CN112795666B - Multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for simultaneously detecting three echinococcus - Google Patents
Multiplex real-time fluorescent quantitative PCR (polymerase chain reaction) primer, probe and kit for simultaneously detecting three echinococcus Download PDFInfo
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Abstract
The invention discloses a multiplex real-time fluorescent quantitative PCR primer, a probe and a kit for simultaneously detecting three echinococcus granulosus, belonging to the technical field of parasite pathogen detection. The invention simultaneously detects the multiple real-time fluorescence quantitative PCR primers and probes of three kinds of echinococcus, and simultaneously detects three kinds of pathogens of echinococcus multilocularis, echinococcus granulosus and echinococcus shiquiae, thereby greatly improving the detection efficiency, and saving the detection time and the detection cost. The invention has strong specificity, high sensitivity which is up to 10 copies/mul, and has no non-specific amplification to other common related parasite gene samples.
Description
Technical Field
The invention relates to the technical field of parasite pathogen detection, in particular to a multiplex real-time fluorescence quantitative PCR primer, a probe and a kit for simultaneously detecting three echinococcus.
Background
Echinococcosis (Hydatid disease), also known as echinococcosis, is an important zoonosis caused by tapeworm stage larvae in Echinococcus tapeworm, and is one of the 17 overlooked tropical diseases identified by the world health organization. Humans or rodents are infected by eating water or food contaminated with ova by mistake, and there are mainly epidemics of Cystic echinococcosis (Cystic echinococcosis) caused by Echinococcus granulosus (e.g.) and Alveolar Echinococcus multilocularis (e.g. melocularis, em) caused by Echinococcus multilocularis (alveolaris Echinococcus echinococcosis) mainly in northwest animal husbandry areas such as the Qinghai, xinjiang, gansu, tibet and Sichuan. Echinococcosis is a worldwide distribution in cold regions and permafrost regions of high latitude in the northern hemisphere of north america, europe and asia, such as north part of alaska and canada, western russia, germany, france, switzerland, austria, turkey, greece and polish in europe, and mediterranean countries such as irak and iran in the middle east, northwest and northeast regions of asia, and the north sea road in japan, and echinococcosis is also spreading continuously with the development of world livestock resources and the frequency of international import and export trades, and is now a global public health problem.
The number of people affected by only echinococcus granulosus infection is over 100 million per year, with over 30 billion dollars spent on treatment per year, under investigation. It is estimated that at least 100 million Disabled Adjusted Life Years (DALYs) are lost annually worldwide due to echinococcosis cystic disease; about 40 million DALYs are lost in China. In addition, worldwide echinococcosis cysticercosis yearly loses $ 19.2 million, and national losses lose $ 6.6 million; meanwhile, the annual animal husbandry production loss all over the world caused by echinococcosis cyst is as high as 21.9 hundred million dollars, and China has a significant share (about 85%). According to estimation, about 18000 new cases of echinococcosis vesicularis are annually discovered globally, wherein 16000 cases (about 91%) are in China; because the alveolar echinococcosis causes about 66.6 thousand years of DALYs globally, and correspondingly causes about 616000 Years of Life Loss (YLLS), wherein China accounts for 95 percent. National survey of echinococcosis in China from 2012 to 2016 shows that about 5000 ten thousand people in the 9 west provinces have infection risk, and nearly 17 ten thousand people have echinococcosis. At least 368 counties of China have echinococcosis cysticercosis epidemic, wherein the echinococcosis cysticercosis occurs in 119 counties at the same time. According to the survey, although the proportion of the global cases in China is not as high as that estimated in the early stage, china is still the most important epidemic high-occurrence area of echinococcosis cyst and cyst.
Three main species of Echinococcus multilocularis, echinococcus granulosus and Echinococcus shiquicus (e.shiquicu, es) are mainly prevalent in the Qinghai-Tibet plateau of China, and the terminal hosts of the Echinococcus shiquicu are mainly canids (dogs, wolves, foxes and the like), so that the Echinococcus shiquicus and the Echinococcus shiquicus are very important for detecting and repelling insects and preventing the terminal hosts. However, currently, china distinguishes Echinococcus multilocularis (Em), echinococcus granulosus (Eg) and Echinococcus shikokianus (Es) mainly through microscopic examination of morphologies of polypides and eggs, molecular biology and immunology detection methods and the like, and the methods all have the problems of harmfulness to operators and environment, complex operation, low sensitivity, omission, false positive and the like.
Therefore, it is an urgent need of those skilled in the art to provide a multiplex real-time fluorescent quantitative PCR primer, probe and kit for simultaneously detecting three echinococcus.
Disclosure of Invention
In view of this, the invention provides a multiplex real-time fluorescence quantitative PCR primer, a probe and a kit for simultaneously detecting three echinococcus granulosus, which can quickly, accurately and sensitively detect three echinococcus multilocularis (Em), echinococcus granulosus (Eg) and echinococcus shiquicus (Es).
In order to achieve the purpose, the invention adopts the following technical scheme:
the multiplex real-time fluorescent quantitative PCR primer and probe for simultaneously detecting three echinococcus Em/Eg/Es have the following primer sequences:
Em g s-F：5’-TTGTTTGCTATGTTTTCTATAGTGT-3’；SEQ ID NO.1；
Em g s-R：5’-TCTTCACATCYAACCCAACAGT-3’；SEQ ID NO.2；
wherein Y = C/T;
the probes used to detect Em were:
Em probe：5’-TTTAGGGAGTAGTGTTT-3’；SEQ ID NO.3；
wherein, the fluorescent group marked at the 5 'end is FAM, and the quenching group marked at the 3' end is BHQ1;
the probes used to detect Eg were:
Eg probe：5’-TTTGGGTAGCAGGGTT-3’；SEQ ID NO.4；
wherein the 5 'end marked fluorescent group is VIC, and the 3' end marked quenching group is BHQ1;
the probes used to detect Es were:
Es probe：5’-TTTAGGAAGGAGAGTTT-3’；SEQ ID NO.5；
wherein the 5 'end labeled fluorescent group is Cy5, and the 3' end labeled quenching group is BHQ1.
Further, the multiplex real-time fluorescent quantitative PCR kit for simultaneously detecting the three echinococcus Em/Eg/Es comprises the primers and the probe.
Further, the multiplex real-time fluorescence quantitative PCR kit for simultaneously detecting three echinococcus Em/Eg/Es also comprises a standard positive template and a negative control sample; the standard positive template is a positive plasmid standard substance containing target fragments of Echinococcus multilocularis, echinococcus granulosus and Echinococcus shikoensis; the negative control sample is RNase free H 2 O。
Further, the multiplex real-time fluorescence quantitative PCR detection method for simultaneously detecting three echinococcus Em/Eg/Es comprises the following operation steps: preparing an amplification reaction system by using the primer and the probe by taking the sample DNA as a template; performing real-time fluorescent quantitative PCR amplification to obtain an amplification curve; and analyzing the amplification curve and making a judgment.
Further, the amplification reaction system comprises: sample template 2. Mu.l, 10. Mu.m/L Em g s-F1.5. Mu.l, 10. Mu.m/L Em g s-R1.5. Mu.l, 10. Mu.m/L Em Probe 0.2. Mu.l, 10. Mu.m/L Eg Probe 0.2. Mu.l, 10. Mu.m/L Es Probe 0.2. Mu.l, 2 XProbe Master Mix 10. Mu.l, RNA free H 2 O4.4. Mu.l; the amplification procedure is as follows: 10min at 95 ℃;95 ℃ 10s,60 ℃ 30sec,40 cycles; the fluorescence signal was collected starting from the 60 ℃ step.
Further, the principle of analyzing and judging the amplification curve is as follows:
when Ct in the FAM fluorescence channel is less than or equal to 30 and the fluorescence channel has an amplification curve, judging that the sample is positive for Echinococcus multilocularis;
when 30-straw (Ct) in the FAM fluorescence channel is less than or equal to 33 and the fluorescence channel has an amplification curve, repeating the experiment once, if the Ct of the FAM fluorescence channel is still within the range or less than or equal to 33, judging that the sample is positive, otherwise, judging that the sample is negative;
when the Ct in the FAM fluorescence channel is more than 33 or no amplification curve exists, the sample is judged to be negative to echinococcus multilocularis;
when Ct in the VIC fluorescence channel is less than or equal to 30 and the fluorescence channel has an amplification curve, judging that the sample is positive by echinococcus granulosus;
when 30-straw (Ct) in the VIC fluorescence channel is less than or equal to 33 and the fluorescence channel has an amplification curve, repeating the experiment once, if the Ct of the VIC fluorescence channel is still within the range or less than or equal to 33, judging that the sample is positive, otherwise, judging that the sample is negative;
when the Ct in the VIC fluorescent channel is more than 33 or no amplification curve exists, the sample is judged to be negative to echinococcus granulosus;
when Ct in the Cy5 fluorescent channel is less than or equal to 33 and the fluorescent channel has an amplification curve, judging that the sample is positive for echinococcus shiquicus;
when 33-straw Ct in the Cy5 fluorescence channel is less than or equal to 36 and the fluorescence channel has an amplification curve, repeating the experiment once, if the Cy5 fluorescence channel Ct is still within the range or less than or equal to 36, judging that the sample is positive, otherwise, judging that the sample is negative;
when the Ct in the Cy5 fluorescence channel is more than 36 or no amplification curve exists, the sample is judged to be negative to the Echinococcus shikoensis.
Further, the application of the multiplex real-time fluorescence quantitative PCR primer and the probe for simultaneously detecting three kinds of Echinococcus Em/Eg/Es in the preparation of a kit for detecting Echinococcus multilocularis, echinococcus granulosus and Echinococcus shikoensis is provided.
According to the technical scheme, compared with the prior art, the invention discloses the multiple real-time fluorescent quantitative PCR primers, the probe and the kit for simultaneously detecting three echinococcus, overcomes the defect that Em/Eg/Es cannot be rapidly, sensitively and accurately quantitatively detected in the prior art, simultaneously detects three pathogens, greatly improves the detection efficiency, and saves the detection time and the detection cost; the time of a common PCR detection method or a common serological detection method is more than 3 hours, and the detection time is shortened to 1 hour by the method. The invention has strong specificity, high sensitivity which is up to 10 copies/mul, and has no non-specific amplification to other common related parasite gene samples. The kit can simultaneously detect three pathogens, namely echinococcus multilocularis, echinococcus granulosus and echinococcus shiquiae, by using a pair of primer pairs and three probes, and has strong specificity and high sensitivity.
The method has the advantages of short detection time period, high detection efficiency and potential application value; the primer and the probe sequence related by the invention are specifically targeted to echinococcus multilocularis, echinococcus granulosus and echinococcus shikoensis, the probe sequence is as long as 17bp, 16bp and 17bp, so that the probability of generating nonspecific amplification with other organisms is (1/4) 17 And (1/4) 16 And (1/4) 17 Almost is zero, which also ensures the high specificity and accuracy of the invention; the method can perform pathogen quantitative analysis while performing pathogen qualitative analysis, and has good quantitative linear range; the detection sensitivity is high; simple operation and easy popularization.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram showing the triple real-time quantitative PCR amplification results of Em, eg and Es;
wherein, the amplification curves 1-3 are the amplification results of Em, and the concentrations are respectively 2X 10 8 Copy, 2X 10 7 Copy, 2X 10 6 Copying; the amplification curves 4 to 6 show the amplification results of Eg at concentrations of 2X 10, respectively 8 Copy, 2X 10 7 Copy, 2X 10 6 Copying; the amplification curves 7-9 are the results of amplification of Es at concentrations of 2X 10, respectively 8 Copy, 2X 10 7 Copy, 2X 10 6 Copying; amplification curves 10-11 are negative controls (i.e., RNase free H) 2 O as a negative control);
FIG. 2 is a graph showing the FAM mark sensitivity results of the present invention;
FIG. 3 is a graph showing the VIC marker sensitivity results of the present invention;
FIG. 4 is a graph showing the sensitivity results of Cy5 labeling according to the present invention;
wherein, in FIGS. 2-4, the amplification curve 1 is 10 9 Copy concentration, amplification Curve 2 10 8 Copy concentration, amplification Curve 3, 10 7 Copy concentration, amplification Curve 4 is 10 6 Copy concentration, amplification Curve 5 10 5 Copy concentration, amplification Curve 6, 10 4 Copy concentration, amplification Curve 7 is 10 3 Copy concentration, amplification Curve 8 is 10 2 Copy concentration, amplification Curve 9, 10 1 Copy concentration, amplification Curve 10 is a negative control (i.e., RNase free H) 2 O as a negative control);
FIG. 5 is a graph showing the Em standard curve of the present invention;
FIG. 6 is a graph showing the Eg standard curve of the present invention;
FIG. 7 is a graph showing the Es standard curve of the present invention;
FIG. 8 is a graph showing the results of the specificity test of the present invention;
wherein the amplification curve 1 is Echinococcus multilocularis; amplification curve 2 is echinococcus granulosus; the amplification curve 3 is echinococcus shiquicus; the amplification curve 4 is Taenia multiceps (Tm); the amplification curve 5 is Taenia solium (Ts); amplification curve 6 is asian Taenia (Ta); amplification curve 7 is canine complex bore tapeworm (Dc); the amplification curve 8 is Taenia vesiculosus (Th); amplification curve 9 is Toxocara canis (Tc); amplification curve 10 is Fasciola hepatica (Fh); the amplification curve 11 is taenia pisiformis (Tp); the amplification curve 12 is a midline tapeworm (Ml); the amplification curve 13 is Cryptosporidium caninum (Cc).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The sample is a positive plasmid standard containing target fragments of Em, eg and Es, and is subjected to PCR amplification by using a Roche kit (Roche Probe PCRKit).
PCR amplification System: sample template 2. Mu.l, 10. Mu.m/L Em g s-F1.5. Mu.l, 10. Mu.m/L Em g s-R1.5. Mu.l, 10. Mu.m/L Em Probe 0.2. Mu.l, 10. Mu.m/L Eg Probe 0.2. Mu.l, 10. Mu.m/L Es Probe 0.2. Mu.l, 2 XProbe MasterMix 10. Mu.l, RNA free H 2 O 4.4μl。
Em g s-F：5’-TTGTTTGCTATGTTTTCTATAGTGT-3’；SEQ ID NO.1；
Em g s-R：5’-TCTTCACATCYAACCCAACAGT-3’；SEQ ID NO.2；
Wherein Y = C/T;
the probes used to detect Em were:
Emprobe：5’-TTTAGGGAGTAGTGTTT-3’；SEQ ID NO.3；
wherein, the fluorescent group marked at the 5 'end is FAM, and the quenching group marked at the 3' end is BHQ1;
the probes used to detect Eg were:
Eg probe：5’-TTTGGGTAGCAGGGTT-3’；SEQ ID NO.4；
wherein, the fluorescent group marked at the 5 'end is VIC, and the quenching group marked at the 3' end is BHQ1;
the probes used to detect Es were:
Es probe：5’-TTTAGGAAGGAGAGTTT-3’；SEQ ID NO.5；
wherein the 5 'end-labeled fluorescent group is Cy5, and the 3' end-labeled quenching group is BHQ1
GenBank accession numbers for primers and probes are shown in Table 1.
TABLE 1
The real-time fluorescent quantitative PCR reaction program is as follows: 10min at 95 ℃;95 ℃ 10s,60 ℃ 30sec,40 cycles; the fluorescence signal was collected starting from the 60 ℃ step. Detection was performed using a Light Cycle 96 fluorescent quantitative PCR instrument.
The results of triple real-time quantitative PCR amplification of Em, eg and Es are shown in FIG. 1; the results of real-time quantitative PCR amplification of Em, eg and Es are shown in FIGS. 2-4, respectively.
From the results of the sensitivity tests of FIGS. 2-4, it can be seen that the detection is 10 2 Criteria ct for determination of copy, FAM (Echinococcus multilocularis) and VIC (Echinococcus granulosus) markers>33 are negative, 30<ct is less than or equal to 33, suspicious and positive is determined less than or equal to 30; criterion of CY5 (Echinococcus shiquicus) marker, ct>36 is negative, 33<ct is less than or equal to 36, suspicious and positive is determined at 33. The limit of detection can be detected to 10 copies/. Mu.l.
The samples (positive plasmid standard containing target fragments of Em, eg and Es) were diluted to the corresponding concentrations in Table 2, and the amplification cycle numbers of Em, eg and Es were obtained, and the results are shown in Table 2.
TABLE 2
Obtaining Em standard curve (y = -3.1849x + 37.722) of FIG. 5, eg standard curve (y = -3.4127x + 38.821) of FIG. 6 and Es standard curve (y = -3.3464x + 42.481) of FIG. 7 according to the results of Table 2; the abscissa of the standard curve is the Ct value of the standard substance, and the ordinate is the logarithm of the concentration of the standard substance; the amplification correlation degree R of Em/Eg/Es thereof 2 The amplification efficiency was respectively 0.9895/0.9874/0.9847, and 106.1%/96.3%/99.0%.
Example 2 specificity test
Specificity tests (the test process is the same as example 1) are carried out by respectively taking parasite genome DNA (Taenia multiceps, taenia bovis, taenia Asiatica, taenia caninum, taenia vesiculosus, toxobolus caninum, toxoplasma gondii, taenia midline, cryptosporidium caninum) and positive plasmid standard products as templates, and the results are shown in FIG. 8; the results in FIG. 8 show that only the positive samples of Echinococcus multilocularis Em, echinococcus granulosus Eg and Echinococcus shikoensis Es have amplification curves in the detection results of the positive plasmid standard, and are positive, and the other control detection samples are negative, which indicates that the method has good specificity.
Example 3
Ct values of the triple real-time quantification method and the single real-time quantification method were compared, and the results are shown in Table 3.
TABLE 3 comparison of Ct values for triple real-time quantitation method and single real-time quantitation method
Note: (triple: a pair of universal primers plus three probes; and single: a pair of universal primers plus a corresponding probe) for the detection of the same samples 1, 2, and 3.
The results in Table 3 show that the detection values of the triple PCR detection and the single PCR detection on the same sample are not changed greatly, and the detection results are reliable.
Example 4 repeatability test
The diluted plasmid standards Em, eg and Es of three tapeworms with 2 concentrations were mixed equally into two groups of group A and group B, amplification was performed according to the real-time quantitative PCR reaction conditions of example 1, 3 replicates were set for each group, and the repeatability of the method was verified, with the results shown in Table 4.
TABLE 4 repeatability test results of triple real-time quantitative PCR method
Note: the mean and standard deviation of the levels of each test sample calculated in 3 replicates were indicated by a small standard deviation, both <0.32, indicating good reproducibility.
The results in Table 4 show that the variation coefficient (CV value) of the repeated tests of the group A and the group B is below 2%, the amplification efficiency is stable, and the established real-time quantitative PCR method for identifying the three tapeworms has good stability and repeatability.
Example 5 simulated clinical sample testing
Combinations of different species of echinococcus were used to simulate triple, double and single positive samples. At the same concentration, a total of 15 mock clinical canine feces (mixed sample of canine feces negative sample genome and tapeworm genome) were detected using triple RT-PCR detection and single RT-PCR. The results showed that 11 samples were positive for echinococcus, including 1 triple positive sample, 4 double positive samples (Eg + Em (2), eg + Es (1) and Em + Es (1), 6 single samples), positive samples for Eg (2), em (2) and Es (2), and 4 negative samples. The results of the triple RT-PCR method are consistent with those of the single RT-PCR method. The coincidence rate of the two methods is 100%. For field samples, 35 fox fecal samples were also tested using triple/single RT-PCR. In both methods, only one fox excrement sample is detected to be echinococcus multilocularis (Em), and the coincidence rate is also 100%.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
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Claims (7)
1. The multiplex real-time fluorescence quantitative PCR primer and probe for simultaneously detecting three echinococcus Em, eg and Es are characterized in that the primer sequences are as follows:
Em g s-F：5’-TTGTTTGCTATGTTTTCTATAGTGT-3’；SEQ ID NO.1；
Em g s-R：5’-TCTTCACATCYAACCCAACAGT-3’；SEQ ID NO.2；
wherein Y = C or T;
the probes used to detect Em were:
Em probe：5’-TTTAGGGAGTAGTGTTT-3’；SEQ ID NO.3；
wherein, the fluorescent group marked at the 5 'end is FAM, and the quenching group marked at the 3' end is BHQ1;
the probes used to detect Eg were:
Eg probe：5’-TTTGGGTAGCAGGGTT-3’；SEQ ID NO.4；
wherein the 5 'end marked fluorescent group is VIC, and the 3' end marked quenching group is BHQ1;
the probes used to detect Es were:
Es probe：5’-TTTAGGAAGGAGAGTTT-3’；SEQ ID NO.5；
wherein the 5 'end labeled fluorescent group is Cy5, and the 3' end labeled quenching group is BHQ1.
2. A multiplex real-time fluorescent quantitative PCR kit for simultaneously detecting three Echinococcus Em, eg and Es, which is characterized by comprising the primer and probe of claim 1.
3. The multiplex real-time fluorescent quantitative PCR kit for simultaneously detecting three Echinococcus Em, eg and Es according to claim 2, further comprising a standard positive template and a negative control sample; the standard positive template is a positive plasmid standard substance containing target fragments of Echinococcus multilocularis, echinococcus granulosus and Echinococcus shikoensis; the negative control sample is RNase free H 2 O。
4. A multiplex real-time fluorescent quantitative PCR detection method for simultaneously detecting three echinococcus Em, eg and Es, which is a non-disease diagnosis purpose, is characterized by comprising the following operation steps: preparing an amplification reaction system by using the primer and the probe of claim 1 and using the sample DNA as a template; performing real-time fluorescent quantitative PCR amplification to obtain an amplification curve; and analyzing the amplification curve and making a judgment.
5. The multiplex real-time fluorescent quantitative PCR detection method for simultaneously detecting three echinococcus Em, eg and Es according to claim 4, wherein the amplification reaction system comprises: sample template 2. Mu.l, 10 μm/L Em g s-F1.5. Mu.l, 10 μm/L Em g s-R1.5. Mu.l, 10 μm/L Em probe 0.2. Mu.l, 10 μm/L Eg probe 0.2. Mu.l, 10 μm/L Es probe 0.2. Mu.l, 2Probe Master Mix 10μl，RNA free H 2 O4.4. Mu.l; the amplification procedure is as follows: 10min at 95 ℃;95 ℃ 10s,60 ℃ 30sec,40 cycles; the fluorescence signal was collected starting from the 60 ℃ step.
6. The multiplex real-time fluorescent quantitative PCR detection method for simultaneously detecting three Echinococcus Em, eg, and Es according to claim 4, wherein the principle of analyzing and judging the amplification curve is as follows:
when Ct in the FAM fluorescent channel is less than or equal to 30 and the fluorescent channel has an amplification curve, judging that the sample is positive for echinococcus multilocularis;
when 30-straw Ct (total number) in an FAM fluorescent channel is less than or equal to 33 and the fluorescent channel has an amplification curve, repeating the experiment once, if the Ct of the FAM fluorescent channel is less than or equal to 33, judging that the sample is Echinococcus multilocularis positive, otherwise, judging that the sample is Echinococcus multilocularis negative;
when the Ct in the FAM fluorescence channel is more than 33 or no amplification curve exists, the sample is judged to be negative to the Echinococcus multilocularis;
when Ct in the VIC fluorescence channel is less than or equal to 30 and the fluorescence channel has an amplification curve, judging that the sample is positive by echinococcus granulosus;
when 30-straw (Ct) in the VIC fluorescence channel is less than or equal to 33 and the fluorescence channel has an amplification curve, repeating the experiment once, if the Ct of the VIC fluorescence channel is less than or equal to 33, judging that the sample is positive Echinococcus granulosus, otherwise, judging that the sample is negative Echinococcus granulosus;
when the Ct in the VIC fluorescence channel is more than 33 or no amplification curve exists, the sample is judged to be negative by echinococcus granulosus;
when Ct in the Cy5 fluorescence channel is less than or equal to 33 and the fluorescence channel has an amplification curve, judging that the sample is positive for echinococcus shikoensis;
when 33< -Ct in the Cy5 fluorescence channel is less than or equal to 36 and the fluorescence channel has an amplification curve, repeating the experiment once, if the Ct in the Cy5 fluorescence channel is less than or equal to 36, judging that the sample is Echinococcus shikokianus positive, otherwise, judging that the sample is Echinococcus shikokianus negative;
and when the Ct in the Cy5 fluorescence channel is more than 36 or no amplification curve exists, the sample is judged to be negative to echinococcus shikokianus.
7. The use of the multiplex real-time fluorescent quantitative PCR primers and probes for simultaneous detection of three kinds of Echinococcus Em, eg and Es in the preparation of a kit for detection of Echinococcus multilocularis, echinococcus granulosus and Echinococcus shikoensis according to claim 1.
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