value, the higher the reaction sensitivity, and the candidate quality control gene with high sensitivity was preferred.

[0066] Fifteen snails were taken, and 15 DNA samples were obtained by extracting the meat tissue of a single snail. With COX1, 18S rRNA and 28SrRNA as candidate quality control genes of Hubei snails, primers were designed, and SYBR Green qPCR method was used to detect 15 snail DNA samples. The results showed that among the 15 DNA samples, 5 and 4 samples failed to obtain valid Ct values ​​for the detection of COX1 and 18S rRNA genes, respectively, while all samples obtained valid Ct values ​​for the detection of 28S rRNA gene. The average Ct values ​​of samples with effective Ct values ​​detected for COX1, 18SrRNA and 28S rRNA genes were 22.90±1.53, 10.39±0.75 and 12.88±0.79, respectively. The differences among the three genes were statistically significant (H=24.18, P<0.0001). However, the results of pairwise comparison showed that the average Ct value of COX1 gene was significantly higher than that of 18S rRNA and 28S rRNA (P<0.0001, P=0.0095), while the difference between the latter two genes was not statistically significant (P=0.0535), suggesting that 28S rRNA is the best candidate quality control gene for Oncomelania hupensis. In addition, no effective Ct values ​​were obtained for the three candidate quality control genes of Oncomelania hupensis in the blank control (Table 4).

[0067] Table 4 Sensitivity of SYBR Green qPCR method in detecting different quality control genes of Oncomelania hupensis

[0068]

[0069]

[0070] Note: “-” indicates no valid Ct value was measured

[0071] 3.3 Evaluation of the dual probe qPCR reaction system

[0072] 3.3.1 Screening of primer and probe concentration ratios for Schistosoma japonicum target genes and Oncomelania hupensis quality control genes

[0073] Ten negative snails and Schistosoma japonicum infected snails were taken respectively, and genomic DNA was extracted from single snail meat tissue, and nuclease-free water and Schistosoma japonicum adult DNA were used as templates as blank and positive controls. The primers and probes of the optimal Schistosoma target gene and snail quality control gene screened were prepared into three reaction systems (Table 5) according to different concentration ratios, and the above samples were detected by dual qPCR. The total volume of the three reaction systems was 20μl, including 10μl of 2×TaqMan PCR masterMix, 1μl of DNA template, and double distilled water to 20μl.

[0074] Table 5 Primer and probe concentrations for Schistosoma japonicum and Oncomelania hupensis genes

[0075]

[0076] The reaction conditions were 95℃ pre-denaturation for 1min; 95℃ denaturation for 10s, 62℃ annealing / extension for 30s, for a total of 40 cycles. A Ct value of ≤35 and an S-shaped amplification curve were considered positive, while no Ct value and no S-shaped amplification curve were considered negative. Statistical analysis was performed to compare the Ct values ​​of the three reaction systems. The lower the average Ct value, the better the concentration ratio of primers and probes.

[0077] Ten negative Oncomelania snails and Schistosoma japonicum infected Oncomelania hupensis were taken respectively, and 10 negative and positive DNA samples were obtained by extracting the meat tissue of a single snail. The 18S rRNA gene of Schistosoma japonicum was used as the target gene, and the 28S rRNA of Oncomelania hupensis was used as the quality control gene. The primers and probes of the two were prepared into three different reaction systems A, B, and C at different concentrations. The above samples were detected by double probe qPCR method, and nuclease-free water and Schistosoma japonicum adult DNA were used as templates as blank and positive controls. The results showed that in system A, none of the 10 negative snail DNA samples had valid Ct values ​​for the Schistosoma target gene, and all the Oncomelania snail quality control genes had valid Ct values; among the 10 Schistosoma japonicum infected Oncomelania snail DNA samples, one sample had no valid Ct value for the Schistosoma target gene, and all the Oncomelania snail quality control genes had valid Ct values. In the B system, none of the 10 negative snail DNA samples had valid Ct values ​​for the Schistosoma target genes, and one sample of the Oncomelania snail quality control gene did not have a valid Ct value; none of the 10 schistosome-infected Oncomelania snail DNA samples had valid Ct values ​​for the Schistosoma target genes, and all the Oncomelania snail quality control genes had valid Ct values. In the C system, none of the 10 negative snail DNA samples had valid Ct values ​​for the Schistosoma target genes, and all the Oncomelania snail quality control genes had valid Ct values; all of the 10 schistosome-infected Oncomelania snail DNA samples had valid Ct values ​​for the Schistosoma target genes, and all the Oncomelania snail quality control genes had valid Ct values. In addition, the blank and positive controls detected in the three reaction systems were all correct expected results (Table 6).

[0078] The target genes of Schistosoma japonicum in the infected snail DNA samples were detected by using the A, B, and C systems, and the average Ct values ​​of the samples with effective Ct values ​​were 20.76±1.69, 26.71±1.78, and 21.65±0.90, respectively. Although the differences among the three were statistically significant (H=7.872, P=0.0195), the pairwise comparison results showed that the average Ct value detected by the C system was not statistically significant compared with that of the A and B systems (P=0.8185, P=0.2378) (Table 6).

[0079] The snail quality control genes of all DNA samples were detected by A, B, and C systems, and the average Ct values ​​of samples with effective Ct values ​​were 17.07±0.43, 18.36±0.38, and 19.52±0.67, respectively, and the differences among the three were statistically significant (H=12.43, P=0.002). The results of pairwise comparison showed that the average Ct value detected by C system was not statistically significant different from that of B system (P=0.6632), but was significantly higher than that of A system (P=0.0015) (Table 6).

[0080] Table 6 Screening of primer and probe concentration ratios for Schistosoma target genes and Oncomelania snail quality control genes

[0081]

[0082] Note: SJ stands for Schistosoma japonicum; OH stands for Oncomelania hupensis; “-” means no valid Ct value was measured

[0083] From the amplification curve, the peak of Schistosoma target gene detected by C system in infected snail DNA samples is more obvious than that of A and B systems (see Figure 3 ).

[0084] In summary, when dual probe qPCR detection was performed with the 18S rRNA gene of Schistosoma japonicum as the target gene and the 28S rRNA of Oncomelania hupensis as the quality control gene, the primer and probe concentration ratio of the two in the C system was preferred, that is, the primer concentration of the Schistosoma target gene was 400 nM, the probe concentration was 200 nM, and the primer concentration of the Oncomelania hupensis quality control gene was 200 nM, and the probe concentration was 100 nM (Table 5).

[0085] 3.3.2 Evaluation of the effects of Oncomelania snail quality control gene primers and probes on the quantification of Schistosoma japonicum target genes

[0086] Take 10 snails infected with Schistosoma japonicum, extract genomic DNA from single snail meat tissue, and use nuclease-free water and adult DNA as templates as blank and positive controls respectively. The primers and probes of the optimal Schistosoma japonicum target gene and snail quality control gene screened out were used for the above samples according to the optimal concentration ratio in 3.3.1, and double qPCR was used. At the same time, single qPCR was used to detect only the optimal Schistosoma japonicum target gene in the above samples, and the concentrations of primers and probes of Schistosoma japonicum genes were the same as those of double qPCR. The reaction system of double and single qPCR was 20μl, including 10μl of 2×TaqMan PCR master Mix, 1μl of DNA template, and double distilled water to 20μl. The reaction conditions were all 95℃ pre-denaturation for 1min; 95℃ denaturation for 10s, 62℃ annealing / extension for 30s, and 40 cycles in total. Ct value ≤35 and S-shaped amplification curve were judged as positive, and no Ct value and no S-shaped amplification curve were judged as negative. Statistical analysis was performed to compare the average Ct values ​​of Schistosoma japonicum target genes in duplex and singleplex qPCR, and to evaluate the effects of Oncomelania snail quality control gene primers and probes on the quantification of Schistosoma japonicum target genes.

[0087] Ten snails infected with Schistosoma japonicum were taken, and 10 positive DNA samples were obtained by extracting the meat tissue of each snail. The dual probe qPCR method was used to detect the Schistosoma japonicum target genes and the snail quality control genes in the samples according to the primer and probe concentration ratio in the C system. At the same time, the single probe qPCR method (containing only the primers and probes of the Schistosoma japonicum target gene, with the same concentration as the dual probe method) was used to detect the Schistosoma japonicum target gene in the same sample. The average Ct values ​​of the Schistosoma japonicum target gene obtained by the single and dual probe qPCR methods were 20.06±0.31 and 20.81±0.43, respectively, and there was no statistically significant difference between the two (t=2.210, P>0.05) (Table 7), indicating that the presence of the snail quality control gene primers and probes in the dual probe qPCR reaction system had no effect on the quantification of the Schistosoma japonicum target gene.

[0088] Table 7 Comparison of Ct values ​​of single- and dual-probe qPCR for detection of Schistosoma japonicum genes

[0089]

[0090]

[0091] Note: SJ stands for Schistosoma japonicum; OH stands for Oncomelania hupensis

[0092] “-” indicates that no valid Ct value was measured

[0093] 3.4 Laboratory evaluation of the dual-probe qPCR method for detecting Schistosoma japonicum-infected Oncomelania snails

[0094] 3.4.1 Test performance evaluation

[0095] According to the mixed sample principle of snail infection LAMP detection in schistosomiasis endemic areas in my country, 1 snail infected with Schistosoma japonicum was mixed with 49 negative snails to prepare a 1:50 mixed snail sample. DNA was extracted from any 10 snails, and a total of 5 tubes of DNA solution were obtained. 10 μl of DNA was taken from each tube and mixed, which was a 1:50 mixed positive DNA sample, and a total of 15 positive DNA samples were prepared.

[0096] Take 50 negative snails, extract DNA from any 10 of them, and obtain 5 tubes of DNA solution. Take 10 μl of DNA from each tube and mix well, which is 1 negative DNA sample. A total of 15 negative DNA samples were prepared.

[0097] The optimal Schistosoma japonicum target gene and Oncomelania hupensis quality control gene were used as the detection objects. According to the optimal ratio of primer and probe concentration, the dual probe qPCR method was used to detect 15 positive and negative DNA samples. The Schistosoma japonicum target gene Ct value ≤ 35 and the presence of an S-shaped amplification curve were judged as positive, and no Ct value and no S-shaped amplification curve were judged as negative. At the same time, the positive result of the Oncomelania hupensis quality control gene was considered valid. The sensitivity, specificity, positive predictive value, negative predictive value and correct index of the dual probe qPCR method were calculated.

[0098] According to the mixed sampling principle of LAMP detection of snail infection in schistosomiasis-endemic areas in my country, 50 snails were mixed and extracted as one snail DNA sample, and 15 schistosomiasis-infected snail DNA samples (only 1 infected snail among 50 snails) and 15 negative snail DNA samples were prepared. The double probe qPCR method established in the previous experiment was used to detect the above samples. The results showed that in the 15 schistosomiasis-infected snail DNA samples, the schistosomiasis gene had an S-shaped amplification curve, with a Ct value of ≤35, and all the sample snail quality control genes had peaks ( Figure 4 A); In the 15 negative snail DNA samples, the Schistosoma target genes had no S-shaped amplification curve and no effective Ct value was obtained, while the snail quality control genes all had peaks ( Figure 4 B). The sensitivity, specificity, positive predictive value, negative predictive value and correct index of the dual probe qPCR method were all 100%.

[0099] 3.4.2 Minimum cercariae detection capability

[0100] Mix 1, 2, 5, 10, 50, and 100 cercariae with 50 negative snail tissues, and prepare 15 snail DNA samples containing different numbers of cercariae according to the DNA extraction method in 3.4.1. Use the established dual probe qPCR method to detect the above samples, and judge them as positive when the Ct value of the Schistosoma target gene is ≤35 and there is an S-shaped amplification curve, and judge them as negative when there is no Ct value and no S-shaped amplification curve. At the same time, the positive results of the snail quality control gene are considered valid. Calculate the positive detection rate of the dual probe qPCR method for mixed snail samples mixed with different numbers of cercariae.

[0101] DNA was extracted from 1, 2, 5, 10, 50, and 100 schistosome cercariae mixed with 50 negative snail tissues, and 15 DNA samples were extracted from each number group. The established dual probe qPCR method was used to detect snail DNA samples containing different numbers of cercariae. The results showed that the snail quality control genes in all samples had S-shaped amplification curves and effective Ct values ​​were obtained. For the detection of schistosome target genes, the positive detection rate of 1 cercariae group was 26.67% (4 / 15); the positive detection rates of 2, 5, and 10 cercariae groups were all 33.33% (5 / 15); the positive detection rates of 50 and 100 cercariae groups were 53.33% (8 / 15) and 60% (9 / 15), respectively. With the increase of cercariae number, the positive detection rate of schistosome target genes gradually increased.

[0102] 3.4.3 Exploration of snail sample detection with the highest mixed ratio

[0103] The mixing ratio of snail infection detection samples was increased, and three mixing ratios of 1:100, 1:150 and 1:200 were selected, that is, 1 Schistosoma japonicum infected snail was mixed with 99 / 149 / 199 negative snails, and 10 snails were randomly mixed to extract genomic DNA, and a total of 10 / 15 / 20 tubes of DNA solution were obtained. 10μl DNA was taken from each tube and mixed, that is, 1 1:100 / 150 / 200 mixed positive DNA sample, and 20 samples were prepared for each ratio, for a total of 60 samples. The established dual qPCR method was used to detect the above samples, and the Schistosoma target gene Ct value ≤35 and the S-shaped amplification curve were judged as positive, and the absence of Ct value and S-shaped amplification curve was judged as negative. At the same time, the positive snail quality control gene was considered valid. The positive detection rate of the dual probe qPCR method for snail samples with different mixing ratios was calculated.

[0104] The mixing ratio of snail infection detection samples was improved, and three mixing ratios of 1:100, 1:150 and 1:200 were selected. 20 infected snail DNA samples were prepared for each ratio (100 / 150 / 200 snails contained 1 schistosoma infected snail). The established dual probe qPCR method was used to detect the infected snail DNA samples with different mixing ratios. The results showed that in 60 DNA samples, the schistosoma target genes all had S-shaped amplification curves, with Ct values ​​≤35, and the snail quality control genes in each sample had peaks, indicating that this dual probe qPCR method still had a good detection effect on samples mixed at a ratio of 1:100, 1:150 and 1:200 ( Figure 5 ).

[0105] 3.5 Statistical analysis

[0106] GraphPad Prism 9.0 software was used for statistical analysis, and the average Ct value was expressed as "mean ± standard error". The average Ct value between groups was compared using t-test and one-way analysis of variance, or Mann-Whitney, Kruskal-Wallis and Friedman tests, with P < 0.05 as a statistically significant difference.

[0107] In summary, with the 18S rRNA gene of Schistosoma japonicum as the target gene and the 28S rRNA of Oncomelania hupensis as the quality control gene, a dual TaqMan probe-based fluorescence quantitative PCR technology was established to detect Schistosoma japonicum-infected Oncomelania snails. The sensitivity and specificity were both as high as 100%, and it can effectively solve the problem of false negatives caused by low quality of Oncomelania snail DNA that the current LAMP technology in schistosomiasis-endemic areas of my country cannot eliminate, greatly improving the level of accurate detection of Oncomelania schistosome infectivity in snails.

Claims

1. A method for detecting Schistosoma japonicum and its host Oncomelania hupensis by dual probe fluorescence quantitative PCR, characterized in that: The target gene of Schistosoma japonicum is one of 16S rRNA, 18S small subunit rRNA gene, non-LTR retrotransposon SjCHGCS19, and non-LTR retrotransposon SjCHGCS20; When the target gene of Schistosoma japonicum is 16S rRNA, the forward primer of the gene is 5′-GGGTTTGTATTATAAGTTTATGA-3′, the rear primer is 5′-ACAGCACTAATCAATCTATAC-3′, and the fluorescent probe is 5′-TCCAAGAACCTCAACCTAACATCAACA-3′; When the target gene of Schistosoma japonicum is the 18S small subunit rRNA gene, the forward primer of the gene is 5'-GCTACTACCGATTGAATG-3', the rear primer is 5'-TCGTCTTCTCGATATCAG-3', and the fluorescent probe is 5'-CCACTACAACGACACCAATCCAAC-3'; When the target gene of Schistosoma japonicum is the non-LTR retrotransposon SjCHGCS19, the forward primer of the gene is 5′-CAGAGTGTTGTTGAATAGAA-3′, the rear primer is 5′-CCTTCTCATAGTCGATGA-3′, and the fluorescent probe is 5′-TACAGTGACGAGTTCCATTCCATTGA-3′; When the target gene of Schistosoma japonicum is the non-LTR retrotransposon SjCHGCS20, the forward primer of the gene is 5′-GGTGAAGACACTGATAAGA-3′, the rear primer is 5′-CCAGTCCTGAAGTAACAA-3′, and the fluorescent probe is 5′-CTTCTGGTCGCACTGAGCAAC-3′; The host snail quality control gene is one of the COX1 gene, 18S rRNA gene, and 28S rRNA gene; When the host snail quality control gene is COX1 gene, the forward primer of the gene is 5'-CCACTTGCTGGAAATCTA-3', and the rear primer is 5'-CCAGCCAGATGTAAAGAA-3'; When the host snail quality control gene is the 18S rRNA gene, the forward primer of the gene is 5'-GCATCTTTCAAATGTCTGCCCTATC-3', and the rear primer is 5'-TGTTATTTTTCGTCACTACCTCCCC-3'; When the host snail quality control gene is the 28S rRNA gene, the forward primer of the gene is 5'-CGGACTCCCCTCTTGCTTC-3', the rear primer is 5'-ACCTTGCTCACCGCCTTCT-3', and the fluorescent probe is 5'-TTGACGTCGAGCAACCG-3'.

2. The method for detecting Schistosoma japonicum and its host Oncomelania hupensis by dual probe fluorescence quantitative PCR according to claim 1, characterized in that: The target gene of Schistosoma japonicum is the 18S small subunit rRNA gene; the host snail quality control gene is the 28S rRNA gene.

3. The method for detecting Schistosoma japonicum and its host Oncomelania hupensis by dual probe fluorescence quantitative PCR according to claim 1, characterized in that: In the reaction system used in the method, the primer concentration of the schistosome target gene is 400nM, and the probe concentration is 200nM; the primer concentration of the snail quality control gene is 200-800nM, and the probe concentration is 100-400nM, and the presence of the snail quality control gene primers and probes in the system does not affect the quantification of the schistosome target gene.

4. The method for detecting Schistosoma japonicum and its host Oncomelania hupensis by dual probe fluorescence quantitative PCR according to claim 2, characterized in that: In the reaction system used in the method, the primer concentration of the schistosome target gene is 400nM, and the probe concentration is 200nM; the primer concentration of the snail quality control gene is 200nM, and the probe concentration is 100nM, and the presence of the snail quality control gene primers and probes in the system does not affect the quantification of the schistosome target gene.

5. The method for detecting Schistosoma japonicum and its host Oncomelania hupensis by dual probe fluorescence quantitative PCR according to claim 1 or 2, characterized in that: The total volume of the reaction system used in this method is 20 μl, including 10 μl of 2×TaqMan PCRmaster Mix, 1 μl of DNA template, and double distilled water to 20 μl.

6. The method for detecting Schistosoma japonicum and its host Oncomelania hupensis by dual probe fluorescence quantitative PCR according to claim 1 or 2, characterized in that: The reaction conditions used in this method are pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s, and annealing / extension at 62°C for 30 s, for a total of 40 cycles.

Images