Compositions, kits, uses thereof, and methods

Abstract

A composition, kit, use and method thereof. The primer and the probe are designed on the specificity of the regulatory gene SNCA of the Parkinson's disease and on the conservative segment sequences in the wild type and the mutant type, the conditions of leak detection and false positive detection can be effectively avoided, and the primer and the probe have extremely high detection sensitivity which can reach 100 copies / mL. Compared with the traditional technology, the technology can realize early diagnosis of PD, adopts a multiple amplification technology, can effectively reduce the detection cost, avoids crosstalk, and is simple and convenient to operate and good in specificity.

Description

Technical Field

[0001] The present application generally relates to the field of biotechnology, and more particularly to a composition, a kit, uses thereof, and a method for detecting SNCA gene using qPCR. Background Art

[0002] Parkinson's disease (PD) is a chronic progressive disease that is currently incurable. It was first described by the British Parkinson in 1817. With the progress of research on PD, its clinical characteristics have become one of the criteria for its diagnosis. It is clinically manifested as motor symptoms such as resting tremor, bradykinesia, muscle rigidity and posture gait disorders and non-motor symptoms such as constipation, sleep disorders, mental and behavioral abnormalities, etc. The diagnosis of Parkinson's syndrome is based on three core motor symptoms, namely, bradykinesia and at least one of the two symptoms of resting tremor or muscle rigidity. The above symptoms must be obvious and unrelated to other interfering factors. The examination of all core motor symptoms must be carried out according to the method described in the Unified Parkinson's Disease Rating Scale (UPDRS). The specific assessment scale is relatively complex and the symptom assessment is performed by the doctor. Since the pathogenesis of PD is still unclear, it may be related to genetic factors, mitochondrial dysfunction, oxidative stress, immune abnormalities, cell apoptosis, environmental factors, etc. Once PD is diagnosed, it cannot be reversed, so early detection and early intervention are the most effective and urgent methods in the medical community. Finding a method for early diagnosis of PD is an urgent problem to be solved in this field. Summary of the invention

[0003] Based on this, the present application provides a composition comprising one or more of the following:

[0004] An upstream decoy oligonucleotide and a downstream decoy oligonucleotide, wherein the upstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 1, and the downstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 2;

[0005] An upstream decoy oligonucleotide and a downstream decoy oligonucleotide, wherein the upstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:3 and the downstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:4; and

[0006] An upstream bait oligonucleotide and a downstream bait oligonucleotide, wherein the upstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:5, and the downstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:6.

[0007] In another aspect, the present application also provides a kit comprising the composition described herein.

[0008] In another aspect, the present application also provides use of the composition described herein or the kit described herein in SNCA gene detection.

[0009] In another aspect, the present application also provides use of the composition described herein or the kit described herein in early diagnosis of PD.

[0010] In another aspect, the present application also provides a method for detecting the SNCA gene using qPCR, comprising:

[0011] The composition described herein or the kit described herein is used to perform qPCR on the sample to be tested on a qPCR instrument, and the presence or absence of the SNCA gene in the sample is determined based on the result of qPCR.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] (1) This application provides a new combination of primers and probes that can be used for high-sensitivity detection of the SNCA gene, up to 100 copies / mL;

[0014] (2) Compared with traditional technologies, this application can realize early diagnosis of PD, adopt multiple amplification technology, effectively reduce detection costs, avoid crosstalk, and is easy to operate and has good specificity;

[0015] (3) Selecting conserved regions of the genome and designing specific primer probes can effectively avoid missed detections and false positive detections.

[0016] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0018] Figure 1 This is the qPCR amplification result in Example 1 of the present application.

[0019] Figure 2 These are the results of qPCR amplification using different conditions in Example 2 of the present application.

[0020] Figure 3 The results of qPCR amplification of different concentrations of standard products in Example 3 of the present application are shown in Table 1. 3 The samples with 100 copies / mL, 500 copies / mL, 200 copies / mL, and 100 copies / mL were selected. The sample without peak was the negative control of GAPDH gene. DETAILED DESCRIPTION

[0021] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the application belongs. When a certain amount, concentration or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper numerical limit and a preferred lower numerical limit, it should be understood that it is equivalent to specifically revealing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within the range.

[0022] The terms "about" and "approximately" when used with a numerical variable generally refer to the value of that variable and all values ​​of that variable are within experimental error (e.g., within a 95% confidence interval of the mean) or within ±10% of the specified value, or a wider range.

[0023] The expression "comprising" or its synonymous similar expressions "including", "containing" and "having" etc. are open-ended and do not exclude additional unrecited elements, steps or ingredients. The expression "consisting of excludes any element, step or ingredient not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps or ingredients, plus the optional elements, steps or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It should be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of".

[0024] The expression "at least one" or "one or more" means 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0025] Unless otherwise specified, “nM” in this article refers to “n mol / L”, “μM” refers to “μmol / L”, and “mM” refers to “mmol / L”.

[0026] In one aspect, the present application provides a composition comprising one or more of the following:

[0027] An upstream decoy oligonucleotide and a downstream decoy oligonucleotide, the upstream decoy oligonucleotide being derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 1, and the downstream decoy oligonucleotide being derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 2;

[0028] an upstream decoy oligonucleotide and a downstream decoy oligonucleotide, the upstream decoy oligonucleotide being derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence set forth in SEQ ID NO:3 and the downstream decoy oligonucleotide being derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence set forth in SEQ ID NO:4; and

[0029] An upstream bait oligonucleotide and a downstream bait oligonucleotide, the upstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:5, and the downstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:6.

[0030] In some embodiments, the composition includes one or more of the following:

[0031] An upstream bait oligonucleotide and a downstream bait oligonucleotide, the upstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 1, and the downstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 2;

[0032] An upstream bait oligonucleotide and a downstream bait oligonucleotide, the upstream bait oligonucleotide being derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:3, and the downstream bait oligonucleotide being derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:4; and

[0033] An upstream bait oligonucleotide and a downstream bait oligonucleotide, the upstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:5, and the downstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:6.

[0034] In some embodiments, the composition includes one or more of the following:

[0035] An upstream bait oligonucleotide and a downstream bait oligonucleotide, wherein the upstream bait oligonucleotide is the sequence shown in SEQ ID NO: 1, and the downstream bait oligonucleotide is the sequence shown in SEQ ID NO: 2;

[0036] An upstream bait oligonucleotide and a downstream bait oligonucleotide, wherein the upstream bait oligonucleotide is the sequence shown in SEQ ID NO: 3, and the downstream bait oligonucleotide is the sequence shown in SEQ ID NO: 4; and

[0037] The upstream bait oligonucleotide and the downstream bait oligonucleotide, the upstream bait oligonucleotide is the sequence shown in SEQ ID NO:5, and the downstream bait oligonucleotide is the sequence shown in SEQ ID NO:6.

[0038] In some embodiments, the composition includes the following:

[0039] An upstream bait oligonucleotide and a downstream bait oligonucleotide, wherein the upstream bait oligonucleotide is the sequence shown in SEQ ID NO: 1, and the downstream bait oligonucleotide is the sequence shown in SEQ ID NO: 2;

[0040] An upstream bait oligonucleotide and a downstream bait oligonucleotide, wherein the upstream bait oligonucleotide is the sequence shown in SEQ ID NO: 3, and the downstream bait oligonucleotide is the sequence shown in SEQ ID NO: 4; and

[0041] The upstream bait oligonucleotide and the downstream bait oligonucleotide, the upstream bait oligonucleotide is the sequence shown in SEQ ID NO:5, and the downstream bait oligonucleotide is the sequence shown in SEQ ID NO:6.

[0042] In various aspects disclosed elsewhere herein, the decoy oligonucleotide may be referred to as a "primer," "oligonucleotide primer," or "nucleic acid primer." The primer may be a deoxyribonucleic acid or a ribonucleic acid. The primer may comprise one or more non-natural nucleotides. The non-natural nucleotide may be, for example, deoxyinosine. The primer may be a forward primer. The primer may be a reverse primer. The primer may be about 5 to about 50 nucleotides in length. The primer may be at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 or more base pairs in length. The primer may be at most 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 5 nucleotides in length. The primer can be about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 base pairs in length.

[0043] A pair of primers may include a forward primer (upstream primer) and a reverse primer (downstream primer). The forward primer may be configured to hybridize with a first region (e.g., 3' end) of a nucleic acid sequence, and the reverse primer may be configured to hybridize with a second region (e.g., 5' end) of the nucleic acid sequence, thereby being configured to amplify the nucleic acid sequence under conditions sufficient for nucleic acid amplification. Different pairs of primers may be configured to amplify different target nucleic acid sequences.

[0044] In some embodiments, the composition includes one or more of the following:

[0045] An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 1, the downstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 2, and the probe sequence is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 7;

[0046] an upstream bait oligonucleotide, a downstream bait oligonucleotide, and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence set forth in SEQ ID NO:3, the downstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence set forth in SEQ ID NO:4, and the probe sequence is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence set forth in SEQ ID NO:8; and

[0047] An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:5, the downstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:6, and the probe sequence is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:9.

[0048] In some embodiments, the composition includes one or more of the following:

[0049] An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 1, the downstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 2, and the probe sequence is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 7;

[0050] An upstream bait oligonucleotide, a downstream bait oligonucleotide, and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:3, the downstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:4, and the probe sequence is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:8; and

[0051] An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:5, the downstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:6, and the probe sequence is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:9.

[0052] In some embodiments, the composition includes one or more of the following:

[0053] An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is the sequence shown in SEQ ID NO: 1, the downstream bait oligonucleotide is the sequence shown in SEQ ID NO: 2, and the probe sequence is the sequence shown in SEQ ID NO: 7;

[0054] An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is the sequence shown in SEQ ID NO: 3, the downstream bait oligonucleotide is the sequence shown in SEQ ID NO: 4, and the probe sequence is the sequence shown in SEQ ID NO: 8; and

[0055] The upstream bait oligonucleotide, the downstream bait oligonucleotide and the probe sequence, the upstream bait oligonucleotide is the sequence shown in SEQ ID NO:5, the downstream bait oligonucleotide is the sequence shown in SEQ ID NO:6, and the probe sequence is the sequence shown in SEQ ID NO:9.

[0056] In some embodiments, the composition includes the following:

[0057] An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is the sequence shown in SEQ ID NO: 1, the downstream bait oligonucleotide is the sequence shown in SEQ ID NO: 2, and the probe sequence is the sequence shown in SEQ ID NO: 7;

[0058] An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is the sequence shown in SEQ ID NO: 3, the downstream bait oligonucleotide is the sequence shown in SEQ ID NO: 4, and the probe sequence is the sequence shown in SEQ ID NO: 8; and

[0059] The upstream bait oligonucleotide, the downstream bait oligonucleotide and the probe sequence, the upstream bait oligonucleotide is the sequence shown in SEQ ID NO:5, the downstream bait oligonucleotide is the sequence shown in SEQ ID NO:6, and the probe sequence is the sequence shown in SEQ ID NO:9.

[0060] In some embodiments, the present application provides primer and probe combinations as shown in the following table:

[0061]

[0062]

[0063] In some embodiments, the probe sequence is 15-30 bp in length, and its 5' end is not G; the Tm value of the probe sequence is higher than that of the upstream primer and the downstream primer. Preferably, the Tm value of the probe sequence is 68-72° C. In some embodiments, the probe can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more nucleotides in length.

[0064] In this article, "detection probe", "probe" and "oligonucleotide probe" are used interchangeably. The probe can be a nucleic acid (e.g., DNA, RNA, etc.). The probe can include a region complementary to a region of the target nucleic acid. The concentration of the probe can make it excessive relative to other components in the detection mixture. The probe can be a signal generating nucleic acid probe. The signal generating nucleic acid probe can include the characteristics of the probe as described herein, as well as the characteristics of generating a signal after applying a stimulus, modifying the probe or hydrolyzing the probe. For example, the probe can be hydrolyzed by the exonuclease activity of a polymerase, thereby generating a signal. The signal generating nucleic acid probe can generate any signal as described elsewhere in this article. For example, the signal generating probe can generate a fluorescent signal. The signal generating probe can include a signal tag. The signal tag can generate a signal. The signal tag can generate a signal in response to a reaction or stimulus. For example, the signal tag can generate a signal after being degraded by exonuclease activity.

[0065] In some embodiments, a signal can be generated while the probe is hybridized with the nucleic acid region. For example, a probe (e.g., a molecular beacon) can generate a signal (e.g., a fluorescent signal) after hybridizing with a nucleic acid. In some cases, a signal can be generated after the probe is hybridized with the nucleic acid region, after the probe is degraded by a nuclease. In the case where the probe comprises a signal tag, the probe can be degraded when combined with the region of the primer, thereby generating a signal. For example, a probe (e.g., a probe) can generate a signal after the probe is hybridized with a nucleic acid and subsequently degraded by a polymerase (e.g., in an amplification such as a PCR amplification process). The probe can be degraded by the exonuclease activity of a nuclease.

[0066] In some embodiments, both ends of the probe sequence are modified with a fluorescent reporter group and a fluorescent quencher group corresponding to the fluorescent reporter group. The probe modified with a fluorescent reporter group and a fluorescent quencher group at both ends is used to display the relative amount of the synthesized DNA product in qPCR. In the absence of amplification, the fluorescent reporter group and the fluorescent quencher group at both ends of the probe sequence are together, and the fluorescence of the fluorescent reporter group is quenched by the fluorescent quencher group and does not emit fluorescence. When the PCR synthesis begins, the probe sequence is complementary to the target fragment in the genomic DNA and can anneal together. If the target microorganism exists in the sample (i.e., the target fragment exists), the probe sequence can bind to the target fragment. When the PCR reaction starts, if the PCR reaction can occur, the taq enzyme can dissociate the probe sequence bound to the target fragment, thereby separating the fluorescent reporter group from the fluorescent quencher group, and at this time, the fluorescence emitted by the fluorescent reporter group can be detected. As the PCR reaction proceeds, the more DNA amplification products are synthesized, the more fluorescent reporter groups are dissociated. By detecting the fluorescence intensity, the amount of DNA amplification products can be accurately quantified.

[0067] In some embodiments, the fluorescent reporter group and the fluorescent quencher group can be selected from various fluorescent reporter groups and corresponding fluorescent quenchers commonly used by those skilled in the art in qPCR. In some embodiments, the fluorescent reporter group can be selected from FAM, VIC, HEX, ROX or CY5, and correspondingly, the fluorescent quencher group can be selected from BHQ1, BHQ2, BHQ3 or MGB.

[0068] If solid support is used in detection (for example, using probe to capture the amplicon of target nucleic acid), then probe can be connected to solid support in different ways. For example, by connecting the 3' or 5' end nucleotide of probe to solid support, probe can be connected to solid support. More preferably, probe is connected to solid support by playing the joint that separates probe from solid support. Joint is generally the length of at least 15-30 atoms, more preferably, the length of at least 15-50 atoms. The expected length of joint will depend on the specific solid support applied. For example, when using highly cross-linked polystyrene as solid support, 6 atomic joints are usually enough.

[0069] Numerous joints are known in the art, and they can be used to connect oligonucleotide probes to solid supports. Joints can be formed by any compound that does not significantly interfere with the hybridization of the target sequence and the probe connected to the solid support. Joints can be formed by homopolymeric oligonucleotides that can be easily added to the joint by automatic synthesis. Alternatively, polymers such as functionalized polyethylene glycol can be used as joints. Such polymers are preferred than homopolymeric oligonucleotides because they do not significantly interfere with the hybridization of probes and target oligonucleotides. Polyethylene glycol is particularly preferred. Examples of solid supports of preferred types for fixing oligonucleotide probes include controlled pore glass, glass plates, polystyrene, avidin-coated polystyrene beads, cellulose, nylon, acrylamide gel and activated dextran.

[0070] In certain embodiments, an internal control (IC) or internal standard is added as a control to illustrate that any negative result is not caused by a detection failure. The use of IC enables control of the separation process, amplification process, and detection system, and enables monitoring of detection behavior and quantification of the sample (one or more). The IC can be included at any suitable point, for example, in a lysis buffer. In one embodiment, the IC includes a phage nucleic acid. When a solid support is used in the detection, the solid support can additionally include a probe specific for the internal standard (IC probe) to assist in capture when the IC probe is used. The IC probe can optionally be coupled to a detectable label different from the detectable label of the target sequence. In an embodiment where the detectable label is a fluorophore, the IC can be quantified by spectrophotometry and by limit of detection studies.

[0071] In another aspect, the present application also provides a kit comprising the composition described herein.

[0072] In some embodiments, the kit may include primers (or "bait oligonucleotides") as described herein. Primers may include paired primers. Paired primers may include forward primers and reverse primers. Primers can be configured to amplify a nucleic acid sequence corresponding to a specific target. For example, a forward primer can be configured to hybridize with a first region (e.g., 3' end) of a nucleic acid sequence, and a reverse primer can be configured to hybridize with a second region (e.g., 5' end) of the nucleic acid sequence, thereby being configured to amplify the nucleic acid sequence. Primers configured to amplify nucleic acid molecules can be used in performing the disclosed methods. In some cases, all primers in the kit are lyophilized.

[0073] The kit may include one or more nucleases. The nuclease may be a nucleic acid polymerase. The nucleic acid polymerase may be a deoxyribonucleic acid polymerase (DNA enzyme). The DNA enzyme may be Taq polymerase or a variant thereof. The nuclease may be a ribonucleic acid polymerase (RNA enzyme). The RNA enzyme may be RNA enzyme III. The nuclease may be an endonuclease. The endonuclease may be an endonuclease I. The endonuclease I may be a T7 endonuclease I. The nuclease may be capable of degrading nucleic acids comprising non-natural nucleotides. The nuclease may be an endonuclease V, such as an E. coli endonuclease V. The nuclease may be a polymerase (e.g., a DNA polymerase). The polymerase may be a Taq polymerase or a variant thereof. The non-limiting examples of DNA polymerases include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, PlatinumTaq polymerase, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, and their variants, modified products and derivatives. For a certain hot start polymerase, a denaturation step of 2 minutes to 10 minutes at 94°C-95°C may be required, which may change the thermal curve according to different polymerases. Nucleases can degrade probes under appropriate conditions. For example, the nuclease may be a polymerase and have exolytic activity and degrade the probe, thereby generating a detectable signal. The nuclease may be able to release the quencher from the probe under appropriate conditions.

[0074] In some embodiments, the kit may also include instructions for using any of the foregoing substances in the methods described herein. Detection reagents, including primers, probes, solid supports with bound probes, and other detection reagents, and appropriate instructions and other required reagents may be provided in the kit to perform the detection as described herein. Typically, the kit contains in separate containers: a combination of primers and probes (either already bound to a solid support; or separately, with reagents to bind them to a matrix), control formulations (positive and / or negative), labeling reagents—when the detection format requires, and signal generating reagents (e.g., enzyme substrates)—if the label does not directly generate a signal. Instructions for performing the detection (e.g., written, tape, VCR, CD-ROM, etc.) are typically included in the kit. Depending on the specific detection applied, the kit may also include other packaging reagents and materials (i.e., elution buffer, etc.). Standard detection, such as the detection described herein, can be performed with these kits. The instructions are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. Similarly, the instructions can be present in the kit as a package insert, on a label of a container of the kit or its components (e.g., associated with a package or subpackage), etc. In other embodiments, the instructions are present as an electronically stored data file on a suitable computer-readable storage medium, such as a CD-ROM, disk, etc., including the same medium on which the program is present. The kit can also contain instructions for using the kit to detect BKV in a sample using a method, including a PCR method. Buffers, dNTPs, and controls (e.g., positive and negative control nucleic acids) can also be included in the kit for practicing the method. The primers in the reagent can be detectably labeled or unlabeled.

[0075] In some embodiments, the kit further comprises one or more of an enzyme, a dNTP, a metal cation, a protein protectant, and a buffer.

[0076] In some embodiments, the enzyme comprises a DNA polymerase and a UNG enzyme, the DNA polymerase is a hot-start Taq enzyme, the concentration of the hot-start Taq enzyme is 0.5-8U / reaction, preferably 3-5U / reaction; the dNTP comprises dATP, dCTP, dGTP and dTTP; the metal cation comprises a magnesium ion and a potassium ion, preferably the metal cation is a magnesium ion with a concentration of 2.5-5mM and a potassium ion with a concentration of 40-50mM; the protein protectant comprises mannitol with a concentration of 0.2%-1.5% W / V (g / ml) and / or sucrose with a concentration of 10.5%-13.5% W / V (g / ml); the buffer is a buffer with a pH of 7.8-8.6, preferably the buffer is a 20-30mM Tris-HCl buffer.

[0077] In another aspect, the present application also provides use of the composition described herein or the kit described herein in SNCA gene detection.

[0078] In another aspect, the present application also provides use of the composition described herein or the kit described herein in early diagnosis of PD.

[0079] In another aspect, the present application also provides a method for detecting the SNCA gene using qPCR, comprising:

[0080] The composition described herein or the kit described herein is used to perform qPCR on the sample to be tested on a qPCR instrument, and the presence or absence of the SNCA gene in the sample is determined based on the result of qPCR.

[0081] In some embodiments, the sample to be tested can be a biological sample. The sample can be derived from a biological sample. The biological sample can be, for example, blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces or tears. The biological sample can be a fluid sample. The fluid sample can be blood or plasma.

[0082] In some embodiments, the extraction of the sample to be tested can be performed by magnetic bead extraction, column extraction, boiling lysis, etc. In some embodiments, the sample can be processed simultaneously with, before, or after the method of the present disclosure. The sample can be processed to purify or enrich nucleic acids (e.g., purify nucleic acids from a plasma sample). The sample containing nucleic acids can be processed to purify or enrich the target nucleic acid.

[0083] The primers and probes of the present application are designed on the specific and conserved segment sequences of the wild-type and mutant-type SNCA gene, a regulatory gene of Parkinson's disease (PD), which can effectively avoid missed detection and false positive detection, have extremely high detection sensitivity and specificity, and are simple to operate.

[0084] The present application describes multiple embodiments, but the description is exemplary rather than restrictive, and it is obvious to those skilled in the art that there may be more embodiments and implementations within the scope of the embodiments described in the present application. Although many possible feature combinations are shown in the drawings and discussed in the specific embodiments, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature or element of any embodiment may be used in combination with any other feature or element in any other embodiment, or may replace any other feature or element in any other embodiment.

[0085] The present application includes and contemplates combinations of features and elements known to those of ordinary skill in the art. The embodiments, features and elements disclosed in the present application may also be combined with any conventional features or elements to form a unique invention scheme. Any features or elements of any embodiment may also be combined with features or elements from other invention schemes to form another unique invention scheme. Therefore, it should be understood that any feature shown and / or discussed in the present application may be implemented individually or in any appropriate combination. Therefore, except for the limitations made according to the attached claims and their equivalents, the embodiments are not subject to other restrictions. In addition, various modifications and changes may be made within the scope of protection of the attached claims.

[0086] In addition, when describing representative embodiments, the specification may have presented the method and / or process as a specific sequence of steps. However, to the extent that the method or process does not rely on the specific order of the steps described herein, the method or process should not be limited to the steps of the specific order described. As will be understood by those of ordinary skill in the art, other sequences of steps are also possible. Therefore, the specific sequence of the steps set forth in the specification should not be interpreted as a limitation to the claims. In addition, the claims for the method and / or process should not be limited to the steps of performing them in the order written, and those skilled in the art can easily understand that these sequences can be changed and still remain within the spirit and scope of the embodiments of the present application.

[0087] The experimental methods in the following examples without specifying specific conditions are usually measured according to national standards. The experimental materials in the following examples without specifying the source are all commercially available raw materials. The equipment used in each step in the following examples is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise defined or specified, all professional and scientific terms used in this application have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of this application.

[0088] Example

[0089] Example 1. Design of primer-probe combination

[0090] The SNCA gene sequence was downloaded from NCBI, and the wild-type and mutant genomic sequences were analyzed using the MegAlign software in the DNAStar software package. The specific and conserved segments in the wild-type and mutant types were found in the corresponding intervals. With the assistance of the primer and probe design software Primer Express 3.0, primer and probe combinations were designed for PD-related exton1, exton3 and exton4, respectively. At the same time, the internal standard primer probe was designed using the human GAPDH gene.

[0091] The optimal primer and probe combination was screened

[0092]

[0093] This set of primers and probes was used for qPCR amplification, and another set of randomly selected designed primers in the table below was used as a control. The strategy of mixed primers and probes was adopted during amplification, that is, the three upstream primers in the three sets of primers were mixed as upstream primers, the three downstream primers were mixed as downstream primers, and the three probes were mixed as probes; the amplification template was 1×10 3 SNCA gene standard with copies / mL:

[0094] Control primers and probes:

[0095]

[0096] The amplification results are as follows Figure 1 The three peak lines are the optimal primer and probe combination screened by the present application; the line without peak is the control primer and probe combination, which has a later peak and is not shown in the figure. It can be seen that compared with the control primer and probe combination, the optimal primer and probe combination screened by the present application has a higher sensitivity.

[0097] Example 2. Optimization of the detection reagent system

[0098] The nucleic acid amplification reagent includes: enzyme, dNTPs, metal cations, protein protective agent, SNCA specific primer, SNCA specific probe and buffer system. The enzyme includes DNA polymerase and UNG enzyme; the DNA polymerase is preferably a hot start Taq enzyme (concentration is 0.5-8U / reaction, preferably 3-5U / reaction), dNTPs include dATP, dCTP, dGTP and dTTP, the metal cation includes magnesium ion and potassium ion (the concentration of magnesium ion is 0.5-6mM, preferably 2.5-5mM; the concentration of potassium ion is 40-50mM, preferably 50mM), the protein protective agent includes mannitol and / or sucrose (the concentration of mannitol is 0.2%-1.5% W / V, and the concentration of sucrose is 10.5%-13.5% W / V) and the buffer system includes a buffer system with a pH of 7.8-8.6, preferably including a Tris-HCl buffer system (20-30mM).

[0099] The amplification system under these different conditions was verified: the SNCA gene standard was diluted in a gradient manner to obtain 1×10 5 The samples were stored at -20℃ for future use. 10 μL of 1×10 5The sample with a concentration of 100 copies / mL was capped (check whether it is tight), and centrifuged instantly. The tube was placed in the real-time fluorescence quantitative PCR and amplified according to the parameters in the table below.

[0100]

[0101] The results of verification of different systems are as follows Figure 2 The optimal reaction system was confirmed.

[0102] The optimal combination of reaction reagent system configuration is as follows:

[0103] Components concentration 10×PCR Buffer 2.5 μL / reaction <![CDATA[25mM MgCl2 / AlCl3]]> 3mM / reaction (50mM / reaction) dNTP (2.5 mM each) 0.2mM / reaction Upstream primer 0.2 μM / reaction Upstream primer 0.2 μM / reaction Probe 0.1 μM / reaction Taq enzyme 5U / reaction <![CDATA[dH2O]]> Make up to 30 μL

[0104] During the detection, the system only needs to add the sample to be tested and the sample treatment solution directly to the nucleic acid amplification reagent to perform PCR amplification detection. It not only eliminates the complicated nucleic acid extraction and purification or sample lysis steps, but also eliminates the preparation steps of the PCR reaction solution, effectively achieving the effect of being ready to use, greatly simplifying the entire process of the kit operation. In addition, adding the sample to be tested directly to the nucleic acid amplification reagent avoids the loss of nucleic acid caused by multiple steps or technical problems during the nucleic acid extraction and purification process to the greatest extent, and the operation steps are simple and convenient. It does not require large-scale and sophisticated nucleic acid extraction and purification equipment, and only requires a conventional pipette to complete it, which greatly reduces the requirements for equipment. At the same time, the simplification of the extraction steps greatly shortens the operation time and manpower consumption.

[0105] Example 3. Sensitivity and specificity verification

[0106] The SNCA genotype standard was diluted in series with ultrapure water to obtain a concentration of 1×10 3 Samples with 100 copies / mL, 500 copies / mL, 200 copies / mL, and 100 copies / mL were tested according to the detection method of the optimal reaction system in Example 2. At the same time, the GAPDH gene was added as a negative control for specific detection.

[0107] like Figure 3 As shown, it can be seen that the detection sensitivity is up to 100 copies / mL.

[0108] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A composition comprising one or more of the following: An upstream decoy oligonucleotide and a downstream decoy oligonucleotide, wherein the upstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 1, and the downstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 2; an upstream decoy oligonucleotide and a downstream decoy oligonucleotide, wherein the upstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:3 and the downstream decoy oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:4; and An upstream bait oligonucleotide and a downstream bait oligonucleotide, wherein the upstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:5, and the downstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:

6.

2. The composition according to claim 1, characterized in that The composition comprises one or more of the following: An upstream decoy oligonucleotide and a downstream decoy oligonucleotide, wherein the upstream decoy oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 1, and the downstream decoy oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 2; An upstream decoy oligonucleotide and a downstream decoy oligonucleotide, wherein the upstream decoy oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:3, and the downstream decoy oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:4; as well as An upstream bait oligonucleotide and a downstream bait oligonucleotide, wherein the upstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:5, and the downstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:

6.

3. The composition according to claim 2, characterized in that The composition comprises one or more of the following: An upstream bait oligonucleotide and a downstream bait oligonucleotide, wherein the upstream bait oligonucleotide is the sequence shown in SEQ ID NO: 1, and the downstream bait oligonucleotide is the sequence shown in SEQ ID NO: 2; An upstream bait oligonucleotide and a downstream bait oligonucleotide, wherein the upstream bait oligonucleotide is the sequence shown in SEQ ID NO: 3, and the downstream bait oligonucleotide is the sequence shown in SEQ ID NO: 4; and The upstream bait oligonucleotide and the downstream bait oligonucleotide, the upstream bait oligonucleotide is the sequence shown in SEQ ID NO:5, and the downstream bait oligonucleotide is the sequence shown in SEQ ID NO:

6.

4. The composition according to claim 1, characterized in that The composition comprises one or more of the following: An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 1, the downstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 2, and the probe sequence is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 7; An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:3, the downstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:4, and the probe sequence is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:8; as well as An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:5, the downstream bait oligonucleotide is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:6, and the probe sequence is derived from at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:

9.

5. The composition according to claim 4, characterized in that The composition comprises one or more of the following: An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 1, the downstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 2, and the probe sequence is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO: 7; An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:3, the downstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:4, and the probe sequence is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:8; as well as An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:5, the downstream bait oligonucleotide is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:6, and the probe sequence is derived from at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the sequence shown in SEQ ID NO:

9.

6. The composition according to claim 5, characterized in that The composition comprises one or more of the following: An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is the sequence shown in SEQ ID NO: 1, the downstream bait oligonucleotide is the sequence shown in SEQ ID NO: 2, and the probe sequence is the sequence shown in SEQ ID NO: 7; An upstream bait oligonucleotide, a downstream bait oligonucleotide and a probe sequence, wherein the upstream bait oligonucleotide is the sequence shown in SEQ ID NO: 3, the downstream bait oligonucleotide is the sequence shown in SEQ ID NO: 4, and the probe sequence is the sequence shown in SEQ ID NO: 8; and The upstream bait oligonucleotide, the downstream bait oligonucleotide and the probe sequence, the upstream bait oligonucleotide is the sequence shown in SEQ ID NO:5, the downstream bait oligonucleotide is the sequence shown in SEQ ID NO:6, and the probe sequence is the sequence shown in SEQ ID NO:

9.

7. The composition according to any one of claims 4 to 6, characterized in that Both ends of the probe sequence are modified with a fluorescent reporter group and a fluorescent quencher group corresponding to the fluorescent reporter group; the fluorescent reporter group is FAM, VIC, HEX, ROX or CY5, and the fluorescent quencher group is BHQ1, BHQ2, BHQ3 or MGB.

8. A kit comprising the composition according to any one of claims 1 to 7.

9. The kit according to claim 8, characterized in that The kit also comprises one or more of an enzyme, a dNTP, a metal cation, a protein protectant, and a buffer.

10. The kit according to claim 9, characterized in that The enzyme comprises DNA polymerase and UNG enzyme, the DNA polymerase is a hot start Taq enzyme, the concentration of the hot start Taq enzyme is 0.5-8U / reaction, preferably 3-5U / reaction; the dNTP comprises dATP, dCTP, dGTP and dTTP; the metal cation comprises magnesium ion and potassium ion, preferably the metal cation is magnesium ion with a concentration of 2.5-5mM and potassium ion with a concentration of 40-50mM; the protein protectant comprises mannitol with a concentration of 0.2%-1.5% W / V (g / ml) and / or sucrose with a concentration of 10.5%-13.5% W / V (g / ml); the buffer is a buffer with a pH of 7.8-8.6, preferably the buffer is a 20-30mM Tris-HCl buffer.

11. Use of the composition according to any one of claims 1 to 7 or the kit according to any one of claims 8 to 10 in SNCA gene detection.

12. Use of the composition according to any one of claims 1 to 7 or the kit according to any one of claims 8 to 10 in the early diagnosis of PD.

13. A method for detecting SNCA gene by qPCR, comprising: qPCR is performed on the sample to be tested using the composition according to any one of claims 1 to 7 or the kit according to any one of claims 8 to 10 on a qPCR instrument, and the presence or absence of the SNCA gene in the sample is determined by the result of qPCR.

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