CN117867172A - Amplification-free detection method and kit for target viruses - Google Patents
Amplification-free detection method and kit for target viruses Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6816—Hybridisation assays characterised by the detection means
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Abstract
The application provides an amplification-free detection method and a kit for target viruses. The detection method comprises the following steps: s1, preparing a nucleic acid-metal ion probe; s2, respectively adding the sample to be detected and the control sample with a nucleic acid-metal ion probe into a nitrate buffer solution to carry out DNA probe hybridization to serve as a detection group and a control group; s3, adding urease and urea into the products obtained in the step S2 to react; s4, adding fluorogenic substrate phthalic aldehyde and sodium sulfite into each group of products obtained in the step S3 to react; s5, detecting fluorescent signals of the products of the step S4, and judging the content of the target virus in the sample to be detected according to the intensity of the fluorescent signals. The detection method does not depend on nucleic acid amplification, and has high detection sensitivity, strong recognition specificity and high detection efficiency.
Description
Technical Field
The application relates to the field of molecular detection, in particular to an amplification-free detection method and a kit for target viruses.
Background
Up to now, the world experiences multiple serious respiratory tract viral infections and serious harm to life and property safety. Patients infected with different types of respiratory viruses (e.g., SARS-CoV-2, influenza A, influenza B, and RSV) have similar clinical symptoms, but are treated and managed differently for different types of respiratory virus infections. In addition, the affinity of different variant strains of the same type of virus for receptor binding and susceptibility to vaccines are also quite different. Thus, accurate identification of viruses is a critical prerequisite for controlling respiratory viral infections.
The current methods available for respiratory virus detection are nucleic acid detection, antigen detection and antibody detection. The accuracy and sensitivity of nucleic acid detection are higher than those of antigen-antibody detection, wherein the quantitative reverse transcription PCR (RT-qPCR) method is most commonly used. However, RT-qPCR is not a real-time diagnostic tool due to its reliance on temperature swing instrumentation. In recent years, a large number of normal temperature detection means such as reverse transcription loop-mediated isothermal amplification (RT-LAMP), rolling Circle Amplification (RCA) and other technologies are also used for virus detection, but the methods all require nucleic acid amplification, so that the operation steps are complex, and the detection process is longer.
Thus, there is a need to propose a visual nucleic acid detection kit that is independent of nucleic acid amplification.
Disclosure of Invention
The present application solves at least one of the problems of the related art from the following aspects.
Embodiments of the first aspect of the present application provide an amplification-free detection method of a target virus, comprising the steps of:
s1, chimeric a single-stranded DNA probe containing P1 complementary to a target sequence, P2 complementary to part of P1 and two P3 containing mismatched bases with metal ions to prepare a nucleic acid-metal ion probe,
wherein two of said P3 s are located at both ends of said single-stranded DNA probe, respectively;
Two of the P3's complement each other to form a first double-stranded region by bases other than the mismatched base contained therein;
the metal ion is chimeric in a mismatched base pair of the non-complementary pairing of the first double-stranded region;
said P2 is complementary to a portion of said P1 to form a second double-stranded region;
the length of P1 is greater than the sum of the lengths of P2 and P3; and is also provided with
The target sequence is selected from a specific gene of the target virus;
s2, respectively hybridizing a sample to be detected and a control sample with the nucleic acid-metal ion probe to obtain a DNA probe hybrid, wherein the DNA probe hybrid is used as a detection group and a control group;
s3, adding urease and urea into the products obtained in the step S2 to react;
s4, adding fluorogenic substrate phthalic aldehyde and sodium sulfite into each group of products obtained in the step S3 to react; and
s5, detecting fluorescent signals of the products of the step S4, and judging the content of the target virus in the sample to be detected according to the intensity of the fluorescent signals.
In some embodiments, wherein the target virus is a novel coronavirus and the target sequence is selected from the group consisting of an N gene and/or an S gene of the novel coronavirus.
In some embodiments, the target virus is wild-type SARS-CoV-2 and the target sequence is selected from the group consisting of the N gene of the target virus.
In some embodiments, the target virus is a ba.5.2 variant or bf.7 variant of SARS-CoV-2, and the target sequence is selected from the S gene of the target virus.
In some embodiments, the target virus is wild-type SARS-CoV-2 and the target sequence is the sequence shown as SEQ ID NO.8 or a sequence having greater than 85% homology thereto.
In some embodiments, the target virus is a ba.5.2 variant of SARS-CoV-2, the target sequence is the sequence shown as SEQ ID No.9 or a sequence having greater than 85% homology thereto; or a sequence shown as SEQ ID NO.10 or a sequence with the homology of more than 85 percent.
In some embodiments, the target virus is a bf.7 variant of SARS-CoV-2, and the target sequence is the sequence set forth in SEQ ID No.11 or a sequence having greater than 85% homology thereto; or a sequence shown as SEQ ID NO.12 or a sequence with the homology of more than 85 percent; or a sequence shown as SEQ ID NO.13 or a sequence with the homology of more than 85 percent.
In some embodiments, when the metal ion is silver, the P3 is 3' -C- (AC) n -5 'and 5'-C-(TG) n -3', n is 1 to 4.
In some embodiments, when the metal ion is mercury ion, the P3 is 3' -T- (CA) n -5 'and 5' -T- (GT) n -3', n is 1 to 4.
In some embodiments, the target virus is wild-type SARS-CoV-2; the target sequence is a sequence shown as SEQ ID NO.8 or a sequence with the homology of more than 85 percent; and the single-stranded DNA probe is a sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
In some embodiments, the target virus is a ba.5.2 variant of SARS-CoV-2, the target sequence is the sequence shown as SEQ ID No.9 or a sequence having greater than 85% homology thereto and the single stranded DNA probe is the sequence shown as SEQ ID No. 3; or the target sequence is a sequence shown as SEQ ID NO.10 or a sequence with homology of more than 85% and the single-stranded DNA probe is a sequence shown as SEQ ID NO. 4.
In some embodiments, the target virus is a bf.7 variant of SARS-CoV-2, the target sequence is the sequence set forth in SEQ ID No.11 or a sequence having greater than 85% homology thereto and the single-stranded DNA probe is the sequence set forth in SEQ ID No. 5; or the target sequence is a sequence shown as SEQ ID NO.12 or a sequence with homology of more than 85% and the single-stranded DNA probe is a sequence shown as SEQ ID NO. 6; or the target sequence is a sequence shown as SEQ ID NO.13 or a sequence with homology of more than 85% and the single-stranded DNA probe is a sequence shown as SEQ ID NO. 7.
In some embodiments, step S1 comprises incubating the single-stranded DNA probe and metal ions in a nitrate buffer.
In some embodiments, step S1 further comprises cooling to room temperature and standing after the incubation is completed.
In some embodiments, in step S2, the DNA probe hybridization includes annealing and extension steps.
In some embodiments, the control group comprises a positive control group and/or a negative control group, wherein the negative control group does not contain the target sequence, and the positive control group contains the target sequence.
In some embodiments, the reaction system of step S1 comprises sodium nitrate and/or magnesium nitrate in a final concentration of 10 to 200 mM.
In some embodiments, in step S1, the molar ratio of the single-stranded DNA probe to the metal ion is 1 (0.1 to 100), preferably 1:1 to 10.
In some embodiments, in step S1, the incubation time is 2 to 10min.
In some embodiments, in step S1, the incubation temperature is 60 to 90 ℃.
In some embodiments, in the step S1, the standing time is 5 to 90min.
In some embodiments, the reaction system of step S2 comprises sodium nitrate and/or magnesium nitrate in a final concentration of 10 to 200 mM.
In some embodiments, in step S2, the temperature of the annealing step is 65 to 90 ℃.
In some embodiments, in the step S2, the annealing step is performed for 0.5 to 10 minutes.
In some embodiments, in step S2, the temperature of the extending step is 20 to 60 ℃.
In some embodiments, in the step S2, the time of the extending step is 5 to 60min.
In some embodiments, the ratio of the concentration of urease to the concentration of metal ions chimeric in the nucleic acid-metal ion probe in the reaction system of step S3 is 1 (1 to 100).
In some embodiments, the final concentration of urea in the reaction system of step S3 is 0.1 to 100mM.
In some embodiments, the reaction temperature of step S3 is 20 to 45 ℃.
In some embodiments, the reaction time of step S3 is 2 to 60 minutes.
In some embodiments, the final concentration of the phthalic aldehyde in the reaction system of step S4 is 1 to 100mM.
In some embodiments, the final concentration of sodium sulfite in the reaction system of step S4 is 1 to 200mM.
In some embodiments, the reaction temperature of step S4 is 20 to 65 ℃.
In some embodiments, the reaction time of step S4 is 2 to 90min.
In some embodiments, the fluorescent signal is detected at a wavelength of 365 nm.
In some embodiments, the control group comprises a negative control group, wherein the negative control group does not contain the target sequence; and when the fluorescence signal intensity of the detection group is smaller than or equal to that of the negative control group, judging that the sample to be detected contains the target virus.
In some embodiments, the control group comprises a plurality of positive control groups containing different concentrations of target sequences; preparing a standard curve according to the target sequence concentration and the fluorescence signal intensity of each positive control group; and calculating the content of the target virus in the sample to be detected according to the standard curve and the fluorescence signal intensity of the detection group.
Embodiments of the second aspect of the present application provide an amplification-free kit for detecting a target virus comprising a nucleic acid-metal ion probe, urease, urea, and a fluorogenic substrate,
wherein the nucleic acid-metal ion probe is prepared by chimerism of a single-stranded DNA probe comprising P1 complementary to the target sequence, P2 complementary to part of P1 and two P3 comprising mismatched bases with a metal ion,
Wherein two of said P3 s are located at both ends of said single-stranded DNA probe, respectively;
two of the P3's complement each other to form a first double-stranded region by bases other than the mismatched base contained therein;
the metal ion is chimeric in a mismatched base pair of the non-complementary pairing of the first double-stranded region;
said P2 is complementary to a portion of said P1 to form a second double-stranded region;
the length of P1 is greater than the sum of the lengths of P2 and P3; and is also provided with
The target sequence is selected from genes specific for the target virus.
In some embodiments, the target virus is a novel coronavirus and the target sequence is selected from the group consisting of an N gene and/or an S gene of the novel coronavirus.
In some embodiments, the target virus is wild-type SARS-CoV-2 and the target sequence is selected from the group consisting of the N gene of the target virus.
In some embodiments, the target virus is a ba.5.2 variant or bf.7 variant of SARS-CoV-2, and the target sequence is selected from the S gene of the target virus.
In some embodiments, the target virus is wild-type SARS-CoV-2 and the target sequence is the sequence shown as SEQ ID No.8 or a sequence having greater than 85% homology thereto;
in some embodiments, the target virus is a ba.5.2 variant of SARS-CoV-2, the target sequence is the sequence shown as SEQ ID No.9 or a sequence having greater than 85% homology thereto; or a sequence shown as SEQ ID NO.10 or a sequence with the homology of more than 85 percent.
In some embodiments, the target virus is a bf.7 variant of SARS-CoV-2, and the target sequence is the sequence set forth in SEQ ID No.11 or a sequence having greater than 85% homology thereto; or a sequence shown as SEQ ID NO.12 or a sequence with the homology of more than 85 percent; or a sequence shown as SEQ ID NO.13 or a sequence with the homology of more than 85 percent.
In some embodiments, when the metal ion is silver, the P3 is 3' -C- (AC) n -5 'and 5' -C- (TG) n -3', n is 1 to 4.
In some embodiments, when the metal ion is mercury ion, the P3 is 3' -T- (CA) n -5 'and 5' -T- (GT) n -3', n is 1 to 4.
In some embodiments, the target virus is wild-type SARS-CoV-2 and the single stranded DNA probe has the sequence shown in SEQ ID NO. 1; and the metal ion is silver ion, or the sequence SEQ ID NO.2 of the single-stranded DNA probe; and the metal ion is a mercury ion.
In some embodiments, the target virus is a BA.5.2 variant of SARS-CoV-2, and the single stranded DNA probe has the sequence shown in SEQ ID NO.3 or SEQ ID NO. 4; and the metal ion is silver ion.
In some embodiments, the target virus is a BF.7 variant of SARS-CoV-2, and the single stranded DNA probe has a sequence as shown in SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO. 7; and the metal ion is silver ion.
In some embodiments, the fluorogenic substrate comprises phthalaldehyde and sodium sulfite.
In some embodiments, the ratio of the concentration of urease to the concentration of metal ions chimeric in the nucleic acid-metal ion probe is 1 (1 to 100).
Embodiments of the third aspect of the present application provide the use of a kit according to the second aspect above for detecting a viral infection.
The embodiment of the application realizes the following beneficial effects:
1) Nucleic acid amplification is avoided. In the detection method provided by the embodiment of the application, the nucleic acid-metal ion probe directly recognizes the viral RNA, so that the viral load is quantified, the operation is simple, and the nucleic acid amplification is not depended.
2) The detection sensitivity is high. The detection method provided by the embodiment of the application is based on OPA and Na 2 SO 3 And urease-catalyzed NH production 4+ The reaction generates a strong fluorescent product Isoindole-1-sulfonate, and SARS-CoV-2RNA with the low frequency of 1.7fM can be detected on the premise of no amplification due to the high catalytic activity of urease.
3) The recognition specificity is strong. The detection method provided by the embodiment of the application can accurately identify various viruses, has high specificity, and can even distinguish single base mutation, thereby distinguishing viruses (such as wild SARS-CoV-2) and variants thereof.
4) The detection method is flexible and has high detection speed. The nucleic acid-metal ion probe related to the detection method provided by the embodiment of the application is a section of short-chain DNA which can be rapidly edited and synthesized, and a detection scheme can be formulated within 12-48 h for different viruses and continuously appeared SARS-CoV-2 variants, so that a detection result can be obtained.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic diagram of an amplification-free detection method of a target virus according to an embodiment of the present application.
FIG. 2 is a schematic illustration of the catalytic NH supply by urease in example 1 4+ Is a test result of (a).
FIG. 3 is the results of the test in example 2 in which Ag (I) inhibits urease activity, resulting in fluorescence quenching.
FIG. 4 shows the results of a test for Ag (I) intercalation into single-stranded DNA to form a nucleic acid-metal ion probe in example 3.
FIG. 5 is the result of the test in example 4, wherein the-N gene is a control group to which the wild-type SARS-CoV-2N gene was not added, and wherein the +N gene is an experimental group to which the wild-type SARS-CoV-2N gene was added.
FIG. 6A is a standard curve for quantitative detection of viruses obtained in example 5 using a single-stranded DNA probe (shown as SEQ ID NO. 1) according to the amplification-free detection method of a target virus provided in the example of the present application, and FIG. 6B is a standard curve for quantitative detection of viruses obtained in comparative example 2 using one of the visualized nucleic acid detection kits disclosed previously.
FIG. 7 results of detection of the BA.5.2 variants using single stranded DNA probes (shown as SEQ ID NO.3 and SEQ ID NO. 4) in example 6.
Fig. 8 is a schematic step diagram of an amplification-free detection method of a target virus according to an embodiment of the present application.
Detailed Description
The invention will now be described in further detail with reference to the following specific embodiments, which are given by way of illustration only and are not intended to limit the scope of the invention. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The present application is made based on the following knowledge of the inventors:
up to now, the world experiences multiple serious respiratory tract viral infections and serious harm to life and property safety. Influenza virus (Influenza virus) is the most common respiratory virus, its strain is numerous and changes every year, and thus continuous vaccination with Influenza vaccine is required. Respiratory syncytial virus (Respiratory syncytial virus, RSV) is another common winter-epidemic virus that is highly infectious and can infect children and adults. RSV, in adults and older children, usually causes mild cough, a mild illness very similar to the common cold. However, in infants and the elderly, the symptoms are more serious, and serious infections such as bronchiolitis and pneumonia are occasionally caused.
In addition, patients infected with respiratory viruses such as SARS-CoV-2, influenza A, influenza B and RSV have similar clinical symptoms, but the methods of treatment and management of infection by these viruses are quite different. Thus, accurate identification of these viruses is beneficial for reducing the risk of viral epidemics. Except for the difficulty in virus detection and management caused by similar clinical manifestations of infection. Viral infection is also driven by emerging variant strains and by the weakening of immunity caused by the variant strains. It was found that the affinity of the variant for receptor binding and the sensitivity to the vaccine were different. In particular, these variants are mostly caused by several single base gene mutations, which also puts new demands on the method of variant detection.
In the related art, methods that can be used for respiratory virus detection include nucleic acid detection, antigen detection, and antibody detection. The accuracy and sensitivity of nucleic acid detection are higher than those of antigen-antibody detection, wherein the quantitative reverse transcription PCR (RT-qPCR) method is most commonly used. Due to the reliance of RT-qPCR on temperature changing instruments, RT-qPCR cannot be used as a real-time diagnostic tool. In recent years, a large number of normal temperature detection means such as reverse transcription loop-mediated isothermal amplification (RT-LAMP), rolling Circle Amplification (RCA) and other technologies are also used for virus detection, but the methods all require nucleic acid amplification, so that the operation steps are complex, and the detection process is longer.
Previously, applicants developed a nucleic acid amplification independent visual nucleic acid detection kit (CN 202011644377.2) and successfully used for SARS-CoV-2 detection. The kit is characterized in that metal ions, DNA double-stranded molecules and urease interact on paper folding, and viruses are detected by monitoring pH change brought by urease catalysis. However, the kit cannot quantitatively detect the viral load, and hybridization of the DNA double-strand recognition probe embedded with the metal ions adopted in the kit is not stable enough, so that the kit has high operation requirements and is unfavorable for standardized quantitative detection.
In this regard, the inventors have made extensive studies to propose a single-stranded DNA probe based on intercalating metal ions, urease and fluorogenic detection substrates of phthalic acid (OPA) and sodium sulfite (Na 2 SO 3 ) Is a kit for detecting nucleic acid without amplification. As shown in FIG. 1, in order to detect viruses in a quasi-determined amount, ammonia was introduced as fluorogenic substrates of phthalic aldehyde (OPA) and sodium sulfite (Na 2 SO 3 ) Methods have been developed for detecting viral RNA using a fluorogenic substrate that binds ammonia with the inhibitory effect of metal ions on urease. The improved nucleic acid-metal ion probe is a single-stranded DNA embedded with metal ions. Furthermore, the fluorogenic substrates of ammonia, phthalic aldehyde (OPA) and sodium sulfite (Na 2 SO 3 ) The method is used for nucleic acid detection, and also provides a new thought for identifying viral RNA by metal ion-nucleic acid probes, and provides an effective strategy for virus management, treatment and control.
As shown in fig. 8, an embodiment of the first aspect of the present application proposes an amplification-free detection method of a target virus, which includes the following steps S1 to S5.
S1, chimeric a single-stranded DNA probe containing P1 complementary to a target sequence, P2 complementary to part of P1 and two P3 containing mismatched bases with metal ions to prepare a nucleic acid-metal ion probe,
wherein two of said P3 s are located at both ends of said single-stranded DNA probe, respectively;
two of the P3's complement each other to form a first double-stranded region by bases other than the mismatched base contained therein;
the metal ion is chimeric in a mismatched base pair of the non-complementary pairing of the first double-stranded region;
said P2 is complementary to a portion of said P1 to form a second double-stranded region; and is also provided with
The length of P1 is greater than the sum of the lengths of P2 and P3, i.e. P1 > P2+P3,
the target sequence is selected from genes specific for the target virus.
In the embodiment of the application, the binding force between P1 completely complementary to the target sequence of the target virus and the target sequence of the target virus is greater than the binding force between the double-stranded portion (consisting of the first double-stranded region and the second double-stranded region) of the nucleic acid-metal ion probe, so that when the target virus exists in the detection system, P1 is complementarily paired with the target sequence of the target virus, the double-stranded portion of the nucleic acid-metal ion probe is opened, and the metal ion embedded in the double-stranded portion of the DNA probe is released.
In some embodiments, both of said P3 comprise the same type and number of mismatched bases.
In some embodiments, the target virus is a novel coronavirus and the target sequence is selected from the group consisting of an N gene and/or an S gene of the novel coronavirus.
In some embodiments, the target virus is wild-type SARS-CoV-2 and the target sequence is selected from the group consisting of the N gene of the target virus.
In some embodiments, the target virus is a ba.5.2 variant or bf.7 variant of SARS-CoV-2, and the target sequence is selected from the S gene of the target virus.
In some embodiments, the target virus is wild-type SARS-CoV-2 and the target sequence is the sequence shown as SEQ ID No.8 or a sequence having greater than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology thereto.
In some embodiments, the target virus is a ba.5.2 variant of SARS-CoV-2, the target sequence is the sequence shown as SEQ ID No.9 or a sequence having greater than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology thereto; or a sequence as shown in SEQ ID No.10 or a sequence having a homology of more than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) thereto.
In some embodiments, the target virus is a bf.7 variant of SARS-CoV-2, the target sequence is the sequence set forth in SEQ ID No.11 or a sequence having greater than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology thereto; or a sequence as set forth in SEQ ID NO.12 or a sequence having greater than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology thereto; or a sequence as set forth in SEQ ID NO.13 or a sequence having more than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology thereto.
In some embodiments, when the metal ion is silver, the P3 is 3' -C- (AC) n -5 'and 5' -C- (TG) n -3', n is 1 to 4.
In some embodiments, when the metal ion is mercury ion, the P3 is 3' -T- (CA) n -5 'and 5' -T- (GT) n -3', n is 1 to 4.
In some embodiments, the target virus is wild-type SARS-CoV-2; the target sequence is the sequence shown in SEQ ID NO.8 or a sequence with homology of more than 85% (such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) with the target sequence; the single-stranded DNA probe is a sequence shown as SEQ ID NO.1 or SEQ ID NO. 2;
In some embodiments, the target virus is a ba.5.2 variant of SARS-CoV-2, the target sequence is the sequence set forth in SEQ ID No.9 or a sequence having greater than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology thereto and the single stranded DNA probe is the sequence set forth in SEQ ID No. 3; or the target sequence is a sequence as shown in SEQ ID NO.10 or a sequence having homology of more than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) thereto and the single-stranded DNA probe is a sequence as shown in SEQ ID NO. 4;
in some embodiments, the target virus is a bf.7 variant of SARS-CoV-2, the target sequence is the sequence set forth in SEQ ID No.11 or a sequence having greater than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology thereto and the single stranded DNA probe is the sequence set forth in SEQ ID No. 5; or the target sequence is the sequence shown as SEQ ID NO.12 or a sequence having homology of more than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) thereto and the single-stranded DNA probe is the sequence shown as SEQ ID NO. 6; or the target sequence is the sequence shown as SEQ ID NO.13 or a sequence having homology of more than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) thereto and the single-stranded DNA probe is the sequence shown as SEQ ID NO. 7.
In some embodiments, step S1 comprises incubating the single-stranded DNA probe and metal ions in a nitrate buffer.
In some embodiments, the reaction system of step S1 comprises sodium nitrate and/or magnesium nitrate in a final concentration of 10 to 200nM (e.g., 20nM, 50nM, 70nM, 100nM, 120nM, 150nM, 170 nM). In some embodiments, in step S1, the molar ratio of the single-stranded DNA probe to the metal ion is 1 (0.1 to 100) (e.g., 1:0.5, 1:1, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90), preferably 1 (1 to 10) (e.g., 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9).
In some embodiments, in step S1, the incubation time is 2 to 10min (e.g., 3min, 4min, 5min, 6min, 7min, 8min, 9 min).
In some embodiments, in step S1, the incubation temperature is 60 to 90 ℃ (e.g., 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃).
In some embodiments, step S1 further comprises cooling to room temperature and standing after the incubation is completed.
In some embodiments, in the step S1, the standing time is 5 to 90min (e.g., 10min, 20min, 30min, 40min, 50min, 60min, 70min, 80 min).
S2, respectively hybridizing the sample to be detected and the control sample with the nucleic acid-metal ion probe to obtain a DNA probe hybrid, and taking the DNA probe hybrid as a detection group and a control group.
In some embodiments, the reaction system of step S2 comprises sodium nitrate and/or magnesium nitrate in a final concentration of 10 to 200nM (e.g., 20nM, 50nM, 70nM, 100nM, 120nM, 150nM, 170 nM).
In some embodiments, in step S2, the DNA probe hybridization includes annealing and extension steps.
In some embodiments, in step S2, the temperature of the annealing step is 65 to 90 ℃ (e.g., 70 ℃, 75 ℃, 80 ℃, 85 ℃).
In some embodiments, in step S2, the annealing step is performed for a time of 0.5 to 10min (e.g., 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9 min).
In some embodiments, in step S2, the temperature of the extending step is 20 to 60 ℃ (e.g., 30 ℃, 40 ℃, 50 ℃).
In some embodiments, in the step S2, the time of the extending step is 5 to 60min (e.g., 10min, 20min, 30min, 40min, 50 min).
S3, adding urease and urea into the products obtained in the step S2 to react.
In some embodiments, the ratio of the concentration of urease to the concentration of metal ions chimeric in the nucleic acid-metal ion probe in the reaction system of step S3 is 1 (1 to 100) (e.g., 1:0.5, 1:1, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90).
In some embodiments, the final concentration of urea in the reaction system of step S3 is 0.1 to 100mM (e.g., 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90 mM).
In some embodiments, the reaction temperature of step S3 is 20 to 45 ℃ (e.g., 25 ℃, 30 ℃, 35 ℃, 40 ℃).
In some embodiments, the reaction time of step S3 is 2 to 60min (e.g., 10min, 20min, 30min, 40min, 50 min).
S4, adding fluorogenic substrate phthalic aldehyde and sodium sulfite into each group of products obtained in the step S3 to react.
In some embodiments, the final concentration of the phthalic aldehyde in the reaction system of step S4 is 1 to 100mM (e.g., 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90 mM).
In some embodiments, the final concentration of sodium sulfite in the reaction system of step S4 is 1 to 200mM (e.g., 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190 mM).
In some embodiments, the reaction temperature of step S4 is 20 to 65 ℃ (e.g., 30 ℃, 40 ℃, 50 ℃).
In some embodiments, the reaction time of step S4 is 2 to 90min (e.g., 10min, 20min, 30min, 40min, 50min, 60min, 70min, 80 min).
S5, detecting fluorescent signals of the products of the step S4, and judging the content of the target virus in the sample to be detected according to the intensity of the fluorescent signals.
In some embodiments, the fluorescent signal is detected at a wavelength of 365 nm.
In some embodiments, the control group comprises a positive control group and/or a negative control group, wherein the negative control group does not contain the target sequence, and the positive control group contains the target sequence.
In some embodiments, when the fluorescence signal intensity of the detection group is less than or equal to that of the negative control group, it is determined that the target virus is contained in the sample to be tested.
In some embodiments, the control group comprises a plurality of positive control groups containing different concentrations of target sequences; preparing a standard curve according to the target sequence concentration and the fluorescence signal intensity of each positive control group; and calculating the content of the target virus in the sample to be detected according to the standard curve and the fluorescence signal intensity of the detection group.
In some embodiments, the target sequence is the sequence shown as SEQ ID No.8 or a sequence having greater than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology thereto; and the single-stranded DNA probe is shown as SEQ ID NO.1, and the control group comprises a plurality of target sequences with the final concentration of 10 -12.5 And 10 -14 Positive control group between M; and the standard curve is y= -392.1x+3003, r 2 = 0.9905, where x is SARS-CoV-2RNA concentration and y is the corresponding fluorescence intensity.
In some embodiments, a method of amplification-free detection of a target virus comprises the following steps S1 to S5.
S1, adding a single-stranded DNA probe containing P1 complementary to a target sequence, P2 complementary to part of P1 and two P3 containing mismatched bases into a nitrate buffer solution at a molar ratio of 1 (1) to 100) for incubation at 60 ℃ to 90 ℃ for 2 min to 10min, cooling to room temperature and standing for 5 min to 90min to prepare a nucleic acid-metal ion probe,
wherein two of said P3 s are located at both ends of said single-stranded DNA probe, respectively;
two of the P3's complement each other to form a first double-stranded region by bases other than the mismatched base contained therein;
The metal ion is chimeric in a mismatched base pair of the non-complementary pairing of the first double-stranded region;
said P2 is complementary to a portion of said P1 to form a second double-stranded region; and is also provided with
The length of P1 is greater than the sum of the lengths of P2 and P3, i.e. P1 > P2+P3,
the target sequence is selected from genes specific for the target virus.
S2, adding the sample to be detected and the control sample (positive control sample and/or negative control sample) and the nucleic acid-metal ion probe into a nitrate buffer solution to conduct DNA probe hybridization (comprising the steps of annealing at 65-90 ℃ for 5-10 min and extending at 20-60 ℃ for 5-60 min) to serve as a detection group and a control group.
S3, adding urease with the final concentration of 0.1-100 mM and urea into each group of products obtained in the step S2, and reacting at 20-45 ℃ for 2-60 min, wherein the concentration ratio of the urease to the concentration of the metal ions is 1 (1-100).
S4, adding a fluorogenic substrate phthalaldehyde with the final concentration of 1-100 mM and sodium sulfite with the final concentration of 1-200 mM into each group of products obtained in the step S3, and reacting for 2-90 min at 20-65 ℃.
S5, detecting fluorescent signals of the products of the step S4, and judging the content of the target virus in the sample to be detected according to the intensity of the fluorescent signals.
Judging the content of the target virus in the sample to be tested according to the following method 1 or method 2:
method 1: and when the fluorescence signal intensity of the detection group is smaller than or equal to that of the negative control group, judging that the sample to be detected contains the target virus.
Method 2: preparing a standard curve according to the target sequence concentration and the fluorescence signal intensity of each positive control group; and calculating the content of the target virus in the sample to be detected according to the standard curve and the fluorescence signal intensity of the detection group.
An embodiment of the second aspect of the present application proposes an amplification-free kit for detecting a target virus, comprising a nucleic acid-metal ion probe, urease, urea and a fluorogenic substrate,
wherein the nucleic acid-metal ion probe is prepared by chimerism of a single-stranded DNA probe comprising P1 complementary to the target sequence, P2 complementary to part of P1 and two P3 comprising mismatched bases with a metal ion,
wherein two of said P3 s are located at both ends of said single-stranded DNA probe, respectively;
two of the P3's complement each other to form a first double-stranded region by bases other than the mismatched base contained therein;
the metal ion is chimeric in a mismatched base pair of the non-complementary pairing of the first double-stranded region;
Said P2 is complementary to a portion of said P1 to form a second double-stranded region; and is also provided with
The length of P1 is greater than the sum of the lengths of P2 and P3, namely P1 > P2+P3,
wherein the target sequence is selected from a specific gene of the target virus.
In some embodiments, when the metal ion is silver, the P3 is 3' -C- (AC) n -5 'and 5' -C- (TG) n -3', n is 1 to 4.
In some embodiments, when the metal ion is mercury ion, the P3 is 3' -T- (CA) n -5 'and 5' -T- (GT) n -3', n is 1 to 4.
In some embodiments, the target virus is wild-type SARS-CoV-2.
In some embodiments, the target virus is a BA.5.2 variant or a BF.7 variant of SARS-CoV-2.
In some embodiments, the target virus is wild-type SARS-CoV-2 and the target sequence is the sequence shown as SEQ ID No.8 or a sequence having greater than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology thereto.
In some embodiments, the target virus is a ba.5.2 variant of SARS-CoV-2, the target sequence is the sequence shown as SEQ ID No.9 or a sequence having greater than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology thereto; or a sequence as shown in SEQ ID No.10 or a sequence having a homology of more than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) thereto.
In some embodiments, the target virus is a bf.7 variant of SARS-CoV-2, the target sequence is the sequence set forth in SEQ ID No.11 or a sequence having greater than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology thereto; or a sequence as set forth in SEQ ID NO.12 or a sequence having greater than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology thereto; or a sequence as set forth in SEQ ID NO.13 or a sequence having more than 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology thereto.
In some embodiments, the target virus is wild-type SARS-CoV-2 and the single stranded DNA probe has the sequence shown in SEQ ID NO. 1; and the metal ion is silver ion, or the sequence SEQ ID NO.2 of the single-stranded DNA probe; and the metal ion is a mercury ion.
In some embodiments, the target virus is a BA.5.2 variant of SARS-CoV-2, and the single stranded DNA probe has the sequence shown in SEQ ID NO.3 or SEQ ID NO. 4; and the metal ion is silver ion.
In some embodiments, the target virus is a BF.7 variant of SARS-CoV-2, and the single stranded DNA probe has a sequence as shown in SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO. 7; and the metal ion is silver ion.
In some embodiments, the fluorogenic substrate comprises phthalaldehyde and sodium sulfite.
In some embodiments, the ratio of the concentration of urease to the concentration of metal ions chimeric in the nucleic acid-metal ion probe is 1 (1 to 100).
Embodiments of the third aspect of the present application provide the use of a kit according to the embodiments of the second aspect described above for detecting a viral infection.
In this application, the term "comprising" is used in an open-ended fashion, i.e., including what is indicated by the invention, but not excluding other aspects.
The term "sequence identity" means: two or more sequences are identical if they have the same length and order of nucleotides or amino acids. The percentage of identity generally describes the degree to which two sequences are identical, i.e., it generally describes the percentage of nucleotides at their sequence positions that correspond to the same nucleotides of the reference sequence. To determine the degree of identity, the sequences to be compared are considered to have the same length, i.e. the length of the longest sequence of the sequences to be compared. This means that a first sequence consisting of 8 nucleotides/amino acids has 80% identity with a second sequence consisting of 10 nucleotides/amino acids comprising the first sequence. In other words, in the context of the present invention, sequence identity preferably relates to the percentage of nucleotides/amino acids of a sequence having the same position in two or more sequences having the same length. Caps are generally considered to be in different positions, regardless of their actual position in the alignment.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples and are carried out according to the techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, code Huang Peitang et al, molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Unless otherwise indicated, the quantitative analysis tests in the following examples were all set up in triplicate, and the results averaged.
TABLE 1 nucleic acid-metal ion probe sequences used in the examples of the present application
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Example 1 test urease catalyzed NH production 4 + Results of formation of strongly fluorescent products
In this example, the urease concentration was adjusted and tested for catalytic NH production by urease 4 + With fluorogenic substrates OPA and Na 2 SO 3 The reaction produced the result of the strong fluorogenic substrate Isoindole-1-sulfonate.
The specific test method is as follows:
concentration of 4. Mu.L to 10 -14 ～10 -11 M urease, 4. Mu.L of 10mM urea solution and 4. Mu.L of nitrate solution (200 mM NaNO) 3 ，50mM Mg(NO 3 ) 2 pH=7.0) was placed in a PCR tube and reacted at 42℃for 10min, after which 4. Mu.L of OPA (10 mM) and 4. Mu.L of Na were sequentially added 2 SO 3 (10 mM), 20. Mu.L of deionized water was added thereto and the mixture was made up to 40. Mu.L, 60℃and reacted for 10 minutes. Excitation at 365nm wavelength, fluorescence signal acquisition, and test results are shown in FIG. 2.
Example 2 results of testing for fluorescence quenching by inhibition of urease activity by Ag (I)
In this example, ag (I) was tested for inhibition of urease activity, inhibition of NH 4 + And the fluorescence signal is quenched.
The specific test method is as follows:
concentration of 4. Mu.L to 10 -11 ～10 -8 M Ag (I) and 4. Mu.L concentration of 10 -11 M urease was mixed and 4. Mu.L of nitrate solution (200 mM NaNO was added 3 ，50mM Mg(NO 3 ) 2 pH=7.0), followed by adding 4. Mu.L of 10mM urea solution and placing in a PCR tube, reacting at 42℃for 10min, followed by adding 4. Mu.L of OPA (10 mM), 4. Mu.L of Na in sequence 2 SO 3 (10 mM), 16. Mu.L of deionized water was added thereto and the mixture was made up to 40. Mu.L, 60℃and reacted for 10 minutes. Excitation at 365nm wavelength, fluorescence signal acquisition, and test results are shown in FIG. 3.
Example 3 results of testing Ag (I) intercalating Single-stranded DNA Probe into nucleic acid-Metal ion Probe
In this example, ag (I) was tested for intercalation into single-stranded DNA to form a D nucleic acid-metal ion probe, thereby blocking the inhibitory activity of Ag (I) on urease and ensuring smooth synthesis of a strong fluorogenic substrate.
The specific test method is as follows:
4. Mu.L of single-stranded DNA probe SEQ ID NO.1 (10) -11 ～10 -8 M) (experimental group) or without addition of single stranded DNA (as control) and 4. Mu.L concentration of 10 -10 M Ag (I) was added to 4. Mu.L of nitrate solution (200 mM NaNO) 3 ，50mM Mg(NO 3 ) 2 Ph=7.0), annealing at 90 ℃ for 3min, room temperature reaction for 17min, followed by the addition of 4 μl of 10% strength -11 M urease was mixed, added, followed by 4. Mu.L of 10mM urea solution and placed in a PCR tube for 10min at 42℃followed by 4. Mu.L of OPA (10 mM), 4. Mu.L of Na 2 SO 3 (10 mM), 12. Mu.L of deionized water was added thereto and the mixture was made up to 40. Mu.L, 60℃and reacted for 10 minutes. Excitation at 365nm wavelength, fluorescence signal acquisition, and test results are shown in FIG. 4.
EXAMPLE 4 detection of wild-type SARS-CoV-2N Gene Using Single-stranded DNA Probe SEQ ID NO.1
In this example, the SARS-CoV-2N gene was detected using single stranded DNA probe SEQ ID NO. 1.
The specific test method is as follows:
before adding urease, 4. Mu.L of the solution with a concentration of 10 was added -8 The SARS-CoV-2N gene of M (experimental group, labeled +N gene) or SARS-CoV-2N gene (control group, labeled-N gene) was not added to the reaction system of example 3, and the procedure of example 3 was repeated.
The test fluorescence spectrum results are shown in figure 5, and the experimental results prove that the nucleic acid-metal ion probe successfully recognizes target viral RNA, a double-chain structure in the probe is opened, ag (I) is released into a reaction system, and the formation of Isoindole-1-sulfonate is inhibited, so that fluorescence quenching is caused.
EXAMPLE 5 Standard Curve for detecting wild-type SARS-CoV-2N Gene Using Single-stranded DNA Probe SEQ ID NO.1
In this example, a standard curve for detecting SARS-CoV-2N gene was prepared.
The specific test method is as follows:
before adding urease, 4. Mu.L of the concentration series was 10 -14 ～10 -7 The SARS-CoV-2N gene of M was added to the reaction system of example 3 (concentration of single-stranded DNA probe: 10) -11 M) and repeating the operation of example 3, the test results are shown in fig. 6A. And (3) establishing a standard curve with the viral RNA concentration as an abscissa and the fluorescence intensity as an ordinate according to the detection result, wherein a curve equation is y= -392 < 1x+3003, and R 2 = 0.9905, where x is SARS-CoV-2RNA concentration and y is the corresponding fluorescence intensity. Experimental results demonstrate that as viral RNA concentration increases, quenching of the fluorescent signal increases gradually and at 10 -15 ～10 -9 The detection limit of the kit is calculated to be as low as 1.7fM in linear relation with the fluorescence signal under M.
Extracting RNA of SARS-CoV-2 virus sample, repeating the operation of example 3 with RNA solution, collecting fluorescence intensity value under excitation wavelength 365nm and emission wavelength 395-475 nm, and taking into standard curve equation to obtain RNA content of SARS-CoV-2.
EXAMPLE 6 detection of SARS-CoV-2BA.5.2 variant Using Single-stranded DNA probes SEQ ID NO.3 and SEQ ID NO.4
In this example, single stranded DNA probes SEQ ID NO.03 and SEQ ID NO.04 were used to detect SARS-CoV-2BA.5.2 variant.
The specific test method is as follows:
before adding urease, 4. Mu.L of the solution was added at a concentration of 10 -8 And 10 -9 The SARS-CoV-2BA.5.2 variant and wild type of M are respectively added into the reaction system of the example 3 (wherein the single-stranded DNA probes are SEQ ID NO.3 or SEQ ID NO.4 respectively), the operation of the example 3 is repeated, the test result is shown in figure 7, and the experimental result proves that the nucleic acid-metal ion probe provided by the embodiment of the application has high specificity and can accurately distinguish the SARS-CoV-2 variant from the wild type.
Comparative example 1 comparative example the kit provided in the example of the present invention was compared to the previously disclosed visual urease-based kit for experimental design differences
In this comparative example, the experimental design difference between the kit provided in the example of the present invention and the previously disclosed invention patent based on urease, which catalyzes urease to monitor pH change, namely a visual nucleic acid detection kit (application publication number: CN 112501248A) was compared.
The method proposed by the patent of the invention (a visual nucleic acid detection kit) is mainly characterized by the following two design differences:
(1) The embodiment of the invention provides a nucleic acid-metal ion probe recognition target sequence embedded with metal ions, which has simpler design and more stable hybridization compared with a DNA double-strand recognition probe embedded with metal ions related to the prior disclosed patent (a visual nucleic acid detection kit). Compared with the ratio of the final concentration substances of the substrate chain and the mismatched chain of the prior patent is 1:1, the operation process does not need to normalize the ratio of the two chains, and the operation is simpler.
(2) The invention provides the preparation method of the crystal structure by OPA, na 2 SO 3 Fluorescent detection kit for fluorogenic substrate is prepared by catalyzing NH generated by urea with urease 4 + With fluorogenic substrates OPA and Na 2 SO 3 The reaction is performed to generate a chemical reaction of a strong fluorogenic substrate Isoindole-1-sulfonate, and a fluorescent signal is detected. Is OPA and Na 2 SO 3 As an ammonia fluorogenic substrate for the first use in nucleic acid detection.
Comparative example 2 comparative example the kit provided in the example of the present invention provides a difference in detection sensitivity over the previously disclosed urease-based visualization kit
In this comparative example, the difference in detection sensitivity of the visual nucleic acid detection kit (application publication number: CN 112501248A) provided by the examples of the present invention was compared with that of the previously disclosed invention based on urease, which catalyzes the detection of pH changes by urease.
The minimum detection limit of the method proposed by the patent of the invention (a visual nucleic acid detection kit) is 3.23pM as shown in figure 6B, and OPA and Na are adopted in the invention 2 SO 3 Minimum detection limit for fluorescence detection kit for fluorogenic substrate As shown in example 5, FIG. 6A, target sequences as low as 1.7fM can be detected. The detection sensitivity is obviously improved, and more detection can be realizedLow load of virus, which would be beneficial for early detection of virus, for management control of the pandemic of viral infection.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. An amplification-free detection method of a target virus, comprising the steps of:
s1, chimeric a single-stranded DNA probe containing P1 complementary to a target sequence, P2 complementary to part of P1 and two P3 containing mismatched bases with metal ions to prepare a nucleic acid-metal ion probe,
wherein two of said P3 s are located at both ends of said single-stranded DNA probe, respectively;
two of the P3's complement each other to form a first double-stranded region by bases other than the mismatched base contained therein;
the metal ion is chimeric in a mismatched base pair of the non-complementary pairing of the first double-stranded region;
said P2 is complementary to a portion of said P1 to form a second double-stranded region;
the length of P1 is greater than the sum of the lengths of P2 and P3; and is also provided with
The target sequence is selected from a specific gene of the target virus;
s2, respectively hybridizing a sample to be detected and a control sample with the nucleic acid-metal ion probe to obtain a DNA probe hybrid, wherein the DNA probe hybrid is used as a detection group and a control group;
S3, adding urease and urea into the products obtained in the step S2 to react;
s4, adding fluorogenic substrate phthalic aldehyde and sodium sulfite into each group of products obtained in the step S3 to react; and
s5, detecting fluorescent signals of the products of the step S4, and judging the content of the target virus in the sample to be detected according to the intensity of the fluorescent signals.
2. The detection method according to claim 1, wherein the target virus is a novel coronavirus and the target sequence is selected from the group consisting of an N gene and/or an S gene of the novel coronavirus;
alternatively, the target virus is wild-type SARS-CoV-2 and the target sequence is selected from the group consisting of the N gene of the target virus;
alternatively, the target virus is a ba.5.2 variant or bf.7 variant of SARS-CoV-2, and the target sequence is selected from the S gene of the target virus;
alternatively, the target virus is wild-type SARS-CoV-2, and the target sequence is the sequence as shown in SEQ ID NO.8 or a sequence having more than 85% homology thereto;
alternatively, the target virus is a BA.5.2 variant of SARS-CoV-2, and the target sequence is a sequence as shown in SEQ ID NO.9 or a sequence having homology of more than 85% thereto; or a sequence shown as SEQ ID NO.10 or a sequence with the homology of more than 85 percent;
Alternatively, the target virus is a BF.7 variant of SARS-CoV-2, and the target sequence is a sequence as shown in SEQ ID NO.11 or a sequence having homology of more than 85% thereto; or a sequence shown as SEQ ID NO.12 or a sequence with the homology of more than 85 percent; or a sequence shown as SEQ ID NO.13 or a sequence with the homology of more than 85 percent;
alternatively, when the metal ion is silver, the P3 is 3' -C- (AC) n -5 'and 5' -C- (TG) n -3', n is 1 to 4;
alternatively, when the metal ion is mercury ion, the P3 is 3' -T- (CA) n -5 'and 5' -T- (GT) n -3', n is 1 to 4;
alternatively, the target virus is wild-type SARS-CoV-2; the target sequence is a sequence shown as SEQ ID NO.8 or a sequence with the homology of more than 85 percent; the single-stranded DNA probe is a sequence shown as SEQ ID NO.1 or SEQ ID NO. 2;
alternatively, the target virus is a ba.5.2 variant of SARS-CoV-2, the target sequence is the sequence shown as SEQ ID No.9 or a sequence having greater than 85% homology thereto and the single stranded DNA probe is the sequence shown as SEQ ID No. 3; or the target sequence is a sequence shown as SEQ ID NO.10 or a sequence with homology of more than 85 percent and the single-stranded DNA probe is a sequence shown as SEQ ID NO. 4;
Alternatively, the target virus is a bf.7 variant of SARS-CoV-2, the target sequence is the sequence shown as SEQ ID No.11 or a sequence having greater than 85% homology thereto and the single-stranded DNA probe is the sequence shown as SEQ ID No. 5; or the target sequence is a sequence shown as SEQ ID NO.12 or a sequence with homology of more than 85% and the single-stranded DNA probe is a sequence shown as SEQ ID NO. 6; or the target sequence is a sequence shown as SEQ ID NO.13 or a sequence with homology of more than 85% and the single-stranded DNA probe is a sequence shown as SEQ ID NO. 7.
3. The detection method according to claim 1, wherein the step S1 comprises adding the single-stranded DNA probe and metal ions to a nitrate buffer for incubation;
optionally, the step S1 further comprises cooling to room temperature and standing after the incubation is completed;
optionally, in the step S2, the DNA probe hybridization includes annealing and extension steps;
optionally, the control group comprises a positive control group and/or a negative control group, wherein the negative control group does not contain the target sequence, and the positive control group contains the target sequence.
4. The detection method according to claim 3, wherein the reaction system of step S1 comprises sodium nitrate and/or magnesium nitrate in a final concentration of 10 to 200 mM;
Optionally, in the step S1, the molar ratio of the single-stranded DNA probe to the metal ion is 1 (0.1 to 100), preferably 1 (1 to 10);
optionally, in the step S1, the incubation time is 2 to 10min;
optionally, in the step S1, the incubation temperature is 60 to 90 ℃;
optionally, in the step S1, the standing time is 5 to 90min;
optionally, the reaction system of step S2 comprises sodium nitrate and/or magnesium nitrate in a final concentration of 10 to 200 mM;
optionally, in the step S2, the temperature of the annealing step is 65 to 90 ℃;
optionally, in the step S2, the annealing step is performed for 0.5 to 10min;
optionally, in the step S2, the temperature of the extending step is 20 to 60 ℃;
optionally, in the step S2, the time of the extending step is 5 to 60min;
alternatively, in the reaction system of step S3, the ratio of the concentration of urease to the concentration of metal ions chimeric in the nucleic acid-metal ion probe is 1 (1 to 100);
optionally, in the reaction system of step S3, the final concentration of urea is 0.1 to 100mM;
optionally, the reaction temperature of step S3 is 20 to 45 ℃;
Optionally, the reaction time of the step S3 is 2 to 60min;
alternatively, in the reaction system of step S4, the final concentration of the phthalic aldehyde is 1 to 100mM;
alternatively, the final concentration of sodium sulfite in the reaction system of step S4 is 1 to 200mM;
optionally, the reaction temperature of step S4 is 20 to 65 ℃;
optionally, the reaction time of the step S4 is 2 to 90min;
optionally, the fluorescent signal is detected at a wavelength of 365 nm.
5. The detection method according to claim 1, wherein the control group comprises a negative control group, wherein the negative control group does not contain the target sequence; when the fluorescence signal intensity of the detection group is smaller than or equal to that of the negative control group, judging that the sample to be detected contains target viruses;
optionally, the control group comprises a plurality of positive control groups containing different concentrations of target sequences; preparing a standard curve according to the target sequence concentration and the fluorescence signal intensity of each positive control group; and calculating the content of the target virus in the sample to be detected according to the standard curve and the fluorescence signal intensity of the detection group.
6. An amplification-free kit for detecting a target virus comprises a nucleic acid-metal ion probe, urease, urea and a fluorogenic substrate,
Wherein the nucleic acid-metal ion probe is prepared by chimerism of a single-stranded DNA probe comprising P1 complementary to the target sequence, P2 complementary to part of P1 and two P3 comprising mismatched bases with a metal ion,
wherein two of said P3 s are located at both ends of said single-stranded DNA probe, respectively;
two of the P3's complement each other to form a first double-stranded region by bases other than the mismatched base contained therein;
the metal ion is chimeric in a mismatched base pair of the non-complementary pairing of the first double-stranded region;
said P2 is complementary to a portion of said P1 to form a second double-stranded region;
the length of P1 is greater than the sum of the lengths of P2 and P3; and is also provided with
The target sequence is selected from genes specific for the target virus.
7. The kit of claim 6, wherein the target virus is a novel coronavirus and the target sequence is selected from the group consisting of an N gene and/or an S gene of the novel coronavirus;
alternatively, the target virus is wild-type SARS-CoV-2 and the target sequence is selected from the group consisting of the N gene of the target virus;
alternatively, the target virus is a ba.5.2 variant or bf.7 variant of SARS-CoV-2 and the target sequence is selected from the S gene of the target virus.
8. The kit of claim 6, wherein the target virus is wild-type SARS-CoV-2 and the target sequence is the sequence shown as SEQ ID No.8 or a sequence having greater than 85% homology thereto;
Alternatively, the target virus is a BA.5.2 variant of SARS-CoV-2, and the target sequence is a sequence as shown in SEQ ID NO.9 or a sequence having homology of more than 85% thereto; or a sequence shown as SEQ ID NO.10 or a sequence with the homology of more than 85 percent;
alternatively, the target virus is a BF.7 variant of SARS-CoV-2, and the target sequence is a sequence as shown in SEQ ID NO.11 or a sequence having homology of more than 85% thereto; or a sequence shown as SEQ ID NO.12 or a sequence with the homology of more than 85 percent; or a sequence shown as SEQ ID NO.13 or a sequence with the homology of more than 85 percent;
alternatively, when the metal ion is silver, the P3 is 3' -C- (AC) n -5 'and 5' -C- (TG) n -3', n is 1 to 4;
alternatively, when the metal ion is mercury ion, the P3 is 3' -T- (CA) n -5 'and 5' -T- (GT) n -3', n is 1 to 4.
9. The kit of claim 6, wherein the target virus is wild-type SARS-CoV-2 and the single stranded DNA probe has the sequence shown in SEQ ID NO. 1; and the metal ion is silver ion, or the sequence SEQ ID NO.2 of the single-stranded DNA probe; and the metal ion is mercury ion;
Alternatively, the target virus is BA.5.2 variant of SARS-CoV-2, and the sequence of the single-stranded DNA probe is shown as SEQ ID NO.3 or SEQ ID NO. 4; and the metal ion is silver ion;
alternatively, the target virus is BF.7 variant of SARS-CoV-2, and the sequence of the single-stranded DNA probe is shown as SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO. 7; and the metal ion is silver ion;
optionally, the fluorogenic substrate comprises phthalaldehyde and sodium sulfite;
alternatively, the ratio of the concentration of the urease to the concentration of the metal ion chimeric in the nucleic acid-metal ion probe is 1 (1 to 100).
10. Use of a kit according to any one of claims 6 to 9 for detecting a viral infection.
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