CN111304340B - RPA-EC schistosoma japonicum detection kit and detection method - Google Patents

RPA-EC schistosoma japonicum detection kit and detection method Download PDF

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CN111304340B
CN111304340B CN202010264434.8A CN202010264434A CN111304340B CN 111304340 B CN111304340 B CN 111304340B CN 202010264434 A CN202010264434 A CN 202010264434A CN 111304340 B CN111304340 B CN 111304340B
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CN111304340A (en
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周晓农
邓王平
胡薇
李石柱
许静
陈军虎
徐斌
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National Institute of Parasitic Diseases of Chinese Center for Disease Control and Prevention
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Abstract

The invention discloses an RPA-EC schistosoma japonicum detection kit, which comprises: each channel of the multichannel sensing electrode array comprises a working electrode, a counter electrode and a reference electrode, an RPA probe is fixed on the surface of the working electrode, the RPA probe is an amino modified RPA probe capable of amplifying SjR gene fragments, the RPA probe is fixed on the surface of the working electrode through bonding with the surface of the working electrode, and a non-specific binding site is blocked by bovine serum albumin and casein; an RPA primer comprising an RPA upstream primer and an RPA downstream primer; a lyophilized powder comprising an RPA component; magnesium acetate; a reaction buffer; avidin-HRP enzyme conjugate; a substrate solution and a washing solution; the invention also discloses a detection method based on the kit.

Description

RPA-EC schistosoma japonicum detection kit and detection method
Technical Field
The invention relates to the technical field of biological detection, in particular to a schistosoma japonicum detection kit combining RPA amplification with an electrochemical sensor (RPA-EC) and a detection method thereof.
Background
Schistosomiasis is a serious human and veterinary zoonosis. Through positive prevention and treatment for 70 years, the prevention and treatment work of schistosomiasis in China at present enters a key stage of elimination, the schistosome infection rate and infection degree of residents and domestic animals in a popular area are reduced to the historic extremely low level, the positive oncomelania infected by the schistosomiasis microscopic examination is not found for 4 years continuously in China, and the current situation brings higher requirements to schistosomiasis diagnosis and monitoring technology. Meanwhile, the defects of immunology and etiology diagnosis technology adopted in China are increasingly obvious, and the requirements of schistosomiasis epidemic situation monitoring under the low epidemic situation are not satisfied, and the method is mainly characterized in that: the traditional etiology diagnosis method has low detection sensitivity to patients with low infection degree, high omission ratio, time and labor waste and low mass compliance. The serological antibody detection method has high sensitivity and simple operation, is widely applied to large-scale screening and serum epidemiological investigation of epidemic areas on chemotherapy subjects, but is easy to cause false positive, and the detection of the antibody level is difficult to distinguish between the symptomatic infection and the past infection.
The molecular diagnosis method for detecting the schistosome nucleic acid sequence is expected to provide an effective technical means for accurate monitoring of schistosome epidemic situation due to the advantages of high sensitivity, strong specificity and the like. In recent years, there have been many reports on Japanese blood fluke (S.japonica) nucleic acid detection methods, including PCR, nested PCR, real-time quantitative PCR, digital PCR, loop-mediated isothermal amplification (LAMP) by recombinant polymerase isothermal amplification (recombinase polymerase amplification, RPA), etc., which are mostly based on PCR, and partly on isothermal amplification techniques. Among them, the RPA technology is an isothermal amplification technology which has been attracting attention in recent years and has been rapidly developed, and can amplify nucleic acid to a detectable level at a constant temperature of 25 to 43 ℃ for 5 to 20 minutes, and the operation is simple and rapid, and the method can read results in combination with various methods, and is particularly suitable for basic-layer application. However, the sensitivity of the currently reported Schistosoma japonicum nucleic acid detection method based on RPA is still not ideal, and a more sensitive detection technology needs to be explored and combined with the RPA, so that the sensitivity and the specificity of nucleic acid detection are improved, and the timeliness and the repeatability of detection are improved through simplifying equipment and operation, so that the method is more suitable for field application.
Disclosure of Invention
The invention aims to combine an RPA technology and an electrochemical biological sensing technology and provides a schistosoma japonicum detection kit combining an RPA amplification technology and an electrochemical sensor (RPA-EC) and a detection method thereof.
In order to achieve the above object, the present invention provides an RPA-EC schistosoma japonicum detection kit comprising: each channel of the multichannel sensing electrode array comprises a working electrode, a counter electrode and a reference electrode, wherein an RPA probe is fixed on the surface of the working electrode, the RPA probe is an amino-modified RPA probe capable of amplifying SjR gene fragments, the RPA probe is fixed on the surface of the working electrode through bonding with the surface of the working electrode, and non-specific binding sites on the surface of the working electrode are blocked by bovine serum albumin and casein so as to avoid non-specific adsorption of irrelevant DNA and proteins in subsequent reactions; an RPA primer comprising an RPA upstream primer and an RPA downstream primer; a lyophilized powder comprising an RPA component; magnesium acetate, which has the effect of initiating the RPA reaction; a reaction buffer which acts to solubilize the RPA lyophilized components and provide a stable buffer system in the RPA reaction; avidin-HRP enzyme conjugate; substrate solution and washing solution.
As a specific example, the working electrode is a carbon working electrode, and an amino-modified RPA probe is immobilized on the surface of the carbon working electrode, and the amino-modified RPA probe is immobilized on the surface of the carbon working electrode by a carboxyl-amide bond interaction with the surface of the carbon working electrode.
As a specific example, the working electrode is a gold working electrode, a thiol-modified RPA probe is fixed on the surface of the gold working electrode, and the thiol-modified RPA probe is fixed on the surface of the gold working electrode through gold-sulfur bonding with the gold working electrode.
As a preferred example, the multi-channel sensing electrode array is a 16-channel three-electrode printed carbon electrode array.
As a preferred example, the detection kit further comprises a negative control, which is double distilled water, and a positive control, which is schistosoma japonicum genomic DNA.
As a specific example, the lyophilized powder containing the RPA component includes: phage recombinases, DNA polymerases, single-stranded DNA binding proteins, dntps, and exonucleases.
As a specific example, the reaction buffer is Rehydration Buffer.
As a specific example, the substrate solution is TMB solution containing hydrogen peroxide.
As a specific example, the wash solution is PBS, typically 10mM PBS.
As a specific example, the RPA probe sequence is shown in a sequence table SEQ ID No. 1.
As a specific example, the sequence of the RPA upstream primer is shown as SEQ ID NO.2, and the sequence of the RPA downstream primer is shown as SEQ ID NO. 3.
The invention also provides a detection method based on the detection kit, which mainly comprises the following steps: in the RPA reaction process, one end of a biotinylated amplification product is formed, then the amplification product is complementarily combined with a probe fixed on an electrode interface, the RPA reaction on the electrode interface is carried out, double-stranded DNA of which one end is marked with biotin is formed on the electrode interface, a compound is formed by the double-stranded DNA and an avidin-HRP enzyme conjugate, and signal acquisition is realized by detecting the reduction current of an HRP catalytic substrate.
The method comprises the following steps:
1) Interface RPA reaction: adding reaction buffer solution, RPA upstream primer, RPA downstream primer, double distilled water, magnesium acetate and DNA template into the freeze-dried powder containing RPA component, uniformly mixing and centrifuging, taking the mixed liquid, dripping the mixed liquid on the surface of the multichannel sensing electrode array, incubating for 10-50min at 37 ℃, and flushing the electrode with a washing liquid.
2) Current signal acquisition: dripping avidin-HRP enzyme conjugate on the surface of the multi-channel sensing electrode array, incubating for 5-15min at room temperature, flushing with a washing solution, dripping a substrate solution into the multi-channel sensing electrode array, scanning a current time curve by using a multi-channel electrochemical instrument, and recording a steady-state current value as a detection signal.
3) And (3) result judgment: the sum of the average negative control current and the 3-fold standard deviation (+3SD) is used as a threshold value, and the sample average current exceeds the threshold value to judge that the positive sample containing the schistosoma japonicum DNA is lower than the threshold value to judge that the negative sample is obtained.
As a specific example, the RAP reaction system is typically a 50ul reaction system: adding 29.5 mu l Rehydration Buffer,2-2.5 mu l (10 mu M) of upstream primer, 2-2.5 mu l (10 mu M) of downstream primer, 2-2.5 mu l (10 mu M) of magnesium acetate and 1-5 mu l of DNA template into the freeze-dried powder containing RPA component, supplementing 50 mu l with double distilled water, uniformly mixing and centrifuging, dripping 10 mu l of mixed liquid onto the surface of a multichannel sensing electrode array, incubating for 20min at 37 ℃, and flushing the electrode with PBS; : and (3) dropwise adding 5 mu l of avidin-HRP enzyme conjugate on the surface of the multi-channel sensing electrode array, incubating for 10min at room temperature, flushing with PBS, dropwise adding 50 mu l of TMB solution to the multi-channel sensing electrode array, scanning a current time curve by using a multi-channel electrochemical instrument, and recording a steady-state current value at 100s as a detection signal.
The invention relates to an electrochemical nucleic acid sensing technology (RPA-EC) based on an interface RPA, which combines the RPA with an electrochemical DNA sensor, the schistosoma japonicum detection kit adopts SjR fragments with better sensitivity and specificity as targets, the lowest detection limit of the schistosoma japonicum genomic DNA is 10ag, the reaction can be carried out at 37 ℃ even room temperature, positive signals can be detected after 10 minutes, sjR fragments in the schistosoma japonicum genomic DNA can be detected within 20 minutes, and the sensitivity is improved by 2 orders of magnitude compared with the previously reported RPA detection method. The kit can detect DNA fragments in serum of mice infected with schistosome, has early detection value, does not have cross reaction with schistosoma mansoni, schistosoma aegypti, paragonium weigii, fasciola gigantica and clonorchis sinensis, and has ideal nucleic acid detection specificity of schistosoma japonicum. Therefore, the method has the beneficial effects of simplicity, rapidness and sensitivity in operation, low requirements on samples and suitability for field application.
Drawings
Fig. 1: the kit provided by the invention is used for detecting the feasibility analysis of the schistosoma japonicum genomic DNA. Wherein A is double distilled water; b is 1fg of Schistosoma japonicum genomic DNA; c is 1pg of Schistosoma japonicum genomic DNA; d is 1ng of Schistosoma japonicum genomic DNA; e is 1ng of Schistosoma mansoni genomic DNA.
Fig. 2: the reaction time of the kit for detecting the schistosoma japonicum genomic DNA is optimized. Wherein 1 is double distilled water; 2 is 1fg schistosoma japonicum genomic DNA; the reaction time of A-E is 10min,20min,30min,40min and 50min respectively.
Fig. 3: the kit of the invention detects the results of the schistosoma japonicum genomic DNA with different concentrations. Wherein A: negative control: double steaming; B-F: respectively 10 -8 ng～10 -4 ng schistosoma japonicum genomic DNA.
Fig. 4: the kit detects the specificity evaluation of the genome DNA of a plurality of flukes. Wherein A is a negative control: sterilizing water; b: schistosoma japonica; c: schistosoma hallucium; d: schistosoma mansoni; e, paragonimiasis weigii; f: schistosoma japonicum; g, clonorchis sinensis.
Fig. 5: the kit of the invention detects the results of DNA of blood samples of mice infected at different time points. Wherein A: negative control: pre-infection (0 d) murine serum DNA; b: infecting 7d mouse serum DNA; c: infecting 21d mouse serum DNA; d: 35d mouse serum DNA was infected.
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: preparation of template DNA, primer and Probe
(1) Genomic DNA preparation: tissue DNA extraction kit Blood&Tissue kit, available from Qiagen) to extract Schistosoma japonicumAdult schistosoma mansoni, schistosoma japonicum ovum, schistosoma japonicum metacercaria, clonorchis sinensis adult and fasciola gigantica genome DNA.
(2) Primer and probe design: and (3) taking SjR gene fragments as target sequences, and designing RPA primers and probes. All primer and probe DNA were synthesized by Shanghai Biotechnology Co.
The upstream primer is as follows: 5'-CCCAAGTCTCAGTGAAGTTGTGAAGGCTAT-3';
the downstream primer is: 5'-GTTAGTGTTCGAGACCAGTCAGATGGGATT-3' (5 ' end biotinylation);
the probe sequence is as follows: NH2-CTTAAAGCGAGGGAGAGCGGCAGGACCAGATG [ THF ] ATTGACCCCTGAGATAT-ph (5' amino modification); wherein [ THF ] is an abbreviation for tetrahydrofuran (tetrahydrofuran), a recognition site for exonuclease; ph represents a phosphate group to block the extension of the DNA strand.
Example 2: multi-channel electrochemical sensing electrode array construction
The surface of the printing electrode was cleaned and scanned by cyclic voltammetry, and 10. Mu.l of a mixed solution containing 400mmol/L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 100mmol/L N-N hydroxysuccinimide was added dropwise thereto, and the carboxyl group on the surface of the electrode was activated at room temperature for 15 minutes. Then, 10. Mu.l of RPA probe solution (1. Mu. Mol/L) was added dropwise to the electrode surface, and the electrode surface was incubated at 37℃for 1 hour and washed with 0.01mmol/L PBS. 50 μl of blocking solution containing 1% bovine serum albumin and 1% casein was added dropwise, incubated at 37deg.C for 1h to block nonspecific sites, and then the electrode was cleaned and placed in a refrigerator at 4deg.C for use.
Example 3: establishment of schistosoma japonica genome DNA detection method
1) Interface RPA reaction: to the RPA lyophilized particles were added 29.5. Mu. l Rehydration Buffer, 2.1. Mu.l of the upstream primer, 2.1. Mu.l of the downstream primer, 12.8. Mu.l of double distilled water, 2.5ul of magnesium acetate, 1.0. Mu.l of DNA template, mixed well and centrifuged, 10. Mu.l of the RPA mixture was dropped onto the electrode sensing surface, incubated for 20min at 37℃and the electrode was rinsed with 1 XPBS.
2) Current signal acquisition: and 5. Mu.l of SA-HRP enzyme is dripped on the surface of the electrode, incubated at room temperature for 10min,1XPBS is washed, 50. Mu.l of TMB solution is dripped on the electrode array, a multichannel electrochemical instrument is used for current time curve scanning, and a steady state current value at 100s is recorded as a detection signal.
3) And (3) result judgment: the negative control current average value +3SD is used as a threshold value, the current exceeds the threshold value to judge that the positive sample containing the schistosoma japonicum DNA is lower than the threshold value to judge that the negative sample is obtained.
Example 4: feasibility evaluation method for kit detection method
And (3) selecting 37 ℃ and 50min as initial reaction conditions, respectively taking 1fg, 1pg, 1ng of schistosoma japonicum genomic DNA and 1ng of schistosoma mansoni genomic DNA as experimental groups, and taking double distilled water as blank control, and carrying out feasibility analysis of RPA amplification and detection of SjR gene fragments on an electrochemical sensing interface. As a result, as shown in FIG. 1, the current signals of 1fg, 1pg and 1ng of the genomic DNA of Schistosoma japonicum were 13.713.3.+ -. 662.7, 15.396.7.+ -. 194.3 and 16 026.7.+ -. 310.1nA, respectively, and the current signals of the DNA groups of double distilled water and 1ng of Schistosoma mansoni were 232.0.+ -. 82.2 and 212.3.+ -. 60.7nA, respectively, at 37℃for 50min. The difference between the schistosoma japonicum DNA group current signal and the double distilled water group current signal is statistically significant (t=35.0, p < 0.01), and the difference between the schistosoma japonicum group current signal and the double distilled water group current signal is not statistically significant (t=35.1, p < 0.01). It is known that the established detection method can detect the genomic DNA of schistosoma japonicum with high sensitivity at 37 ℃ for 50min.
Example 5: kit detection time optimization
At 37 ℃,1fg of schistosoma japonicum genomic DNA is taken as an experimental group, double distilled water is taken as a blank control, and the optimal reaction time of the detection method of the kit is examined. As a result, as shown in FIG. 2, when RPA was amplified for 10, 20, 30, 40, and 50min, the current signals of 1fg Japanese blood fluke genomic DNA groups were 331.5.+ -. 55.9, 1.125.0.+ -. 18.4, 3.5.+ -. 249.6, 6.411.0.+ -. 131.5, and 13.0.+ -. 586.9nA, respectively, and the double distilled water group current signals were 87.0.+ -. 36.8, 189.0.+ -. 24.0, 227.0.+ -. 46.7, 257.0.+ -. 38.2, and 277.5.+ -. 33.2nA, respectively. It can be seen that the current signal of the experimental group is gradually enhanced along with the extension of the amplification time, the positive signal of the experimental group reaches the highest value at 50min, and the difference of the positive current signals of different time groups has statistical significance (F=350.9, P < 0.01). The double distilled water group signal does not obviously increase along with the amplification time and is maintained below 300 nA. After 20min, the signal-to-noise ratio (the signal ratio of the DNA group to the double distilled water group) of the detected current is obviously improved, and the signal-to-noise ratio is the highest at 50min, but the signal stability is poor. From the viewpoints of reaction stability and timeliness, the reaction condition was selected as the optimum reaction condition for the detection of a template having a low concentration of 37 ℃.
Example 6: kit detection sensitivity evaluation
The detection sensitivity of the detection method of the kit is evaluated by adopting the optimized reaction condition (37 ℃ C., 30 min) of the kit and taking double distilled water as a negative control and detecting the schistosoma japonicum genomic DNA (10-4, 10-5, 10-6, 10-7 and 10-8 ng) with different concentrations, and the result is shown in figure 3, and the current signals obtained by 10-4, 10-5, 10-6, 10-7 and 10-8ng schistosoma japonicum genomic DNA based on the RPA-EC method under the condition of 37 ℃ C., 30min are 5 779.5+/-137.5, 3.504.5+/-419.5, 2 372.0+/-213.0, 815.0 +/-43.0 and 392.5+/-6.5 nA respectively. It was found that the detection current signal was increased in gradient with the increase in the amount of DNA. When the sum of the average value of the current signal (227.5 nA) of the control group and the 3-fold standard deviation thereof is set as a positive threshold (Cut off=268 nA), the lowest detection limit of the genomic DNA of schistosoma japonicum can reach 10-8ng (10 ag).
Example 7: kit detection specificity evaluation
The genome DNA (10 ng) of the same concentration of Schistosoma japonicum, schistosoma mansoni, schistosoma japonicum and clonorchis sinensis was detected with sterilized water as negative control, and the specificity of the detection method of the kit was evaluated. As shown in FIG. 4, under the condition of 37 ℃ and 30min, the current signals obtained by the genomic DNA of 10ng of schistosoma japonicum, schistosoma aegypti, schistosoma mansoni, schistosoma hygienii, schistosoma japonicum and clonorchis sinensis are respectively 18 757.5+/-265.5, 230.5 +/-24.6, 378.5+/-2.5, 482+/-9.0, 327+/-29.0 and 264+/-31.0 nA, wherein the schistosoma japonicum group detects stronger signals, and the current signals of other insect species groups are very weak and have little difference with the current signals of the control group (222.5 nA+/-19.5 nA), so that the detection specificity of the kit detection method is better.
Example 8: evaluation of effect of detecting dynamic serum DNA of schistosoma japonicum infected mice by kit
And (3) detecting serum DNA of 0, 7, 21 and 35d after the mice are infected with the schistosoma japonicum by adopting the optimized detection conditions of the kit, and evaluating the feasibility of detecting animal dynamic serum DNA by the method, wherein each group of experiments are repeated 3 times. As a result, as shown in FIG. 5, the current signals obtained from the serum DNA of 0, 7, 21, 35d after infection of the mouse model with Schistosoma japonicum at 37℃for 30min were 655.5.+ -. 24.5, 7.5.+ -. 197.5, 6.723.5.+ -. 129.5, 8 389.5.+ -. 239.5nA, respectively. It was found that the current signal was highest in the 35d infected group, and was lowest in the 7d infected group, and that the current signal difference at three time points was statistically significant (f=55.8, p < 0.01). When the sum of the average value of the current of the 0d group and the standard deviation of 3 times of the average value is set as a positive threshold value (cut off value=729nA), the current signals of the 7, 21 and 35d groups are all larger than the threshold value, which indicates that the detection method of the kit can detect SjR DNA fragments in 7, 21 and 35d serum after infection.
In summary, the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, but any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the scope of the present invention.
Sequence listing
<110> center for prevention and control of diseases in China
<120> an RPA-EC schistosoma japonicum detection kit and detection method
<130> CPC-NP-20-101901
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<170> SIPOSequenceListing 1.0
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<213> probe sequence (probe sequence)
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<212> DNA
<213> RPA upstream primer (RPA upstream primer)
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<213> RPA downstream primer (RPA downstream primer)
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gttagtgttc gagaccagtc agatgggatt 30

Claims (4)

1. An RPA-EC schistosoma japonicum detection kit, comprising: each channel of the multichannel sensing electrode array comprises a working electrode, a counter electrode and a reference electrode, wherein the working electrode is a carbon working electrode, an amino-modified RPA probe is fixed on the surface of the carbon working electrode, the RPA probe is an amino-modified RPA probe capable of amplifying SjR gene fragments, the amino-modified RPA probe is fixed on the surface of the carbon working electrode through the action of carboxyl amide bond with the surface of the carbon working electrode, and non-specific binding sites on the surface of the working electrode are blocked by bovine serum albumin and casein; an RPA primer comprising an RPA upstream primer and an RPA downstream primer; a lyophilized powder comprising an RPA component; magnesium acetate; a reaction buffer; avidin-HRP enzyme conjugate; a substrate solution and a washing solution;
the RPA upstream primer is as follows: 5'-CCCAAGTCTCAGTGAAGTTGTGAAGGCTAT-3';
the RPA downstream primer is as follows: 5'-GTTAGTGTTCGAGACCAGTCAGATGGGATT-3', 5' end biotinylation;
the RPA probe sequence is as follows: NH2-CTTAAAGCGAGGGAGAGCGGCAGGACCAGATG [ THF ]
ATTGACCCCTGAGATAT-ph, 5' amino modification; wherein THF is an abbreviation for tetrahydrofuran, a recognition site for exonuclease; ph represents a phosphate group to block the extension of the DNA strand;
the freeze-dried powder containing the RPA component comprises the following components: phage recombinases, DNA polymerases, single-stranded DNA binding proteins, dntps, and exonucleases;
the substrate solution is TMB solution containing hydrogen peroxide, and the washing solution is PBS.
2. The kit of claim 1, wherein the multi-channel sensing electrode array is a 16-channel three-electrode printed carbon electrode array.
3. The kit of claim 1, further comprising a negative control that is double distilled water and a positive control that is schistosoma japonicum genomic DNA.
4. The kit of claim 1, wherein the reaction buffer is Rehydration Buffer.
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