阅读说明：本技术 可用于微量样本分型检测多房棘球蚴和细粒棘球蚴的一管法巢式实时定量pcr检测试剂盒 (One-tube method nest type real-time quantitative PCR detection kit for detecting echinococcus multilocularis and echinococcus granulosus by parting trace samples ) 是由 汤锋 格日力 樊海宁 李润乐 赵连娣 郑佳 张玉英 冯琳 于 2020-05-12 设计创作，主要内容包括：本发明公开了一种可用于微量样本分型检测多房棘球蚴和细粒棘球蚴的一管法巢式实时定量PCR检测试剂盒,设计内外引物、探针和PCR扩增条件的选择达到巢式PCR一管法完成,操作过程中避免开盖产生气溶胶污染,提高检测结果准确性。检测试剂盒内含有PCR内、外引物对序列、MGB分型检测探针序列、用于内质控的人源内参比基因引物对序列及探针序列。本发明可针对患者血清循环DNA等微量样本分型检测出含有多房棘球蚴和/或细粒棘球蚴的样品,准确度高,可准确地分型检测出多房棘球蚴与细粒棘球蚴源特异DNA序列；可以大批量快速分型鉴定多房棘球蚴与细粒棘球蚴；操作简便,可以对样本中微量虫源DNA进行扩增并达到分型检出的水平。(The invention discloses a one-tube nest real-time quantitative PCR detection kit for trace sample parting detection of echinococcus multilocularis and echinococcus granulosus, which designs the selection of internal and external primers, probes and PCR amplification conditions to be completed by a nest PCR one-tube method, avoids aerosol pollution caused by uncovering in the operation process and improves the accuracy of detection results. The detection kit contains PCR inner and outer primer pair sequences, MGB typing detection probe sequence, humanized internal reference gene primer pair sequence for internal quality control and probe sequence. The method can detect the samples containing the echinococcus multilocularis and/or the echinococcus granulosus aiming at the typing of the trace samples such as the serum circulating DNA of the patients, has high accuracy, and can accurately detect the source specific DNA sequences of the echinococcus multilocularis and the echinococcus granulosus by typing; the mass rapid typing identification of echinococcus multicavis and echinococcus granulosus can be realized; simple operation, and can amplify the DNA of micro insect sources in the sample and reach the level of typing detection.)

1. A one-tube nest type real-time quantitative PCR detection kit for detecting echinococcus multilocularis and echinococcus granulosus by parting a trace sample, which is characterized in that: contains an outer primer, an inner primer and a probe for typing detection of echinococcus multilocularis and echinococcus granulosus, and a primer and a probe of a human endogenous quality control gene, wherein,

the primer sequences are as follows:

an upstream outer primer WF: 5 '-GGWCACAGTGCCAGCATCTGCGGTTARTCT-3' (SEQ ID NO: 1);

downstream outer primer WR: 5'-GAGGGTGACGGGCGGTGTGTACCTGAG-3' (SEQ ID NO: 2);

an upstream inner primer NF: 5'-GACAGGGATTAGATACCCCATT-3' (SEQ ID NO: 3);

the downstream inner primer NR: 5 '-GTGGACCATCCTTYACTATGC-3' (SEQ ID NO: 4);

an upstream endoplasmic control primer CF: 5'-CTCTGAAGCTGACCACACCA-3' (SEQ ID NO: 5);

downstream internal quality control primer CR: 5'-GCCAGGCATAAAGAAATGGA-3' (SEQ ID NO: 6);

the probe sequence is as follows:

AE probe：5’–FAM-CAGTGAGTGATTCTTGT-MGB–3’(SEQ ID NO：7)；

CE probe：5’–HEX-CAGTGAGCGATTCTTAT-MGB–3’(SEQ ID NO：8)；

C probe：5’–TAMRA-CTACGGGGATCTGAACCACCTTGTCTC-BHQ2-3’(SEQ ID NO：9)。

2. the one-tube nested real-time quantitative PCR detection kit for the typing detection of echinococcus multilocularis and echinococcus granulosus according to claim 1, wherein: the detection kit also comprises dNTPs, Taq enzyme and Mg²⁺And one, two or more of PCR reaction buffers.

3. The one-tube nested real-time quantitative PCR detection kit for the typing detection of echinococcus multilocularis and echinococcus granulosus according to claim 1, wherein: the detection kit also comprises a standard positive template.

4. The one-tube nested real-time quantitative PCR detection kit for the typing detection of echinococcus multilocularis and echinococcus granulosus according to claim 1, wherein: and performing typing PCR detection on echinococcus multilocularis and echinococcus granulosus source DNA in plasma circulating free DNA by using an outer primer pair sequence, an inner primer pair sequence and a typing detection probe sequence of the detection kit, and simultaneously evaluating the extraction quality of the human source sample by using an inner quality control primer pair sequence and a probe sequence.

5. The one-tube nested real-time quantitative PCR detection kit for the typing detection of echinococcus multilocularis and echinococcus granulosus according to claim 4, wherein: comprises the following steps of (a) carrying out,

1) extracting total DNA in a body fluid sample;

2) taking the total DNA in the step 1) as a template, WR and WF as primer pairs to carry out nested PCR pre-amplification, NR and NF as inner primer pairs, CF and CR as inner quality control primer pairs, and AE-probe, CE-probe and C-probe as probes to carry out real-time quantitative PCR amplification reaction;

3) DNA products were analyzed by fluorescent quantitative PCR.

6. The one-tube nested real-time quantitative PCR detection kit for the typing detection of echinococcus multilocularis and echinococcus granulosus in the micro-sample according to claim 5, wherein: the real-time quantitative PCR amplification reaction system which takes WR and WF as outer primer pairs, NR and NF as inner primer pairs, CF and CR as inner quality control primer pairs and AE-probe, CE-probe and C-probe as probes is calculated by 20 mu L:

2 × Taq PCR MasterMix, 10 μ L; 10 μ M outer primer WF, 0.2 μ L; 10 μ M outer primer WR, 0.2 μ L; 10 μ M inner primer NF, 0.40 μ L; 10 μ M inner primer NR, 0.4 μ L; 10 μ M of the endoplasmic control primer CF, 0.4 μ L; 10 μ M of an endoplasmic control primer CR, 0.4 μ L; AE-probe, 0.4. mu.L; CE-probe, 0.4. mu.L; c-probe, 0.4. mu.L; DNF buffer2 uL

DNA template, 2. mu.L; ddH₂O, 2.8 μ L; the total reaction system was 20. mu.L.

7. The one-tube nested real-time quantitative PCR detection kit for the typing detection of echinococcus multilocularis and echinococcus granulosus in the micro-sample according to claim 5, wherein: the real-time quantitative PCR amplification reaction conditions of WR and WF as an outer primer pair, NR and NF as an inner primer pair, CF and CR as an inner quality control primer pair and AE-probe, CE-probe and C-probe as probes are as follows: enzyme activation at 94 deg.C for 3 min; denaturation at 94 ℃ for 15s, annealing at 70 ℃ for 15s, extension at 72 ℃ for 30s, and 20 cyclic preamplification without collecting fluorescence signals; denaturation at 94 ℃ for 15s, annealing at 50 ℃ for 15s, and extension at 72 ℃ for 30s, for 40 cycles of fluorescence signal acquisition; the FAM channel detects AE probe in real time, the VIC channel detects CE probe amplification curve in real time, and the TAMRA channel detects C probe amplification curve.

Technical Field

The invention relates to the technical field of biological detection, in particular to a real-time quantitative PCR detection kit for type-division detection of echinococcus multilocularis and echinococcus granulosus.

Background

Echinococcosis (Echinococcosis) is a zoonosis parasitic disease, also called Echinococcosis, which is a juvenile disease mainly caused by echinococcus granulosus in our country: echinococcosis uniatrial/granulosa (Cystic Echinococcosis, CE) and echinococcus multilocularis caused juvenile disease: echinococcosis multiatrial (Alveolar echinococosis, AE). The echinococcosis infection focus grows slowly in the human body, the clinical manifestation is complex, and no obvious symptoms and physical signs exist for a long time. At present, effective and sensitive methods are not available for diagnosis and typing identification of echinococcosis, and patients who are diagnosed by imaging mainly rely on imaging diagnosis (B ultrasound, nuclear magnetism and CT) and often have substantive lesions of organs such as liver, so that the patients lose the best cure time window and even miss the chance of surgical treatment. Although there is a molecular biological diagnosis method based on serum antibody, the diagnosis result is inaccurate due to factors such as high protein similarity of echinococcus multicavis and echinococcus granulosus and cross antigen existence with other pathogenic microorganisms.

With the development of molecular biology, the accuracy of pathogen-based nucleic acid detection is now well recognized. Real-time fluorescent Quantitative PCR (Quantitative Real-time PCR) is a method for measuring the total amount of products after each Polymerase Chain Reaction (PCR) cycle by using fluorescent chemical substances in DNA amplification reaction. The fluorescence labeled probe can improve the efficiency, sensitivity and specificity of real-time quantitative PCR results, and the quantitative PCR can simultaneously detect a plurality of target genes in one reaction. Currently, genes such as mitochondrial genes ND5, COX2 and 12SrRNA and the like are used for detecting the grouping of echinococcus granulosus and echinococcus pluvialis, and the prior invention patent application of 'PCR diagnosis method for detecting echinococcus pluvialis and echinococcus granulosus samples based on MGB probe grouping' is provided, and the application number is 201611198588.1.

"liquid biopsy" as a branch of in vitro diagnosis refers to a non-invasive blood test. At present, the detection of extracellular free DNA aiming at samples such as blood, urine, saliva and the like is applied in the related fields of disease monitoring, fetal parturition detection, tumor staging and typing and the like. However, whether the detection of circulating free DNA can be used for the diagnosis of echinococcosis typing is a problem, French scientists in 2018 detect the expression of ND5 and U1 of free DNA in a pluriparian echinococcus infected gerbil animal model and alveolar echinococcosis patients, and the results show that ND5 and U1 widely exist in the gerbil model (8/9) and only 22.58 percent (7/31) of the patients can detect the genes in human body studies. Therefore, the inventors also examined the primers and probes of the previously filed patent application (application No. 201611198588.1) in free plasma of patients for the detection of circulating DNA of insect origin, but the results were not satisfactory. For analytical reasons, two factors may cause the above method to be undesirable: 1) at present, the mechanism of the insect source DNA entering a host is not clear, and the growth modes of echinococcus in human bodies and animals are different, so that the number and the types of genes of the insect source DNA entering organisms are different; 2) the expression level of insect-derived DNA in a patient is very low, and the conventional PCR method cannot detect trace DNA due to competitive influence on PCR reaction caused by a variety of circulating DNA.

Aiming at the first problem, the inventor discovers that DNA information of insect sources exists by sequencing circulating DNA of 10 patients with alveolar echinococcosis, but the DNA information exists in a fragment form and is not complete genome DNA. And it was found that the genes for echinococcosis typing detection, such as EM18, EMY162, ELP, AgB and mitochondrial codes ND5, COX1, are not widely available, and a 1260bp mitochondrial DNA sequence (CBLO020001206,18820 to 10080) was detected in 90% (9/10) of patients, indicating that the sequence can be used for diagnosis of free DNA in CBLO020001206. The primers and probes of the invention patent (application No. 201611198588.1) of the prior application are found to be exactly located in the sequence by sequence alignment.

In the second problem, although the primers and probes of the previously applied invention patent (application No. 201611198588.1) are located in the sequence, the primers and probes of the previously applied patent cannot detect echinococcus nucleic acid information from the patient due to low copy number, etc., the inventors further consider that the detection limit of the original method is increased by designing outer primers to perform nested PCR, and the outer primers are pre-amplified first to increase the amount of the target sequence in the template, and then perform the second round of amplification using the first pre-amplification product as the template. However, the nested PCR needs to be uncapped for sample loading in the two-step operation process, and the risk of aerosol pollution is generated, which is also the reason that the nested PCR is difficult to be used for clinical diagnosis.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a one-tube method nest type real-time quantitative PCR detection method and a kit for typing detection of plasma free DNA echinococcus multilocularis and echinococcus granulosus, wherein a gene segment for detecting echinococcus infection is discovered by sequencing the plasma free DNA of a patient; by designing the outer primer pair and the inner primer pair, nested PCR amplification is carried out by controlling annealing temperature, so that aerosol pollution caused by uncovering in the midway of reaction is avoided; the method comprises the steps of designing primers and typing detection probes aiming at echinococcus specific DNA, typing and detecting the echinococcus multicavicularis and echinococcus granulosus by adopting a real-time quantitative PCR detection technology, and evaluating the quality of extracted DNA by designing and adding a human-derived reference gene primer pair and a human-derived reference gene probe.

In order to solve the technical problems, the invention adopts the following technical scheme: a one-tube nest type real-time quantitative PCR detection kit for detecting echinococcus multilocularis and echinococcus granulosus by parting a trace sample, which is characterized in that: contains an outer primer, an inner primer and a probe for typing detection of echinococcus multilocularis and echinococcus granulosus, and a primer and a probe of a human endogenous quality control gene, wherein,

the primer sequences are as follows:

an upstream outer primer WF: 5 '-GGWCACAGTGCCAGCATCTGCGGTTARTCT-3' (SEQ ID NO: 1);

downstream outer primer WR: 5'-GAGGGTGACGGGCGGTGTGTACCTGAG-3' (SEQ ID NO: 2);

an upstream inner primer NF: 5'-GACAGGGATTAGATACCCCATT-3' (SEQ ID NO: 3);

the downstream inner primer NR: 5 '-GTGGACCATCCTTYACTATGC-3' (SEQ ID NO: 4);

an upstream endoplasmic control primer CF: 5'-CTCTGAAGCTGACCACACCA-3' (SEQ ID NO: 5);

downstream internal quality control primer CR: 5'-GCCAGGCATAAAGAAATGGA-3' (SEQ ID NO: 6);

the probe sequence is as follows:

AE probe：5’–FAM-CAGTGAGTGATTCTTGT-MGB–3’(SEQ ID NO：7)；

CE probe：5’–HEX-CAGTGAGCGATTCTTAT-MGB–3’(SEQ ID NO：8)；

c probe: 5 '-TAMRA-CTACGGGGATCTGAACCACCTTGTCTC-BHQ 2-3' (SEQ ID NO: 9). (see sequence listing)

The detection kit also comprises dNTPs, Taq enzyme and Mg²⁺And one, two or more of PCR reaction buffers.

The detection kit also comprises a standard positive template.

And performing typing PCR detection on echinococcus multilocularis and echinococcus granulosus source DNA in plasma circulating free DNA by using an outer primer pair sequence, an inner primer pair sequence and a typing detection probe sequence of the detection kit, and simultaneously evaluating the extraction quality of the human source sample by using an inner quality control primer pair sequence and a probe sequence.

The detection method comprises the following steps of,

1) extracting total DNA in a body fluid sample;

2) taking the total DNA in the step 1) as a template, WR and WF as primer pairs to carry out nested PCR pre-amplification, NR and NF as inner primer pairs, CF and CR as inner quality control primer pairs, and AE-probe, CE-probe and C-probe as probes to carry out real-time quantitative PCR amplification reaction;

3) DNA products were analyzed by fluorescent quantitative PCR.

Preferably, WR and WF are used as outer primer pairs, NR and NF are used as inner primer pairs, CF and CR are used as inner quality control primer pairs, AE-probe, CE-probe and C-probe are used as probes, and the real-time quantitative PCR amplification reaction system is calculated by 20 muL:

2 × Taq PCR MasterMix, 10 μ L; 10 μ M outer primer WF, 0.2 μ L; 10 μ M outer primer WR, 0.2 μ L; 10 μ M inner primer NF, 0.40 μ L; 10 μ M inner primer NR, 0.4 μ L; 10 μ M of the endoplasmic control primer CF, 0.4 μ L; 10 μ M of an endoplasmic control primer CR, 0.4 μ L; AE-probe, 0.4. mu.L; CE-probe, 0.4. mu.L; c-probe, 0.4. mu.L; DNF buffer2 uL

DNA template, 2. mu.L; ddH₂O, 2.8 μ L; the total reaction system was 20. mu.L.

Preferably, the real-time quantitative PCR amplification reaction conditions for WR and WF as external primer pairs, NR and NF as internal primer pairs, CF and CR as internal quality control primer pairs and AE-probe, CE-probe and C-probe as probes are as follows: enzyme activation at 94 deg.C for 3 min; denaturation at 94 ℃ for 15s, annealing at 70 ℃ for 15s, extension at 72 ℃ for 30s, and pre-amplification for 20 cycles (no fluorescent signal collected); denaturation at 94 ℃ for 15s, annealing at 50 ℃ for 15s, and extension at 72 ℃ for 30s for 40 cycles (fluorescence signal acquisition); the FAM channel detects AE probe in real time, the VIC channel detects CE probe amplification curve in real time, and the TAMRA channel detects C probe amplification curve.

Compared with the traditional detection tool, the kit has the advantages that firstly, the accuracy is high, primers and probes are designed according to the specific distribution of echinococcus multilocularis and echinococcus granulosus pathogen DNA in the plasma of a patient, and the echinococcus multilocularis and echinococcus granulosus specific DNA can be accurately detected in a typing mode;

secondly, the operation is simple and convenient, the probes for distinguishing the echinococcus multilocularis, the echinococcus granulosus and the human endogenous quality control genes are marked with different fluorescent dyes (AE probe-FAM; CE probe-HEX; C probe-TAMRA), and the three probes are added into a reaction system at the same time, so that the typing detection of the echinococcus granulosus and the echinococcus multilocularis can be completed in one reaction, meanwhile, the DNA extraction quality evaluation can be carried out on the human endogenous sample, and the echinococcus multilocularis and the echinococcus granulosus can be rapidly typed and identified in large batch;

thirdly, the sensitivity is high, the invention extracts DNA from a sample as a template, uses WR and WF as an outer primer pair, NR and NF as an inner primer pair, CF and CR as an inner quality control primer pair, and uses AE-probe, CE-probe and C-probe as probes to carry out nested multiplex real-time quantitative PCR amplification which is improved in comparison with the detection limit of the traditional PCR, and can amplify the micro-insect source mitochondrial DNA in the sample and reach the level of typing detection.

Drawings

FIG. 1 is a graph showing the sequencing result of plasma free DNA of echinococcosis patients;

FIG. 2A shows that the reference gene probe C probe-TAMRA signal in the plasma free DNA is amplified positively; FIG. 2B shows that the signals of the fine particle probe CE probe-VIC and the multi-chamber probe AE-probe-FAM are all negative; FIG. 2C shows that the signals of the echinococcus multilocularis probe AE-probe-FAM are all positive, and the echinococcus granulosus probe CE probe-VIC is negative; FIG. 2D shows that the Echinococcus multiplex-chamber probes AE-probe-FAM are all negative, and the Echinococcus granulosus probe CE probe-VIC is positive;

FIG. 3A is a graph showing the difference between the amplification of the echinococcus polysterori positive quality control plasmid by the "one-tube method" nested real-time quantitative PCR and the real-time quantitative PCR without the outer primer; FIG. 3B is a graph showing the difference between the amplification of the echinococcus granulosus positive quality control plasmid by the "one-tube method" nested real-time quantitative PCR and the real-time quantitative PCR without the outer primer;

FIG. 4A is the result of plasma free DNA detection of cystic echinococcosis patients by quantitative PCR method without outer primer; FIG. 4B is the result of detecting free DNA in plasma of cystic echinococcosis patient by nested real-time quantitative PCR in one-tube method; FIG. 4C is a quality evaluation of plasma free DNA extraction by endoplasmic control primer probe for cystic echinococcosis patients; FIG. 4D shows the result of plasma free DNA detection of alveolar echinococcosis patients by the quantitative PCR method without external primers; FIG. 4E shows the result of detecting plasma free DNA of alveolar echinococcosis patients by nested real-time quantitative PCR in one-tube method; FIG. 4F is an evaluation of the quality of plasma free DNA extraction by endoplasmic control primer probe for patients with alveolar echinococcosis.

Detailed Description

The technical solution of the present invention will be described clearly and completely with reference to the accompanying drawings, and of course, the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments, and are not meant to limit the present invention. 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.

In the embodiment, CBLO020001206 can be used for in vitro diagnosis of echinococcus multicavis and echinococcus granulosus infection based on sequencing of free DNA in plasma of echinococcus patients with echinococcosis, an outer primer pair, an inner quality control gene primer pair and a probe are designed and screened for specific DNA sequences of echinococcus multicavis and echinococcus granulosus, an one-tube method nest real-time quantitative PCR technology is established for amplification through designing and adjusting annealing temperature, and real-time quantitative fluorescence analysis is performed on an amplification product, so that the extraction quality of free DNA of a human sample can be inspected while the levels of echinococcus multicavis and echinococcus granulosus are accurately judged.

Material

1. Sample preparation: blood plasma of echinococcus multilocularis and echinococcus granulosus infected patients

2. DNA extraction: extracting with QIAamp Circulating Nucleic Acid kit (50 times);

3. 2 × Taq PCR MasterMix (Low ROX);

4. a PCR instrument: ABI 7500.

Extraction of plasma DNA

Adding 60 mu L of Protease K into a 15mL centrifuge tube, adding 4mL of plasma sample, adding 4mL of Buffer AL, and vortexing for 15 s;

incubating at 56 deg.C for 10min until lysis is complete, and rapidly centrifuging;

adding 4mL of ethanol (100%), vortexing for 15s, and rapidly centrifuging;

transferring the mixed solution to Spin column (2mL collecting tube) to a vacuum extractor, adding 500 μ L CC to column (activated column), turning on circulating water type vacuum pump, and adjusting to about 0.3 Mpa;

1mL of Buffer VW1 was added until the liquid was drained by suction, and 500. mu.L of WB was added to the column and filtered by suction for 30 s.

The bottom of the centrifuged column was transferred to a 1.5mL EP tube (EP tube needs to be numbered) and centrifuged at 12000rmp for 3 min.

Add 30. mu.L of preheated Elution buffer to the core of the EP tube, after standing at room temperature for 3min, centrifuge at 12000rmp for 3 min.

Sucking out the liquid at the bottom of the EP tube, adding the liquid on the core of the EP tube again, standing at room temperature for 3min, centrifuging at 12000rmp for 3min, discarding the core of the EP tube, and collecting the solution obtained by centrifugation in a centrifuge tube. The temperature was kept at 4 ℃ for a short time and-20 ℃ for a long time.

DNA sequencing analysis of plasma of echinococcosis patient

500X sequencing of plasma free DNA of echinococcosis patients followed by alignment revealed that the insect-derived DNA sequence CBLO020001206.1 was detectable in 90% of patients (FIG. 1).

Third, design of primer and probe

Designing an inner primer pair and an outer primer pair and a probe according to a CBLO020001206.1 sequence:

an upstream outer primer WF: 5 '-GGWCACAGTGCCAGCATCTGCGGTTARTCT-3' (SEQ ID NO: 1);

downstream outer primer WR: 5'-GAGGGTGACGGGCGGTGTGTACCTGAG-3' (SEQ ID NO: 2);

an upstream inner primer NF: 5'-GACAGGGATTAGATACCCCATT-3' (SEQ ID NO: 3);

the downstream inner primer NR: 5 '-GTGGACCATCCTTYACTATGC-3' (SEQ ID NO: 4);

probes designed to discriminate detection of echinococcosis multiplex/granulosa AE probe and CE probe:

AE probe：5’–FAM-CAGTGAGTGATTCTTGT-MGB–3’(SEQ ID NO：7)；

CE probe：5’–HEX-CAGTGAGCGATTCTTAT-MGB–3’(SEQ ID NO：8)；

designing specific primers and probes according to the human endogenous quality control foundation beta-tublin:

an upstream endoplasmic control primer CF: 5'-CTCTGAAGCTGACCACACCA-3' (SEQ ID NO: 5);

downstream internal quality control primer CR: 5'-GCCAGGCATAAAGAAATGGA-3' (SEQ ID NO: 6);

designing a probe C probe for detecting the internal quality control gene:

C probe：5’–TAMRA-CTACGGGGATCTGAACCACCTTGTCTC-BHQ2-3’(SEQ ID NO：9)；

both primers and probes were synthesized by Biotechnology engineering (Shanghai) Inc.

Fourth, the kit detects the free DNA of plasma

The nested real-time quantitative PCR reaction system of 'one-tube method' is that 2 × Taq PCR MasterMix, 10 muL, 10 muM external primer WF, 0.2 muL, 10 muM external primer WR, 0.2 muL, 10 muM internal primer NF, 0.40 muL, 10 muM internal primer NR, 0.4 muL, 10 muM internal quality control primer CF, 0.4 muL, 10 muM internal quality control CR, 0.4 muL, AE-probe, 0.4 muL, CE-probe, 0.4 muL, C-probe, 0.4 muL, buff 2 muL, DNA template, 2 muL, ddH template, ddH probe and probe are used as templates to carry out real-time quantitative PCR amplification by taking external primer pairs WF and WR, internal quality control primer pairs NF and NR as probes₂O, 2.8 μ L; the total reaction system was 20. mu.L. The reaction conditions are as follows: enzyme activation at 94 deg.C for 3 min; denaturation at 94 ℃ for 15s, annealing at 70 ℃ for 15s, extension at 72 ℃ for 30s, and pre-amplification for 20 cycles (no fluorescent signal collected); denaturation at 94 ℃ for 15s, annealing at 50 ℃ for 15s, and extension at 72 ℃ for 30s for 40 cycles (fluorescence signal acquisition); the FAM channel detects AE probe in real time, the VIC channel detects CE probe amplification curve in real time, and the TAMRA channel detects C probe amplification curve. The result is shown in FIG. 2, the positive signal of C probeTAMRA indicates that the humanized endoplasmic control gene in the extracted free DNA is positive, which indicates that the extraction of the plasma free DNA is successful (FIG. 2A); the FAM and VIC fluorescent signals are negative, which indicates that the DNA sample to be detected does not contain insect-derived genes (figure 2B); the positive AE probe FAM fluorescence signal and the negative CE probe VIC fluorescence signal indicate that the DNA to be detected contains echinococcus multilocularis specific DNA (figure 2C); AE probe FAM fluorescence signal negative, CE probe VIC fluorescence signal positive shows that the DNA to be tested contains the fine grain spinesCoccidiosis specific DNA (FIG. 2C);

five, one-tube method nested real-time quantitative PCR and real-time quantitative PCR without external primer are compared for positive quality control products

The nested real-time quantitative PCR amplification program of the one-tube method comprises the following steps:

a nested real-time quantitative PCR reaction system of 'one-tube method' is to perform real-time quantitative PCR amplification by using 104copy/uL multi-chamber/fine-particle control plasmid DNA as a template and using external primer pairs WF and WR, internal primer pairs NF and NR and internal quality control primer pairs CF, CR and AE probe, CE probe and C probe as probes, wherein the nested real-time quantitative PCR reaction system comprises 10 muL of 2 × Taq PCR MasterMix, 10 muM external primer WF, 0.2 muL, 10 muM external primer WR, 0.2 muL, 10 muM internal primer NF, 0.40 muL, 10 muM internal primer NR, 0.4 muL, 10 muM internal quality control primer CF, 0.4 muL, 10 muM internal quality control primer CR, 0.4 muL, AE-DNbe, 0.4 muL, CE-probe, 0.4 muL, C-probe, 0.4 muL, 2 muL of internal quality control primer CR, 0.4 muL, DNA template, 2 muH and 2 muL of DNA₂O, 2.8 μ L; the total reaction system was 20. mu.L. The reaction conditions are as follows: enzyme activation at 94 deg.C for 3 min; denaturation at 94 ℃ for 15s, annealing at 70 ℃ for 15s, extension at 72 ℃ for 30s, and pre-amplification for 20 cycles (no fluorescent signal collected); denaturation at 94 ℃ for 15s, annealing at 50 ℃ for 15s, and extension at 72 ℃ for 30s for 40 cycles (fluorescence signal acquisition); the FAM channel detects AEprobe in real time, the VIC channel detects CE probe amplification curve in real time, and the TAMRA channel detects C probe amplification curve.

Real-time quantitative PCR amplification procedure without outer primers (see earlier patent application: 201611198588.1)

At 10⁴The real-time quantitative PCR reaction system comprises 2 × Taq PCR Mastermix 10 muL, 10 muM primer EF 0.40 muL, 10 muM primer ER 0.4 muL, AE-probe 0.3 muL, CE-probe 0.3 muL, DNFbuffer 2 muL, DNA template 2 muL, and DNH₂O, 4.6 μ L; the total reaction system was 20. mu.L. The reaction conditions of real-time quantitative PCR using AE probe and CE probe as probes were: enzyme activation at 94 ℃ for 3min, denaturation at 94 ℃ for 5s, annealing/extension/fluorescence data collection at 60 ℃ for 5s for 40 cycles; the FAM channel detects AE probe in real time, and the VIC channel detects CE probe amplification curve in real time.

The results are shown in FIG. 3: for echinococcus multilocularis samples, the FAM channel CT value was advanced by 16.28 (FIG. 3A) compared with the real-time quantitative PCR method without the outer primers by the "one-tube method" nested real-time quantitative PCR with the outer primers for nested pre-amplification; for echinococcus granulosus samples, the one-tube method' nested real-time quantitative PCR with external primers advanced the VIC channel CT value by 16.31 (FIG. 3B) compared to the real-time quantitative PCR method without external primers. The results show that the kit can remarkably improve the yield of the amplification product by adding the external primer pair to carry out nested PCR (polymerase chain reaction) by a one-tube method, so that the amplification reaction reaches the stage as early as possible and the detection of trace samples is facilitated.

Six, one-tube method nested real-time quantitative PCR and real-time quantitative PCR clinical patient plasma free DNA detection contrast without external primer

The nested real-time quantitative PCR amplification program of the one-tube method comprises the following steps:

the nested real-time quantitative PCR reaction system of 'one-tube method' is that 2 × Taq PCR MasterMix, 10 muL, 10 muM external primer WF, 0.2 muL, 10 muM external primer WR, 0.2 muL, 10 muM internal primer NF, 0.40 muL, 10 muM internal primer NR, 0.4 muL, 10 muM internal quality control primer CF, 0.4 muL, 10 muM internal quality control CR, 0.4 muL, AE-probe, 0.4 muL, CE-probe, 0.4 muL, C-probe, 0.4 muL, buff 2 muL, DNA template, 2 muL, ddH template, ddH primer pair NF, NR and NR are used as probes to carry out real-time quantitative PCR amplification₂O, 2.8 μ L; the total reaction system was 20. mu.L. The reaction conditions are as follows: enzyme activation at 94 deg.C for 3 min; denaturation at 94 ℃ for 15s, annealing at 70 ℃ for 15s, extension at 72 ℃ for 30s, and pre-amplification for 20 cycles (no fluorescent signal collected); denaturation at 94 ℃ for 15s, annealing at 50 ℃ for 15s, and extension at 72 ℃ for 30s for 40 cycles (fluorescence signal acquisition); the FAM channel detects AE probe in real time, the VIC channel detects CE probe amplification curve in real time, and the TAMRA channel detects C probe amplification curve.

Real-time quantitative PCR amplification procedure without outer primers (see earlier patent application: 201611198588.1)

Taking free DNA of plasma of a patient as a template, EF and ER as primers,the real-time quantitative PCR reaction system comprises 10 muL of 2 × Taq PCR MasterMix, 0.40 muL of 10 muM primer EF, 0.4 muL of 10 muM primer ER, 0.3 muL of AE-probe, 0.3 muL of CE-probe, 2 muL of DNF buffer, 2 muL of DNA template, and ddH₂O, 4.6 μ L; the total reaction system was 20. mu.L. The reaction conditions of real-time quantitative PCR using AE probe and CE probe as probes were: enzyme activation at 94 ℃ for 3min, denaturation at 94 ℃ for 5s, annealing/extension/fluorescence data collection at 60 ℃ for 5s for 40 cycles; the FAM channel detects AE probe in real time, and the VIC channel detects CE probe amplification curve in real time.

The results are shown in fig. 4, the real-time quantitative PCR amplification result without the outer primer for cystic echinococcosis patients shows that the positive amplification is weak, the CT value is 31.00 (fig. 4A), and it is difficult to judge whether the positive amplification is positive or not between the critical value of the fluorescent quantitative PCR needle break, while the one-tube method "nested real-time quantitative PCR reaction result shows that the CT value is 16.47, the positive result is consistent with the CT diagnosis result (fig. 4B), and the internal quality control amplification result shows that the human internal quality control gene amplification is positive (fig. 4C), and the extraction quality of the free DNA in plasma of the sample is qualified. The real-time quantitative PCR amplification result of the echinococcus multilocularis infected alveolar echinococcosis patient without the outer primer shows that the positive amplification is weak, the CT value is 31.56 (figure 4D), and whether the positive amplification is performed or not is difficult to judge, while the nested real-time quantitative PCR reaction result of the one-tube method can also be diagnosed as the positive amplification CT value of 17.19 (figure 4E), and the result of the amplification of the inner quality control gene shows that the sample plasma free DNA extraction quality is qualified (figure 4F).

Seventh, application of the kit to plasma free DNA of echinococcosis patient

Collecting 73 plasma samples of patients with hydatid infection confirmed by imaging method (CT), wherein 41 patients with cystic hydatid and 32 patients with alveolar hydatid are selected; 74 negative control clinical samples were collected. The result of the diagnosis result of the kit is compared with the result of the CT diagnosis as follows:

TABLE comparison of the kit with the imaging results

Sensitivity (true positive rate): the capsule type (CE) was 92.68% and the bubble type (AE) was 87.50%.

Specificity (true negative rate): 100 percent.

Accuracy: 95.24 percent.

The kit and the imaging comparison adopt Kappa consistency test, and the calculated Kappa value is as follows: 0.90, Kappa value >0.75, the consistency of the kit and the imaging diagnosis is higher.

The present invention has been described in detail, and it should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.