Method and kit for multiplex quantitative detection of miRNA for Alzheimer's disease

Abstract

A multiplex reverse transcription primer combination comprises two or more stem-loop reverse transcription primers and exhibits multispecificity. According to the multiple reverse transcription primer combination, two or more kinds of target miRNA can be quantified at the same time, and the target miRNA comprises miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p.

Description

[0001] Cross-references to related patent applications

[0002] This application is a partial continuation application of PCT patent application PCT / CN2023 / 092571 filed on May 6, 2023, and the PCT patent application is incorporated herein by reference in its entirety.

[0003] This application is related to co-pending PCT patent application with attorney docket number 1010469.100WO1, entitled “Method for Screening Multiplex Reverse Transcription Primer Combinations for Quantifying Multiple miRNAs”; co-pending PCT patent application with attorney docket number 1010469.102WO1, entitled “Method and Kit for Multiplex Quantitative Detection of miRNAs for Lung Carcinoma”; and co-pending PCT patent application with attorney docket number 1010469.103WO1, entitled “Multiplex-Reverse Transcription Primer Combinations for Multiplex Reverse Transcription–Quantitative PCR Reactions)"; and co-pending PCT patent application with attorney case number 1010469.104WO1, entitled "Method for Clinical Detection of Multiple Target miRNAs", which are filed on the same day as the present application and by the same applicant as the present application, and any and all of the co-pending PCT patent applications are incorporated herein by reference in their entirety.

[0004] Statement regarding sequence listing

[0005] The sequence listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into this specification. The name of the XML file containing the sequence listing is 1010469_100WO1.xml. The XML file is approximately 225KB and was created on April 26, 2023. Technical Field

[0006] The present invention relates to a multiple reverse transcription primer combination and its application in multiple detection / quantification of multiple target miRNAs including miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p. Background Art

[0007] The background description provided herein is intended to present the context of the present invention in general. The subject matter discussed in the background section of the present invention should not be assumed to be prior art simply because of the reference to the subject matter in the background section of the present invention. Similarly, problems mentioned in the background section of the present invention or associated with the subject matter of the background section of the present invention should not be assumed to have been previously identified in the prior art. The subject matter in the background section of the present invention merely represents different methods, which themselves may also be inventions.

[0008] MicroRNA or miRNA is a non-coding RNA widely present in organisms, and usually its length is 18-25 nucleotides. According to miRBase (22 version), the human genome encodes about 2,600 mature miRNAs. Related studies have shown that these miRNAs are involved in many biological processes and their regulation, such as apoptosis, proliferation, organogenesis, development, tumorigenesis and hematopoiesis. At the same time, miRNA also regulates gene expression in the post-transcriptional stage, and therefore plays an important role in regulating a variety of life processes, such as the occurrence and development of diseases and cell aging. Some studies have shown that miRNA has the potential to be used as a clinical diagnostic tool for diseases.

[0009] The first miRNA was not discovered until 1993, when Lee et al. cloned lin-4 from Caenorhabditis elegans using positional cloning. The lin-4 gene was found to encode a miRNA of approximately 22 nucleotides in length. Later, in 2000, Reinhart et al. discovered let-7, the second gene known to regulate temporal development in worms, and a negative regulator that is also transcribed into a 21-nucleotide miRNA. Since then, people have gradually realized that miRNAs are important evolutionarily conserved molecules that play a regulatory role in disease development and biological evolution.

[0010] Despite the potential for use as a diagnostic tool for disease and its important regulatory role, accurate quantification of miRNAs is extremely difficult. Real-time fluorescence quantitative PCR (qPCR) detection is considered the gold standard for quantitative detection, but it was not suitable for miRNA quantification in the early stages of research. This is due to the fact that miRNAs have very short sequences, low expression levels in cells, and highly homologous sequences. Northern blot hybridization and gene chips were the main methods used to study miRNA expression in the early days, but these methods have limitations in terms of sample requirements, linear range, and sensitivity.

[0011] It was not until 2005 that Chen et al. first used the stem-loop method to implement real-time fluorescence qPCR detection of a single miRNA (C. Chen et al. (2005) Real-time quantification of microRNAs by stem-loop RT-PCR, Nucleic Acids Res., 33, e179). The detection process includes two main steps: reverse transcription and real-time PCR. In the first step, the stem-loop reverse transcription primer is mixed with the miRNA molecule and the PCR product is analyzed using MultiScribe. TM Reverse transcriptase is used for reverse transcription. Conventional Taqman PCR is then used to quantitatively analyze the resulting product. The stem-loop primer used has a complementary sequence of 6 bases at the 3' end of the miRNA to initiate the reverse transcription reaction. In the experiment, it was observed that the stem-loop primer has better specificity and sensitivity than the traditional linear primer. Nucleotide base stacking can effectively improve thermal stability, and the spatial structure of the stem-loop sequence can help it bind to the double-stranded DNA molecules in the genome. It is also proposed that the stem-loop reverse transcription primer has the potential for a variety of reverse transcription reactions and can have better efficiency and specificity.

[0012] In order to simultaneously detect / quantify multiple miRNAs in small samples, Tang et al. (2006) further improved the above method to achieve the simultaneous detection of 220 miRNAs in a single embryonic stem cell (Tang et al. (2006) MicroRNA expression profiling of single whole embryonic stem cells, "Nucleic Acids Research" 24e9). The method mainly involves reverse transcription of all miRNAs in a single embryonic stem cell under cyclic pulse temperature, followed by pre-PCR to improve the detection sensitivity of the resulting product, and then separating the real-time fluorescence quantification of each miRNA expression. Lao et al. (2006) verified Tang's method and believed that as the number of detections increases, the interaction between primers will also increase. The difference between the Ct values ​​of specific miRNAs obtained under single and multiple detection conditions using the above method is observable. Therefore, the results obtained by this method still need to be verified under single detection conditions. Lao et al. also believed that this interaction between primers needs in-depth analysis and research to reduce, and this study requires testing a large number of primer combinations. This task is expensive and difficult for any laboratory to perform in the 326 known human miRNAs. Currently, when studying the relationship between disease and the expression levels of multiple miRNAs, only a single quantitative detection method by Chen et al. is used to detect the expression level of miRNAs individually by fluorescence quantification.

[0013] As the number of tests increases, the interactions between primers gradually increase, and the difficulty increases not only exponentially, but also exponentially. Considering issues such as cross-reactions between probe primers, specificity between systems, sensitivity, and efficiency, it is almost impossible to achieve multiple real-time fluorescence qPCR detection using a single principle. Current technology still cannot use the stem-loop reverse transcription primer method to achieve stable, standardized, and accurate multiple quantitative detection of multiple miRNAs. These shortcomings also hinder its entry into the clinical field.

[0014] Therefore, there is still an urgent need for a multiplex detection method and multiple stem-loop primer combination that enables the simultaneous quantification of multiple target miRNA combinations in a multiplex reverse transcription-fluorescence qPCR (RT-qPCR) process. Summary of the invention

[0015] In view of the above, the present invention discloses a multiplex stem-loop primer combination that enables simultaneous quantification of a combination of multiple target miRNAs in a multiplex RT-qPCR process.

[0016] In one aspect of the present invention, a multiple reverse transcription primer combination for simultaneously quantifying miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p is provided, the multiple reverse transcription primer combination comprising: a first stem-loop reverse transcription primer, the first stem-loop reverse transcription primer comprising a nucleic acid sequence of SEQ ID No.5; a second stem-loop reverse transcription primer, the second stem-loop reverse transcription primer comprising a nucleic acid sequence of SEQ ID No.6; a third stem-loop reverse transcription primer, the third stem-loop reverse transcription primer comprising a nucleic acid sequence of SEQ ID No.7; and a fourth stem-loop reverse transcription primer, the fourth stem-loop reverse transcription primer comprising a nucleic acid sequence of SEQ ID No.8.

[0017] In another aspect of the present invention, a multiple reverse transcription primer combination for simultaneously quantifying multiple target miRNAs, the multiple reverse transcription primer combination comprising multiple stem-loop reverse transcription primers; wherein each of the multiple stem-loop reverse transcription primers has a stem-loop sequence forming a stem-loop structure and an anchor sequence complementary to a unique 3' sequence of one of the multiple target miRNAs, wherein the lengths of the anchor sequences of at least two of the stem-loop reverse transcription primers are different from each other.

[0018] In one embodiment, each stem-loop reverse transcription primer in the plurality of stem-loop reverse transcription primers has a length of about 40-60 nt.

[0019] In one embodiment, the anchor sequence of each of the plurality of stem-loop reverse transcription primers has a length of about 3-12 nt.

[0020] In one embodiment, the multiple stem-loop reverse transcription primers include a first stem-loop reverse transcription primer having a first stem-loop sequence and a first anchor sequence; and a second stem-loop reverse transcription primer having a second stem-loop sequence and a second anchor sequence, wherein each stem-loop sequence in the first stem-loop sequence and the second stem-loop sequence is selected from the group consisting of SEQ ID No.9-12; and the first stem-loop sequence and the second stem-loop sequence are different from each other; and wherein the length of each anchor sequence in the first anchor sequence and the second anchor sequence is selected from one of 4nt, 6nt, 8nt and 11nt.

[0021] In one embodiment, the first anchor sequence of the first stem-loop reverse transcription primer is complementary to a unique 3' sequence of a first target miRNA among the plurality of target miRNAs, and the second anchor sequence of the second stem-loop reverse transcription primer is complementary to a unique 3' sequence of a second target miRNA among the plurality of target miRNAs.

[0022] In one embodiment, the multiple stem-loop reverse transcription primers further include a third stem-loop reverse transcription primer having a third stem-loop sequence and a third anchor sequence, wherein the third stem-loop sequence is selected from the group consisting of SEQ ID No.9-12; and the third stem-loop sequence is different from the first stem-loop sequence and the second stem-loop sequence; wherein the length of the third anchor sequence is selected from one of 4nt, 6nt, 8nt and 11nt; and wherein the third anchor sequence is complementary to the unique 3' sequence of the third target miRNA among the multiple target miRNAs.

[0023] In one embodiment, the multiple stem-loop reverse transcription primers further include a fourth stem-loop reverse transcription primer having a fourth stem-loop sequence and a fourth anchor sequence, wherein the fourth stem-loop sequence is selected from the group consisting of SEQ ID No.9-12; and the fourth stem-loop sequence is different from the first stem-loop sequence, the second stem-loop sequence and the third stem-loop sequence; wherein the length of the fourth anchor sequence is selected from one of 4nt, 6nt, 8nt and 11nt; and wherein the fourth anchor sequence is complementary to the unique 3' sequence of the fourth target miRNA among the multiple target miRNAs.

[0024] In one embodiment, the multiple reverse transcription primer combination has multi-specificity, so that the first stem-loop reverse transcription primer effectively and only reverse transcribes the first target miRNA, the second stem-loop reverse transcription primer effectively and only reverse transcribes the second target miRNA, the third stem-loop reverse transcription primer effectively and only reverse transcribes the third target miRNA, and the fourth stem-loop reverse transcription primer effectively and only reverse transcribes the fourth target miRNA.

[0025] In one embodiment, each of the first target miRNA, the second target miRNA, the third target miRNA and the fourth target miRNA is selected from the group consisting of miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p, wherein each of the first target miRNA, the second target miRNA, the third target miRNA and the fourth target miRNA is different from each other.

[0026] In one embodiment, the first stem-loop reverse transcription primer includes the nucleic acid sequence of SEQ ID No.5, the second stem-loop reverse transcription primer includes the nucleic acid sequence of SEQ ID No.6, the third stem-loop reverse transcription primer includes the nucleic acid sequence of SEQ ID No.7, and the fourth stem-loop reverse transcription primer includes the nucleic acid sequence of SEQ ID No.8.

[0027] In another aspect of the present invention, a kit for simultaneously quantifying the expression levels of multiple target miRNAs, the kit comprising: a first stem-loop reverse transcription primer and a second stem-loop reverse transcription primer, wherein the first stem-loop reverse transcription primer has a first stem-loop sequence and a first anchor sequence, and the second stem-loop reverse transcription primer has a second stem-loop sequence and a second anchor sequence, wherein each of the first stem-loop sequence and the second stem-loop sequence is selected from the group consisting of SEQ ID No. 9-12, and the first stem-loop sequence and the second stem-loop sequence are different from each other, wherein the length of each of the first anchor sequence and the second anchor sequence is selected from one of 4nt, 6nt, 8nt and 11nt; a first forward primer and a second forward primer, wherein each of the first forward primer and the second forward primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. 17-20, and the first forward primer is different from the second forward primer; a first reverse primer and a second reverse primer, wherein each of the first reverse primer and the second reverse primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. No.25-28, and the first reverse primer is different from the second reverse primer; and a first probe and a second probe, wherein the first probe comprises a first probe sequence, a first fluorescent reporter group and a first quencher group, and the second probe comprises a second probe sequence, a second fluorescent reporter group and a second quencher group, wherein each probe sequence in the first probe sequence and the second probe sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID No.21-24.

[0028] In one embodiment, each of the first fluorescent reporter group and the second fluorescent reporter group is selected from the group consisting of VIC, CY5, ROX and FAM; and the first fluorescent reporter group is different from the second fluorescent reporter group.

[0029] In one embodiment, the first anchor sequence is complementary to a 3' sequence of a first target miRNA of the plurality of target miRNAs, and the second anchor sequence is complementary to a unique 3' sequence of a second target miRNA of the plurality of target miRNAs.

[0030] In one embodiment, the kit further comprises: a third stem-loop reverse transcription primer, wherein the third stem-loop reverse transcription primer has a third stem-loop sequence and a third anchor sequence, wherein the third stem-loop sequence is selected from the group consisting of SEQ ID No.9-12, and the third stem-loop sequence is different from the first stem-loop sequence and the second stem-loop sequence, and wherein the length of the third anchor sequence is selected from one of 4nt, 6nt, 8nt and 11nt; a third forward primer, wherein the third forward primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID No.17-20, and the third forward primer is different from the first forward primer and the second forward primer; a third reverse primer, wherein the third reverse primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID No.25-28, and the third reverse primer is different from the first reverse primer and the second reverse primer; and a third probe, wherein the third probe comprises a third probe sequence, a third fluorescent reporter group and a third quencher group, wherein the third probe sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID No.21-24.

[0031] In one embodiment, the third fluorescent reporter group is selected from the group consisting of VIC, CY5, ROX and FAM; and the third fluorescent reporter group is different from the first fluorescent reporter group and the second fluorescent reporter group.

[0032] In one embodiment, the kit further comprises: a fourth stem-loop reverse transcription primer, wherein the fourth stem-loop reverse transcription primer has a fourth stem-loop sequence and a fourth anchor sequence, wherein the fourth stem-loop sequence is selected from the group consisting of SEQ ID No. 9-12, and the fourth stem-loop sequence is different from the first stem-loop sequence, the second stem-loop sequence and the third stem-loop sequence, wherein the length of the fourth anchor sequence is selected from one of 4 nt, 6 nt, 8 nt and 11 nt; a fourth forward primer, wherein the fourth forward primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. 17-20, and the fourth forward primer is different from the first forward primer, the second forward primer and the third forward primer; a fourth reverse primer, wherein the fourth reverse primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. 25-28, and the fourth reverse primer is different from the first reverse primer, the second reverse primer and the third reverse primer; and a fourth probe, wherein the fourth probe comprises a fourth probe sequence, a fourth fluorescent reporter group and a fourth quencher group, wherein the fourth probe sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. 21-24.

[0033] In one embodiment, the fourth fluorescent reporter group is selected from the group consisting of VIC, CY5, ROX and FAM; and the fourth fluorescent reporter group is different from the first fluorescent reporter group, the second fluorescent reporter group and the third fluorescent reporter group.

[0034] In another aspect of the present invention, a kit for simultaneously quantifying the expression levels of multiple target miRNAs, the kit comprising: a multiple reverse transcription primer combination, the multiple reverse transcription primer combination comprising a first stem-loop reverse transcription primer comprising a nucleic acid sequence of SEQ ID No.5, a second stem-loop reverse transcription primer comprising a nucleic acid sequence of SEQ ID No.6, a third stem-loop reverse transcription primer comprising a nucleic acid sequence of SEQ ID No.7, and a fourth stem-loop reverse transcription primer comprising a nucleic acid sequence of SEQ ID No.8; a forward primer combination, the forward primer combination comprising a first forward primer comprising a nucleic acid sequence of SEQ ID No.17, a second forward primer comprising a nucleic acid sequence of SEQ ID No.18, a third forward primer comprising a nucleic acid sequence of SEQ ID No.19, and a fourth forward primer comprising a nucleic acid sequence of SEQ ID No.20; a reverse primer combination, the reverse primer combination comprising a first reverse primer comprising a nucleic acid sequence of SEQ ID No.25, a second reverse primer comprising a nucleic acid sequence of SEQ ID No.26, a third reverse primer comprising a nucleic acid sequence of SEQ ID No.27, and a fourth reverse primer comprising a nucleic acid sequence of SEQ ID No. A fourth reverse primer comprising a nucleic acid sequence of SEQ ID No.28; and a probe combination, wherein the probe combination comprises a first probe comprising a nucleic acid sequence of SEQ ID No.21 and a first fluorescent reporter group of VIC, a second probe comprising a nucleic acid sequence of SEQ ID No.22 and a second fluorescent reporter group of CY5, a third probe comprising a nucleic acid sequence of SEQ ID No.23 and a third fluorescent reporter group of ROX, and a fourth probe comprising a nucleic acid sequence of SEQ ID No.24 and a fourth fluorescent reporter group of FAM.

[0035] In another aspect of the present invention, a method for simultaneously quantifying miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p comprises performing multiplex RT-qPCR using the multiplex reverse transcription primer combination disclosed above.

[0036] In another aspect of the present invention, a method for determining a medical condition of a living subject using the quantitative results of miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p, the method comprising: performing multiplex RT-qPCR using the kit according to claim 19; obtaining the quantitative results of miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p from the multiplex RT-qPCR; and using the quantitative results to determine the medical condition of the living subject, wherein the medical condition of the living subject is Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings illustrate one or more embodiments of the present invention and together with the written description serve to explain the principles of the present invention. Wherever possible, the same reference numerals are used throughout the drawings to refer to the same or similar elements of the embodiments.

[0038] Figure 1A -D shows singleplex RT-qPCR of individual target miRNAs as templates. Figure 1A Shown is singleplex RT-qPCR of miR-16-5p / ddH2O using corresponding stem-loop reverse transcription primers with different anchor sequence lengths (6 / 8 / 11 nt). Figure 1B Shown is singleplex RT-qPCR of miR-34c-5p / ddH2O using corresponding stem-loop reverse transcription primers with different anchor sequence lengths (6 / 8 / 11 nt). Figure 1C Shown is singleplex RT-qPCR of miR-9-3p / ddH2O using corresponding stem-loop reverse transcription primers with different anchor sequence lengths (6 / 8 / 11 nt). Figure 1D Shown is singleplex RT-qPCR of miR-9-5p / ddH2O using corresponding stem-loop reverse transcription primers with different anchor sequence lengths (6 / 8 / 11 nt).

[0039] Figure 2A -F shows the validation results for the specificity of the following two dual systems: miR-34c-5p and miR-9-3p; and miR-34c-5p and miR-16-5p. Specifically, Figure 2A Shown is the amplification plot of singleplex RT-qPCR of a mixed target miRNA template containing miR-34c-5p and miR-9-3p using the corresponding stem-loop reverse transcription primers selected for miR-34c-5p. Figure 2B Shown is the amplification plot of singleplex RT-qPCR of a mixed target miRNA template containing miR-34c-5p and miR-9-3p using the corresponding stem-loop reverse transcription primers selected for miR-9-3p. Figure 2C Amplification plots of singleplex RT-qPCR using selected stem-loop reverse transcription primers corresponding to miR-34c-5p and miR-9-3p in ddH2O are shown. Figure 2D Shown is the amplification plot of singleplex RT-qPCR of a mixed target miRNA template containing miR-34c-5p and miR-16-5p using the corresponding stem-loop reverse transcription primers selected for miR-16-5p. Figure 2E Shown is the amplification plot of singleplex RT-qPCR of a mixed target miRNA template containing miR-34c-5p and miR-16-5p using the corresponding stem-loop reverse transcription primers selected for miR-34c-5p. Figure 2F Amplification plots of singleplex RT-qPCR using selected stem-loop reverse transcription primers corresponding to miR-34c-5p and miR-16-5p in ddH2O are shown.

[0040] Figure 3A -D shows the validation results of the multi-specificity of the following two dual systems: miR-34c-5p and miR-9-3p; and miR-34c-5p and miR-16-5p. Specifically, Figure 3A Amplification plots of dual RT-qPCR of a mixed target miRNA template containing miR-34c-5p and miR-9-3p using corresponding stem-loop reverse transcription primers selected for miR-34c-5p and miR-9-3p are shown. Figure 3B Amplification plots of dual RT-qPCR using selected stem-loop reverse transcription primers corresponding to miR-34c-5p and miR-9-3p in ddH2O are shown. Figure 3C Amplification plots of dual RT-qPCR of a mixed target miRNA template containing miR-34c-5p and miR-16-5p using corresponding stem-loop reverse transcription primers selected for miR-34c-5p and miR-16-5p are shown. Figure 3D Amplification plots of dual RT-qPCR using selected stem-loop reverse transcription primers corresponding to miR-34c-5p and miR-16-5p in ddH2O are shown.

[0041] Figure 4A -D shows the results of sensitivity tests of the following two dual systems: miR-34c-5p and miR-9-3p; and miR-34c-5p and miR-16-5p. Specifically, Figure 4A Sensitivity testing of miR-34c-5p is shown. Figure 4B Sensitivity testing of miR-9-3p is shown. Figure 4CSensitivity testing of miR-34c-5p is shown. Figure 4D Sensitivity testing of miR-16-5p is shown.

[0042] Figure 5A -F shows a comparison between single and double quantification of the following two double systems: miR-34c-5p and miR-9-3p; and miR-34c-5p and miR-16-5p. Specifically, Figure 5A Singleplex quantification of miR-34c-5p is shown. Figure 5B Singleplex quantification of miR-9-3p is shown. Figure 5C Dual quantification of miR-34c-5p and miR-9-3p is shown. Figure 5D Singleplex quantification of miR-34c-5p is shown. Figure 5E Singleplex quantification of miR-16c-5p is shown. Fig. 5F Dual quantification of miR-34c-5p and miR-16-5p is shown.

[0043] Fig. 6A -H shows the validation results for the specificity of the following two triple systems: miR-34c-5p, miR-9-3p, miR-16-5p; and miR-9-5p, miR-16-5p and miR-34c-5p. Specifically, Fig. 6A Shown is the amplification plot of singleplex RT-qPCR of a mixed target miRNA template containing miR-34c-5p, miR-9-3p, and miR-16-5p using the corresponding stem-loop reverse transcription primer selected for miR-16-5p. Figure 6B Shown is the amplification plot of singleplex RT-qPCR of a mixed target miRNA template containing miR-34c-5p, miR-9-3p, and miR-16-5p using the corresponding stem-loop reverse transcription primer selected for miR-9-3p. Figure 6C Shown is the amplification plot of singleplex RT-qPCR of a mixed target miRNA template containing miR-34c-5p, miR-9-3p, and miR-16-5p using the corresponding stem-loop reverse transcription primer selected for miR-34c-5p. Fig.6D Amplification plots of singleplex RT-qPCR using corresponding stem-loop reverse transcription primers of selected miR-34c-5p, miR-9-3p and miR-16-5p in ddH2O are shown. Fig. 6E Shown is the amplification plot of singleplex RT-qPCR of a mixed target miRNA template containing miR-34c-5p, miR-9-5p, and miR-16-5p using the corresponding stem-loop reverse transcription primer selected for miR-9-5p. Fig. 6F Shown is the amplification plot of singleplex RT-qPCR of a mixed target miRNA template containing miR-34c-5p, miR-9-5p, and miR-16-5p using the corresponding stem-loop reverse transcription primer selected for miR-16-5p. Figure 6G Amplification plots of singleplex RT-qPCR of a mixed target miRNA template containing miR-34c-5p, miR-9-5p, and miR-16-5p using the corresponding stem-loop reverse transcription primers selected for miR-34c-5p are shown. Figure 6H Amplification plots of singleplex RT-qPCR using corresponding stem-loop reverse transcription primers of selected miR-34c-5p, miR-9-5p and miR-16-5p in ddH2O are shown.

[0044] Fig. 7A -D shows the validation results of the multi-specificity of the following two triple systems: miR-34c-5p, miR-16-5p and miR-9-3p; and miR-34c-5p, miR-16-5p and miR-9-5p. Specifically, Fig. 7A Shown is the amplification plot of triplex RT-qPCR of a mixed target miRNA template containing miR-34c-5p, miR-16-5p and miR-9-3p using the corresponding stem-loop reverse transcription primers selected for miR-34c-5p, miR-16-5p and miR-9-3p. Figure 7B Amplification plots of triplex RT-qPCR using corresponding stem-loop reverse transcription primers of selected miR-34c-5p, miR-16-5p and miR-9-3p in ddH2O are shown. Figure 7C Shown is the amplification plot of triplex RT-qPCR of a mixed target miRNA template containing miR-34c-5p, miR-16-5p and miR-9-5p using the corresponding stem-loop reverse transcription primers selected for miR-34c-5p, miR-16-5p and miR-9-5p. Fig.7D Amplification plots of triplex RT-qPCR using corresponding stem-loop reverse transcription primers of selected miR-34c-5p, miR-16-5p and miR-9-5p in ddH2O are shown.

[0045] Fig. 8A -F shows the results of sensitivity testing of the following two triple systems: miR-34c-5p, miR-16-5p and miR-9-3p; and miR-34c-5p, miR-16-5p and miR-9-5p. Specifically, Fig. 8AShown is the sensitivity test of miR-9-3p in the triple combination of miR-34c-5p, miR-16-5p and miR-9-3p. Figure 8B Shown is the sensitivity test of miR-16-5p in the triple combination of miR-34c-5p, miR-16-5p and miR-9-3p. Figure 8C Shown is the sensitivity test of miR-34c-5p in the triple combination of miR-34c-5p, miR-16-5p and miR-9-3p. Fig.8D Shown is the sensitivity test of miR-9-5p in the triple combination of miR-34c-5p, miR-16-5p and miR-9-5p. Fig. 8E Shown is the sensitivity test of miR-16-5p in the triple combination of miR-34c-5p, miR-16-5p and miR-9-5p. Figure 8F Shown is the sensitivity test of miR-34c-5p in the triple combination of miR-34c-5p, miR-16-5p and miR-9-5p.

[0046] Fig. 9A -H shows a comparison between single and triple quantification of the following two triple systems: miR-34c-5p, miR-16-5p, and miR-9-3p; and miR-34c-5p, miR-16-5p, and miR-9-5p. Specifically, Fig. 9A Singleplex quantification of miR-34c-5p is shown. Fig. 9B Singleplex quantification of miR-9-3p is shown. Fig. 9C Singleplex quantification of miR-16-5p is shown. Fig.9D Triple quantification of miR-34c-5p, miR-16-5p and miR-9-3p is shown. Fig.9E Singleplex quantification of miR-9-5p is shown. Fig.9F Singleplex quantification of miR-16-5p is shown. Figure 9G Singleplex quantification of miR-34c-5p is shown. Fig.9D Triple quantification of miR-34c-5p, miR-16-5p, and miR-9-5p is shown.

[0047] Fig. 10A -F shows the validation results for the specificity of the following quadruple system: miR-9-3p, miR-9-5p, miR-16-5p and miR-34c-5p. Specifically, Fig. 10A Amplification plots of singleplex RT-qPCR using corresponding stem-loop reverse transcription primers of selected miR-9-3p are shown. Fig. 10BAmplification plots of singleplex RT-qPCR using corresponding stem-loop reverse transcription primers of selected miR-34c-5p are shown. Fig. 10C Amplification plots of singleplex RT-qPCR using corresponding stem-loop reverse transcription primers of selected miR-9-5p are shown. Fig. 10D Amplification plots of singleplex RT-qPCR using corresponding stem-loop reverse transcription primers of selected miR-16-5p are shown. Fig.10E and Fig.10F Amplification plots of singleplex RT-qPCR using corresponding stem-loop reverse transcription primers of selected miR-34c-5p, miR-9-5p, miR-9-3p and miR-16-5p in ddH2O are shown.

[0048] Fig.11A -B shows the validation results of the multi-specificity of the following quadruple system: miR-34c-5p, miR-16-5p, miR-9-5p and miR-9-3p. Specifically, Fig.11A Shown is an amplification plot of quadruple RT-qPCR of a mixed target miRNA template containing miR-34c-5p, miR-16-5p, miR-9-5p and miR-9-3p using the corresponding stem-loop reverse transcription primers selected for miR-34c-5p, miR-16-5p, miR-9-5p and miR-9-3p. Fig. 11B Amplification plots of quadruple RT-qPCR using corresponding stem-loop reverse transcription primers of selected miR-34c-5p, miR-16-5p, miR-9-3p and miR-9-5p in ddH2O are shown.

[0049] Fig. 12A -D shows the results of the sensitivity test of the following four-fold system: miR-34c-5p, miR-16-5p, miR-9-5p and miR-9-3p. Specifically, Fig. 12A Sensitivity testing of miR-9-5p in quadruple panels is shown. Fig. 12B Sensitivity testing of miR-9-3p in quadruple panels is shown. Fig. 12C Sensitivity testing of miR-16-5p in quadruple panels is shown. Fig.12D Sensitivity testing of miR-34c-5p in a quadruple panel is shown.

[0050] Fig.13A -F shows the comparison between single and quadruple quantification of miR-34c-5p, miR-16-5p, miR-9-5p and miR-9-3p. Specifically, Fig.13A Singleplex quantification of miR-34c-5p is shown. Fig. 13BSingleplex quantification of miR-9-5p is shown. Fig. 13C Singleplex quantification of miR-9-3p is shown. Fig.13D Singleplex quantification of miR-16-5p is shown. Fig.13E Quadruple quantification of miR-34c-5p, miR-16-5p, miR-9-5p, and miR-9-3p is shown. Fig.13F Comparison between singleplex and quadruplex quantification of miR-34c-5p, miR-16-5p, miR-9-5p, and miR-9-3p is shown.

[0051] Fig.14A -F shows a quadruple quantitative stability test of miR-34c-5p, miR-16-5p, miR-9-5p and miR-9-3p. Specifically, Fig.14A Singleplex quantification of miR-34c-5p is shown. Fig. 14B Singleplex quantification of miR-9-5p is shown. Fig. 14C Singleplex quantification of miR-9-3p is shown. Fig.14D Singleplex quantification of miR-16-5p is shown. Fig.14E Quadruple quantification of miR-34c-5p, miR-16-5p, miR-9-5p, and miR-9-3p is shown. Fig.14F Comparison between singleplex and quadruplex quantification of miR-34c-5p, miR-16-5p, miR-9-5p, and miR-9-3p is shown.

[0052] Fig.15A -D shows the cross-reaction within a quadruple system including stem-loop reverse transcription primers each having a different anchor length. Specifically, Fig.15A Amplification plots of singleplex RT-qPCR using a stem-loop reverse transcription primer with an anchor sequence length of 8 nt for miR-9-3p are shown. Fig. 15B Amplification plots of singleplex RT-qPCR using a stem-loop reverse transcription primer with an anchor sequence length of 6 nt for miR-9-5p are shown. Fig. 15C Amplification plots of singleplex RT-qPCR using a stem-loop reverse transcription primer with an anchor sequence length of 11 nt for miR-16-5p are shown. Fig.15D Amplification plots of singleplex RT-qPCR using a stem-loop reverse transcription primer with an anchor sequence length of 8 nt for miR-34c-5p are shown. DETAILED DESCRIPTION

[0053] The present invention will now be described more fully below with reference to the accompanying drawings, in which exemplary embodiments of the present invention are shown. However, the present invention can be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that the present invention will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art. Throughout the text, the same reference numerals refer to the same elements.

[0054] The terms used in this specification generally have their general meaning in the art within the context of the present invention and in the specific context in which each term is used. Certain terms used to describe the present invention are discussed below or elsewhere in this specification to provide practitioners with additional guidance on the description of the present invention. For convenience, certain terms may be highlighted, for example, using italics and / or quotation marks. The use of highlighting has no effect on the scope and meaning of the terms; whether or not the terms are highlighted, the scope and meaning of the terms are the same in the same context. It should be understood that the same thing can be described in more than one way. Therefore, for any one or more of the terms discussed herein, alternative language and synonyms can be used, and no special meaning should be given to the terms regardless of whether the terms are described or discussed in detail herein. Synonyms for certain terms are provided. The detailed description of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification (including examples of any terms discussed herein) is illustrative only, and is by no means limiting the scope and meaning of the present invention or any exemplified term. Similarly, the present invention is not limited to the various embodiments given in this specification.

[0055] It will be understood by those skilled in the art that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods and biological methods other than the specifically exemplified starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods and biological methods can be used to practice the present invention without resorting to excessive experiments. All functional equivalents known in the art of any such materials and methods are intended to be included in the present invention. The terms and expressions that have been adopted are used as illustrative rather than limiting terms, and are not intended to use such terms and expressions that exclude any equivalent of the features shown and described or parts thereof, but it should be recognized that various modifications can be made within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been clearly disclosed by preferred embodiments and optional features, those skilled in the art can modify and change the concepts disclosed herein, and such modifications and changes are considered to be within the scope of the present invention as defined by the appended claims.

[0056] Whenever a range is given in the specification, such as a temperature range, time range, or composition or concentration range, all intermediate ranges and subranges, as well as all individual values ​​contained within the given range, are intended to be included in the invention. It should be understood that any subrange or individual value contained within a range or subrange in the description herein may be excluded from the claims herein.

[0057] It should be understood that, as used in this specification and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those of skill in the art. Likewise, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It should also be noted that the terms "including," "comprising," and "having" can be used interchangeably.

[0058] It should be understood that when an element is referred to as being "located on another element," "attached to another element," "connected to another element," "coupled to another element," "contacting another element," etc., the element may be directly located on, attached to, connected to, coupled to, contacting another element, or there may be intervening elements. In contrast, when an element is referred to as being, for example, "directly located on another element," "directly attached to another element," "directly connected to another element," "directly coupled to another element," or "directly contacting another element," there are no intervening elements. Those skilled in the art will also recognize that reference to a structure or feature being disposed "adjacent" to another feature may have portions that overlap or are located below the adjacent feature.

[0059] It should be understood that, although the terms first, second, third, etc. can be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms are only used to distinguish an element, component, region, layer or section from another element, component, region, layer or section. Therefore, without departing from the teachings of the present invention, the first element, component, region, layer or section discussed below can be referred to as the second element, component, region, layer or section.

[0060] In addition, as shown in the accompanying drawings, relative terms such as "lower" or "bottom" and "upper" or "top" may be used herein to describe the relationship of one element to another element. It should be understood that relative terms are intended to cover different orientations of the device other than the orientation depicted in the accompanying drawings. For example, if the device in one of the drawings is turned over, the element described as being located on the "lower side" of the other elements will then be oriented on the "upper side" of the other elements. Therefore, depending on the specific orientation of the drawings, the exemplary term "lower" can cover both the lower orientation and the upper orientation. Similarly, if the device in one of the drawings is turned over, the element described as being located "below" or "below" the other elements will then be oriented "above" the other elements. Therefore, the exemplary term "below" or "below" can cover both the upper orientation and the lower orientation.

[0061] It should be further understood that the terms “comprises” and / or “comprising”, or “includes” and / or “including”, or “has” and / or “having”, or “carry” and / or “carrying”, or “contain” and / or “containing”, or “involve” and / or “involving”, “characterized by” and the like are open ended, i.e., mean including but not limited to. When used in the present disclosure, the terms specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0062] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. It should be further understood that terms (such as those defined in commonly used dictionaries) should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology and the present invention, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0063] As used in this disclosure, "approximately," "about," "roughly," or "substantially" shall generally mean within 20%, preferably within 10%, and more preferably within 5% of a given value or range. The numerical quantities given herein are approximate, meaning that the term "approximately," "about," "roughly," or "substantially" can be inferred without explicitly stating.

[0064] As used in this disclosure, the phrase at least one of A, B, and C should be construed to mean a logical (A or B or C), using a non-exclusive logical or. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0065] As used in the present disclosure, the term "target microRNA" or "target miRNA" refers to a microRNA sequence that is sought to be amplified and / or quantified. The target miRNA can be obtained from any source and can include any number of different constituent components. The target microRNA can be a marker for determining the condition of a subject. The condition can be a physiological or mental condition. Further, it should be understood that "target miRNA" can refer to the target miRNA itself as well as its substitutes, such as amplification products and native sequences. The target miRNA of the present teachings can be derived from any of a variety of sources, including but not limited to viruses, prokaryotes, eukaryotes, such as but not limited to plants, fungi and animals. These sources can include but are not limited to whole blood, tissue biopsies, lymph, bone marrow, amniotic fluid, hair, skin, semen, biological warfare agents, anal secretions, vaginal secretions, sweat, saliva, oral swabs, various environmental samples (e.g., agriculture, water and soil), general research samples, generally purified samples, cultured cells and lysed cells. It should be understood that any of the various procedures known in the art can be used to separate the target miRNA from the sample. Typically, the target miRNA of the present teachings will be single-stranded.

[0066] As used in the present disclosure, the term "reverse transcription reaction" refers to an extension reaction, wherein the 3' target-specific portion of the stem-loop primer is extended to form an extension reaction product comprising a chain complementary to the target miRNA. In certain embodiments, the target miRNA is a miRNA molecule, and the extension reaction is a reverse transcription reaction comprising a reverse transcriptase, wherein the 3' end of the stem-loop primer is extended. In certain embodiments, the extension reaction is a reverse transcription reaction comprising a polymerase derived from true bacteria. In certain embodiments, the extension reaction can include rTth polymerase, such as the polymerase with catalog numbers N808-0192 and N808-0098 commercially available from Applied Biosystems. In certain embodiments, the target miRNA is a miRNA or other RNA molecule, and the use of the polymerase also comprising reverse transcription properties can allow the first reverse transcription reaction to be carried out in the same reaction vessel, followed by an amplification reaction, such as pre-amplification based on multiplex PCR, thereby allowing two reactions to be merged in a single reaction vessel. In certain embodiments, the target miRNA is a DNA molecule, and the extension reaction includes a polymerase and causes the synthesis of a complementary DNA chain. The term reverse transcription also includes the synthesis of the DNA complement of the template DNA molecule. Similarly, a reverse transcription product may be a DNA molecule synthesized in a reverse transcription reaction that is thus complementary to the template.

[0067] As used in the present disclosure, the term "reverse transcription reaction" refers to an extension reaction, in which the 3' target-specific portion of the stem-loop primer is extended to form an extension reaction product comprising a chain complementary to the target miRNA. In certain embodiments, the extension reaction is a reverse transcription reaction comprising a reverse transcriptase, in which the 3' end of the stem-loop primer is extended. In certain embodiments, the extension reaction is a reverse transcription reaction comprising a polymerase derived from true bacteria. In certain embodiments, the extension reaction may include rTth polymerase. In certain embodiments, the use of a polymerase also comprising a reverse transcription property may allow the first reverse transcription reaction to be carried out in the same reaction vessel, followed by an amplification reaction, such as multiplex fluorescence quantitative PCR, thereby allowing two reactions to be merged in a single reaction vessel. The reverse transcription product may be a DNA molecule synthesized in the reverse transcription reaction, so that it is complementary to the target miRNA template.

[0068] As used in the present disclosure, the term "hybridization" refers to the complementary base pairing interaction of a nucleic acid with another nucleic acid, which allows the formation of a duplex, triplex or other higher order structure, and can be used interchangeably with "annealing" in this article. Typically, the main interaction is base specific, such as A / T and G / C, through Watson / Crick and Hoogsteen type hydrogen bonds. Base stacking and hydrophobic interactions can also contribute to duplex stability. The conditions for hybridizing primers to complementary and substantially complementary target sequences are well known, for example, as described in "Nucleic Acid Hybridization, A Practical Approach", edited by B.Hames and S.Higgins, IRL Press (IRLPress), Washington, DC (Washington, DC) (1985) and J.Wetmur and N.Davidson, "Molecular Biology (Mol.Biol.)" 31:349et seq. (1968). Generally, whether such annealing occurs is affected by factors such as the length of the polynucleotide and the complementary polynucleotide, pH, temperature, the presence of monovalent and divalent cations, the ratio of G and C nucleotides in the hybridization zone, the viscosity of the medium, and the presence of denaturants. Such variables affect the time required for hybridization. Therefore, the preferred annealing conditions will depend on the specific application. However, such conditions can be routinely determined by a person of ordinary skill in the art without excessive experimentation. It should be recognized that complementarity is not necessarily perfect; there may be a small amount of base pair mismatches, which will minimally interfere with the hybridization between the target sequence and the single-stranded nucleic acid of this teaching. However, if the number of base pair mismatches is so large that hybridization cannot occur under the conditions of minimum stringency, the sequence is generally not a complementary target sequence. Therefore, complementarity herein refers to the primer and target sequence being fully complementary to hybridize under the selected reaction conditions, thereby achieving the purpose of this teaching.

[0069] As used in the present disclosure, the term "amplification" refers to any means of replicating at least a portion of a target miRNA or its surrogate, usually in a template-dependent manner, including but not limited to a wide range of techniques for amplifying nucleic acid sequences in a linear or exponential manner. In some embodiments, amplification can be achieved in a self-contained integrated method including sample preparation and detection, such as described in U.S. Patents No. 6,153,425 and No. 6,649,378. Amplified nucleic acids can use reversibly modified enzymes, such as but not limited to those described in U.S. Patent No. 5,773,258. This teaching also contemplates various uracil-based decontamination strategies, wherein, for example, uracil can be incorporated into the amplification reaction, and subsequent carryover products are removed using various glycosylase treatments (see, for example, U.S. Patent No. 5,536,649). It will be appreciated by those skilled in the art that any protein with a desired enzymatic activity can be used in the disclosed methods and kits.

[0070] As used in the present disclosure, the term "fluorescent quantitative PCR", "quantitative PCR" or "qPCR" refers to a PCR reaction performed in such a manner and under such controlled conditions that the result of the assay is quantitative, i.e., the assay is able to quantify the amount or concentration of nucleic acid ligands present in the test sample. qPCR is a technique based on polymerase chain reaction and is used to amplify and simultaneously quantify target nucleic acid molecules. qPCR allows the detection and quantification of specific sequences in DNA samples (in the form of absolute copy number or relative amount when normalized to DNA input or another normalizing gene). In some embodiments, the DNA sample is a product containing cDNA generated by reverse transcription of an RNA sample (e.g., miRNA). The procedure follows the general principles of PCR, with the additional feature that after each amplification cycle, the amplified DNA can be quantified as it accumulates in real time in the reaction. qPCR is described, for example, in Kurnit et al. (U.S. Pat. No. 6,033,854), Wang et al. (U.S. Pat. Nos. 5,567,583 and 5,348,853), Ma et al. (The Journal of American Science, 2(3), (2006), Heid et al. (Genome Research, 986-994, (1996), Sambrook and Russell (Quantitative PCR, Cold Spring Harbor Protocols, (2006)), and Higuchi (U.S. Pat. Nos. 6,171,785 and 5,994,056). The contents of these references are incorporated herein by reference in their entirety.

[0071] As used in this disclosure, the term "detection" refers to any of a variety of methods for determining the presence and / or quantity and / or identity of a target miRNA. In some embodiments employing a donor moiety and a signal moiety, certain energy transfer fluorescent dyes may be used. For example, certain non-limiting exemplary pairs of donors (donor moieties) and acceptors (signal moieties) are described in U.S. Patents Nos. 5,863,727, 5,800,996, and 5,945,526. The use of some combinations of donors and acceptors is referred to as FRET (fluorescence resonance energy transfer). In some embodiments, fluorophores that may be used as signaling probes include, but are not limited to, rhodamine, cyanine 3 (Cy 3), cyanine 5 (Cy 5), fluorescein, Vic TM , LiZ TM Tamra TM , 5-Fam TM 、6-Fam TM and Texas Red (Molecular Probes). (Vic TM , Liz TM Tamra TM , 5-Fam TM and 6-Fam TM). In some embodiments, the amount of the probe that gives the fluorescent signal in response to the excitation light is generally related to the amount of the nucleic acid produced in the amplification reaction. Therefore, in some embodiments, the amount of the fluorescent signal is related to the amount of the product produced in the amplification reaction. In such embodiments, the amount of the amplification product can therefore be measured by measuring the intensity of the fluorescent signal from the fluorescent indicator. According to some embodiments, an internal standard can be used to quantify the amplification product indicated by the fluorescent signal. See, for example, U.S. Patent No. 5,736,333. A device has been developed that can utilize a composition containing a fluorescent indicator to perform a thermal cycle reaction, emit a light beam of a specified wavelength, read the intensity of the fluorescent dye, and display the fluorescence intensity after each cycle. Devices including a thermal cycler, a beam emitter, and a fluorescent signal detector have been described in, for example, U.S. Patent Nos. 5,928,907, 6,015,674, and 6,174,670. In some embodiments, the combined thermal cycle and fluorescence detection device can be used to accurately quantify the target nucleic acid sequence in the sample. In some embodiments, the fluorescent signal can be detected and displayed during and / or after one or more thermal cycles, thereby allowing the monitoring of the amplification product when the reaction occurs in "real time". In some embodiments, the amount of amplified product and the number of amplification cycles can be used to calculate how much target nucleic acid sequence is in the sample before amplification. In some embodiments, the amount of amplified product can be simply monitored after a predetermined number of cycles sufficient to indicate the presence of the target nucleic acid sequence in the sample. One skilled in the art can easily determine how many cycles are sufficient to determine the presence of a given target miRNA for any given sample type, primer sequence, and reaction conditions. As used herein, determining the presence of a target can include identifying the target, and optionally quantifying the target. In some embodiments, once a given number of cycles are completed, the amplified product can be scored as positive or negative. In some embodiments, the results can be electronically transmitted directly to a database and tabulated. Therefore, in some embodiments, when such instruments are used, a large number of samples can be processed and analyzed with less time and labor. In some embodiments, different detector probes can distinguish between different target miRNAs. Non-limiting examples of such probes are 5'-nuclease fluorescent probes, such as Probe molecule or MGB probe, wherein the fluorescent molecule is attached to the fluorescent quenching molecule through an oligonucleotide connecting element. In some embodiments, the oligonucleotide connecting element of the 5'-nuclease fluorescent probe is combined with a specific sequence of the identification part or its complement. In some embodiments, different 5'-nuclease fluorescent probes that each fluoresce at different wavelengths can distinguish different amplification products in the same amplification reaction. For example, in some embodiments, two different 5'-nuclease fluorescent probes can be used, and the two probes fluoresce at two different wavelengths (WLA and WLB), and are specific to two different regions of two different extension reaction products (A and B, respectively). If the target miRNA A is in the sample, an amplification product A is formed, and if the target polynucleotide B is in the sample, an amplification product B is formed. After amplification, it can be determined which specific target nucleic acid sequences are present in the sample based on the wavelength of the detected signal and its intensity. Therefore, if only the appropriate detectable signal value of the wavelength WLA is detected, it is known that the sample contains target miRNA A, but does not contain target miRNA B. If the appropriate detectable signal value of both wavelengths WLA and WLB is detected, it is known that the sample contains both target miRNA A and target miRNA B.

[0072] As used in this disclosure, the term "detector probe" or "probe" refers to a molecule that is typically used in quantitative or real-time PCR analysis and endpoint analysis in an amplification reaction. Such probes can be used to monitor the amplification of the reverse transcription product of the target microRNA. In some embodiments, the probe present in the amplification reaction is suitable for monitoring the amount of amplicon produced over time. Such detector probes include, but are not limited to, 5'-exonuclease assays (described herein). probes) (see also U.S. Pat. No. 5,538,848), various stem-loop molecular beacons (see, e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517, and Tyagi and Kramer, 1996, Nature Biotechnology 14:303-308), stemless or linear beacons (see, e.g., WO 99 / 21881), PNA molecular beacons TM (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, e.g., Kubista et al., 2001, SPIE 4264:53-58), non-FRET probes (see, e.g., U.S. Pat. No. 6,150,097), Probes (U.S. Pat. No. 6,548,250), stem-loops, and double-stranded Scorpion TMprobes (Solinas et al., 2001, Nucleic Acids Research 29:E96 and U.S. Patent No. 6,589,743), raised loop probes (U.S. Patent No. 6,590,091), pseudoknot probes (U.S. Patent No. 6,589,250), hybridization-based fluorescent primer probes (U.S. Patent No. 6,383,752), MGB Eclipse TMprobes (Epoch Biosciences), hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA) luminescent probes, self-assembled nanoparticle probes, and ferrocene-modified probes such as those described in U.S. Pat. No. 6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et al., 1999, Nature Biotechnology 17:804-807; lsacsson et al., 2000, Molecular Cell Probes 14:321-328; Svanvik et al., 2000, Analytical Biochemistry 14:331-338. =Biochem. 281:26-35; Wolffs et al., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002, Nucleic Acids Res. 30:4208-4215; Riccelli et al., 2002, Nucleic Acids Res. 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332; Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et al., 2002, Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem. Toxicologica Sinica. Res. Toxicol. 15: 118-126; and Yu et al., 2001, Journal of the American Chemical Society 14: 11155-11161. The probe may also include a quencher, including but not limited to Black Hole Quencher (Biosearch), Iowa Black (IDT), QSY Quencher (Molecular Probes), and Dabsyl and Dabcel Sulfonate / Carboxylate Quenchers (Epoch). The probe may also include two probes, where, for example, the fluorophore is on one probe and the quencher is on the other probe, where the two probes hybridize together on the target to quench the signal, or where hybridization on the target changes the signal characteristics through fluorescence changes. The probe may also include sulfonate derivatives of fluorescein dyes with S03 instead of a carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of CY 5 (commercially available, for example, from Amersham). In some embodiments, an embedded label such as ethidium bromide, Green I (Molecular Probes) and (Molecular Probes), thereby allowing visualization of amplification products in real time or at an endpoint in the absence of a probe. In some embodiments, real-time visualization may include an embedded probe, and a sequence-based detector probe may be employed. In some embodiments, the detector probe is at least partially quenched when it is not hybridized with a complementary sequence in an amplification reaction, and is at least partially unquenched when it is hybridized with a complementary sequence in an amplification reaction. In some embodiments, the probe may further include various modifications, such as a small groove binder (see, e.g., U.S. Patent 6,486,308), to further provide desired thermodynamic properties. In some embodiments, the detector probe may correspond to a zip-code introduced by a stem-loop reverse transcription primer.

[0073] As used in the present disclosure, the term "stem-loop primer" or "stem-loop reverse transcription primer" refers to a molecule that includes an anchor sequence at its 3' end and a stem-loop structure at its 5' end. The stem-loop structure includes a stem portion and a loop portion. The term "anchor sequence" refers to the single-stranded portion of the stem-loop primer that is complementary to the target miRNA. The anchor sequence is located downstream of the stem-loop structure of the stem-loop primer. Typically, the length of the anchor sequence is between 3 and 12 nucleotides. The term "stem" refers to the double-stranded region between the anchor sequence and the loop of the stem-loop structure of the stem-loop primer and is discussed more fully below. The term "loop" refers to the region between the two complementary chains of the stem-loop sequence of the stem-loop sequence. Typically, the loop includes single-stranded nucleotides, but it is also possible to include other parts of modified RNA, carbon spacers such as C18 and / or PEG (polyethylene glycol). Typically, the length of the loop is between 4 and 30 nucleotides. In some embodiments, the length of the loop is between 14 and 18 nucleotides. In some embodiments, the length of the loop is 16 nucleotides. Those skilled in the art will appreciate that loops shorter than 4 nucleotides and longer than 20 nucleotides can be identified in the course of routine methods without undue experimentation, and such shorter and longer loops are contemplated by the present teachings.

[0074] As used in the present disclosure, the term "Tm enhancing tail" refers to a small number of nucleobases, typically between 3 and 10, included at the 5' end of the forward primer used in the qPCR reaction. The Tm enhancing tail is not complementary to the reverse transcription product. In some embodiments, the Tm enhancing tail is 4 bases. In some embodiments, the Tm enhancing tail is 5 bases. In some embodiments, the Tm enhancing tail is 6 bases. In some embodiments, the Tm enhancing tail is 7 bases. Generally, longer Tm enhancing tails are possible, but this will be at the expense of increased oligonucleotide manufacturing costs, will further increase reaction complexity, and may increase Tm to undesirable levels.

[0075] As used in the present disclosure, "multiple" refers to the reaction of multiple target DNA / RNA and / or targets in or from multiple samples that are transcribed, amplified or quantified in the same reaction. In some embodiments of any aspect in aspect, multiple reverse transcription reaction can include the reverse transcription of 1 to 100 target nucleotide sequences. As a non-limiting example, multiple reverse transcription reaction can include the reverse transcription of about 1 sample, about 2 samples, about 3 samples, about 4 samples, about 5 samples, about 6 samples, about 7 samples, about 8 samples, about 9 samples, about 10 samples, about 20 samples, about 30 samples, about 40 samples, about 50 samples, about 60 samples, about 70 samples, about 80 samples, about 90 samples, about 100 samples. "Single weight" refers to the reaction of transcribing, amplifying or quantifying only one target DNA / RNA in the same reaction.

[0076] As used in the present disclosure, a "fragment" or "portion" of a nucleotide sequence refers to a nucleotide sequence that is reduced in length (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) relative to a reference nucleic acid or nucleotide sequence and that includes, consists essentially of, and / or consists of contiguous nucleotides that are identical or nearly identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 990%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 11112, 113, 114, 15 This nucleic acid fragment or part according to the present invention may, where appropriate, be included in a larger polynucleotide of which the nucleic acid fragment or part is a component. Thus, hybridizing with (or hybridizing with, and other grammatical variants thereof, for example, at least a portion of a target miRNA or cDNA refers to hybridizing with a nucleotide sequence that is identical or substantially identical in length to a continuous nucleotide sequence of the target miRNA or cDNA.

[0077] As used in the present disclosure, a "heterologous" or "recombinant" nucleotide sequence is a nucleotide sequence that is not naturally associated with the host cell into which it is introduced, comprising non-naturally occurring multiple copies of a naturally occurring nucleotide sequence. Different nucleic acids or proteins with homology are referred to herein as "homologs". The term homolog comprises homologous sequences from the same and other species and orthologous sequences from the same and other species. "Homology" refers to the level of similarity between two or more nucleic acid and / or amino acid sequences in terms of positional identity (i.e., sequence similarity or identity) percentage. Homology also refers to the concept of similar functional properties between different nucleic acids or proteins. Therefore, the compositions and methods of the present invention further include homologs of the nucleotide sequences and polypeptide sequences of the present invention. "Orthologs" as used herein refers to homologous nucleotide sequences and / or amino acid sequences produced by common ancestral genes in different species during speciation. Homologs of the nucleotide sequences of the invention have substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and / or 100%) to the nucleotide sequences of the invention.

[0078] As used in the present disclosure, the term "percentage of sequence identity" or "percentage of identity" refers to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, "percentage of identity" may refer to the percentage of identical amino acids in an amino acid sequence.

[0079] As used in the present disclosure, in the context of two nucleic acid molecules, nucleotide sequences or protein sequences, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection, the phrases "substantially identical" or "significant identity" refer to two or more sequences or subsequences with at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and / or 100% nucleotide or amino acid residue identity. In some embodiments of the invention, significant identity is present in a region of a sequence of at least about 5 residues to about 150 residues in length. Thus, in some embodiments of the invention, significant identity exists over a region of at least about 3 to about 15 residues in length (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 residues in length, etc., or any value or range therein), at least about 2 to about 30, at least about 5 to about 30, at least about 10 to about 30, at least about 16 to about 30, at least about 18 to at least about 25, at least about 18, at least about 22, at least about 25, at least about 30, at least about 40, at least about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150 or more residues, and any range therein. In representative embodiments, the sequences may be substantially identical over at least about 15 nucleotides. In some specific embodiments, the sequences are substantially identical over at least about 150 residues. In some embodiments, the sequence of the present invention can be about 70% to about 100% identical at least about 15 nucleotides to about 25 nucleotides. In some embodiments, the sequence of the present invention can be about 75% to about 100% identical at least about 15 nucleotides to about 25 nucleotides. In other embodiments, the sequence of the present invention can be about 80% to about 100% identical at least about 15 nucleotides to about 25 nucleotides. In other embodiments, the sequence of the present invention can be about 80% to about 100% identical at least about 7 nucleotides to about 25 nucleotides. In some embodiments, the sequence of the present invention can be about 70% identical at least about 15 nucleotides. In other embodiments, the sequence can be about 85% identical at about 22 nucleotides. In still other embodiments, the sequence can be 100% homologous at about 15 nucleotides. In other embodiments, the sequence is substantially identical over the entire length of the coding region. In addition, in representative embodiments, substantially identical nucleotides or protein sequences perform substantially the same functions, such as reverse transcription.

[0080] In order to compare sequences, usually one sequence serves as a reference sequence to be compared with a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters.

[0081] Optimal alignment of sequences for comparison windows is well known to those skilled in the art and can be performed by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and optionally by computerized implementations of these algorithms, such as those provided by GCG Wisconsin (Accelrys Inc., San Diego, Calif.)). The "identity score" of the comparison segment of the test sequence and the reference sequence is the number of identical components shared by the two comparison sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. The percentage of sequence identity is expressed as the identity score multiplied by 100. The comparison of one or more polynucleotide sequences can be with a full-length polynucleotide sequence or a portion thereof, or with a comparison of a longer polynucleotide sequence. For purposes of the present invention, the "identity percentage" can also be determined using BLASTX 2.0 version for translating nucleotide sequences and BLASTN 2.0 version for polynucleotide sequences.

[0082] Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which, when compared with words of the same length in the database sequence, match or satisfy a positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing the initial neighborhood word hits. The word hits are then extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, cumulative scores are calculated using parameters M (a reward score for a pair of matching residues: always>0) and N (penalty for mismatched residues; always<0). For amino acid sequences, cumulative scores are calculated using a scoring matrix. Extension of the word hits in each direction will cease when: the cumulative alignment score drops by the amount X from its maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both chains as defaults. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0083] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match would occur by chance between two nucleotide or amino acid sequences. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of a test nucleotide sequence to a reference nucleotide sequence is less than about 0.1 to less than about 0.001. Thus, in some embodiments of the invention, the smallest sum probability in a comparison of a test nucleotide sequence to a reference nucleotide sequence is less than about 0.001.

[0084] When two nucleotide sequences hybridize to each other under stringent conditions, the two sequences may also be considered to be substantially complementary. In some representative embodiments, two nucleotide sequences that are considered to be substantially complementary hybridize to each other under highly stringent conditions.

[0085] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments (such as Southern and Northern hybridizations) are sequence-dependent and are different under different environmental parameters. An extensive guide to nucleic acid hybridization is found in the following literature: Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993). Typically, highly stringent hybridization and washing conditions are selected to be higher than the thermal melting point (T) of the specific sequence at a defined ionic strength and pH. m ) is about 5℃ lower.

[0086] The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a specific probe. m. In Southern or northern blotting, an example of stringent hybridization conditions for hybridization of complementary nucleotide sequences with more than 100 complementary residues on the filter is 50% formamide with 1 mg heparin at 42°C, where hybridization is performed overnight. An example of highly stringent washing conditions is 0.15M NaCl at 72°C for about 15 minutes. An example of stringent washing conditions is 0.2×SSC washing at 65°C for 15 minutes (see Sambrook, below for a description of SSC buffer). Typically, a low stringency wash is performed before a high stringency wash to remove background probe signals. For example, an example of a medium stringency wash for a duplex of more than 100 nucleotides is 1×SSC at 45°C for 15 minutes. For example, an example of a low stringency wash for a duplex of more than 100 nucleotides is 4-6×SSC at 40°C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and a temperature of typically at least about 30° C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Typically, a signal-to-noise ratio of 2× (or higher) compared to the signal-to-noise ratio observed for an unrelated probe in a particular hybridization assay indicates that a specific hybridization has been detected. If the proteins encoded by nucleotide sequences that do not hybridize to each other under stringent conditions are substantially identical, the nucleotide sequences are still substantially identical. For example, this occurs when a copy of a nucleotide sequence is created using the maximum codon degeneracy allowed by the genetic code.

[0087] The multiplex real-time fluorescence qPCR (or qPCR) detection / quantification described in the present invention refers to a process in which two or more qPCR systems are configured to perform qPCR reactions simultaneously in one PCR tube, and thus two or more multiplex real-time fluorescence quantifications are performed together.

[0088] The present invention provides a multiplex quantitative method for miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p. The method comprises two steps. The first step involves reverse transcription of each target miRNA in a sample using a stem-loop reverse transcription primer. The stem-loop sequence in each reverse transcription primer is different. Then, in the second step, multiplex qPCR detection / quantification is performed together for all reverse transcription products. In the reverse transcription of the first step, miR-16-5p is reverse transcribed using a first stem-loop reverse transcription primer with 8 anchor bases. miR-34c-5p is reverse transcribed using a second stem-loop reverse transcription primer with 6 anchor bases. miR-9-3p is reverse transcribed using a third stem-loop reverse transcription primer with 6 anchor bases. miR-9-5p is reverse transcribed using a fourth stem-loop reverse transcription primer with 11 anchor bases.

[0089] The present invention also provides a multiplex quantitative / detection kit for miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p, the kit comprising a reverse transcription reagent. The reverse transcription reagent comprises two or more stem-loop reverse transcription primers in the first stem-loop reverse transcription primer, the second stem-loop reverse transcription primer, the third stem-loop reverse transcription primer and the fourth stem-loop reverse transcription primer, wherein the first stem-loop reverse transcription primer is used for the reverse transcription of miR-16-5p and contains 8 anchor bases. The second stem-loop reverse transcription primer is used for the reverse transcription of miR-34c-5p and contains 6 anchor bases. The third stem-loop reverse transcription primer is used for the reverse transcription of miR-9-3p and contains 6 anchor bases. The fourth stem-loop reverse transcription primer is used for the reverse transcription of miR-9-5p and contains 11 anchor bases.

[0090] The length of the anchor sequence in the stem-loop reverse transcription primer is considered to be related to the efficiency of reverse transcription and the specificity of the stem-loop reverse transcription primer itself. Surprisingly, it is found in the present invention that when quantifying multiple miRNAs, the combination of stem-loop reverse transcription primers in which each stem-loop reverse transcription primer has a specific anchor sequence length shows outstanding multi-specificity. That is, each stem-loop reverse transcription primer in the stem-loop reverse transcription primer effectively and only reversely transcribes the target miRNA complementary to its anchor sequence.

[0091] Therefore, in one PCR tube, multiple RT can be performed on a sample containing multiple target miRNAs using a combination of stem-loop reverse transcription primers, and then multiple qPCR can be performed in the same PCR tube. In terms of amplification curves and Ct values, the quantitative results obtained by performing multiple RT-qPCR in the same tube are substantially the same as those of single RT-single qPCR detection. Therefore, the present invention can be used for clinical diagnosis of diseases.

[0092] Examples

[0093] Target miRNA

[0094] In this embodiment, in the multiplex RT-qPCR process, the target miRNA associated with Alzheimer's disease is quantified using the selected multiplex stem-loop reverse transcription primer combination. In one embodiment, the target miRNA comprises miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p. The nucleotide sequences of all four miRNAs are reported in the microRNA database www.mirbase.org. Specifically, the sequence of miR-16-5p is shown as SEQ ID No.1, the sequence of miR-34c-5p is shown as SEQ ID No.2, the sequence of miR-9-3p is shown as SEQ ID No.3, and the sequence of miR-9-5p is shown as SEQ ID No.4.

[0095] RNA template and mixed RNA template

[0096] Each single target miRNA template comprises a single synthetic RNA template of each of miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p, respectively. The mixed target miRNA template comprises a mixture of two or more single target miRNA templates in a desired ratio.

[0097] Principles for designing stem-loop reverse transcription primers

[0098] The design principle of stem-loop reverse transcription primer is described as follows.The stem-loop reverse transcription primer comprises 5' end, stem-loop sequence, anchor base sequence and 3' end from its 5' to 3' end successively.The number of nucleotide bases in every kind of stem-loop reverse transcription primer in the stem-loop reverse transcription primer used is usually between 40 and 65, wherein in the stem-loop sequence, a double-stranded region is formed by a pair of mutually complementary fragments.This double-stranded region is "stem", and usually has 11-14 paired nucleotide bases.The unpaired region between the complementary fragment pairs is prominently formed "ring", and the ring is usually a sequence of 16-36 nucleotide bases.In one embodiment, design has the stem-loop reverse transcription primer with different stem-loop sequences, and its stem-loop sequence is as shown in table 1 below.

[0099] Table 1. Stem-loop sequences of stem-loop reverse transcription primers

[0100]

[0101] Oligo Analyzer (idtdna.com) was used to analyze and compare the differences and interferences between the stem-loop sequences of the designed stem-loop reverse transcription primers. In this example, a stem-loop reverse transcription primer containing a stem-loop structure 1 (SEQ ID No. 9) was selected for quantification / detection of miR-16-5p using multiple RT-qPCR; a stem-loop reverse transcription primer containing a stem-loop structure 2 (SEQ ID No. 10) was selected for quantification / detection of miR-34c-5p using multiple RT-qPCR; a stem-loop reverse transcription primer containing a stem-loop structure 3 (SEQ ID No. 11) was selected for quantification / detection of miR-9-3p using multiple RT-qPCR; a stem-loop reverse transcription primer containing a stem-loop structure 4 (SEQ ID No. 12) was selected for quantification / detection of miR-9-5p using multiple RT-qPCR. Table 2 reflects the stem-loop sequences selected for each stem-loop reverse transcription primer.

[0102] Table 2. Stem-loop sequences selected for each stem-loop reverse transcription primer

[0103]

[0104] The anchor sequence of the stem-loop reverse transcription primer is located downstream of the stem-loop sequence. The anchor sequence is complementary to the 3' sequence of the target miRNA, and the stem-loop reverse transcription primer is designed for reverse transcription of the target miRNA. The length of the anchor sequence is generally between 3-12nt. In one embodiment, the length of the anchor sequence is between 4-11nt. In one embodiment, the length of the anchor sequence is between 4-8nt. In one embodiment, the length of the anchor sequence is between 6-11nt. In one embodiment, the length of the anchor sequence is between 6-8nt.

[0105] Principles used to design forward primers

[0106] The 5' end to 3' end of the forward primer is: 5'-Tm enhancement tail-specific complementary sequence-3', wherein the Tm enhancement tail includes randomly arranged A, T, C or G to increase the Tm value of the forward primer. The GC content of the forward primer is usually between 50% and 60%, and the 3' end of the primer should avoid having three or more consecutive G or C bases. The length of the Tm enhancement tail of the forward primer should be selected to ensure that the difference between the Tm values ​​of the forward primer and the reverse primer should be within 1°C, and the Tm values ​​of the forward primer and the reverse primer should be about 55-60°C. The designed forward primer is shown in Table 3.

[0107] Table 3. Sequences of forward primers used for each target miRNA in qPCR

[0108]

[0109]

[0110] The naming format of the forward primer is: target miRNA-F. For example, if the forward primer is named miR-16-5p-F, the forward primer is used for qPCR amplification of miR-16-5p.

[0111] Principles used to design reverse primers

[0112] The length of the reverse primer is usually about 15-25 nt, the Tm value is 55-60°C, and the GC content is 50%-60%. The 3' end of the primer should avoid having three or more consecutive G or C bases, and the Tm values ​​of the forward primer and the reverse primer should be within 1°C of each other. In order to improve the amplification efficiency, the reverse primer should be designed to be as close to the loop of the stem-loop structure as possible. The sequence of the designed reverse primer is shown in Table 4.

[0113] Table 4. Sequences of reverse primers used for each target miRNA in qPCR

[0114]

[0115] The reverse primer is named in the format of: target miRNA–R. For example, if the reverse primer is named miR-16-5p-R, the reverse primer is used for qPCR amplification of miR-16-5p.

[0116] Principles used to design probes

[0117] In one embodiment, The probe is used as a probe in a qPCR reaction. The length of each probe is typically in the range of 25 nt to 32 nt, and the Tm value is between 65-75°C, 5-10°C higher than the Tm value of the forward / reverse primer. The GC content is 30-80%. The first base at the 5' end of each probe in the probe cannot be G, and should be as close as possible to the 3' end of the target miRNA sequence. The 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group. The fluorescent reporter group can be selected from fluorescent reporter groups such as FAM, VIC, CY5 or ROX. The designed probes are shown in Table 5.

[0118] Table 5. Probe sequences in qPCR

[0119]

[0120] The naming format of the probe is: target miRNA-fluorescent reporter group. For example, if the probe is named miR-16-5p-VIC, the probe is used for the detection of miR-16-5p in qPCR, and VIC is used as the fluorescent reporter group on the 5' end of the probe.

[0121] In one embodiment, in the above-mentioned multiplex qPCR, the design of each forward primer and each probe should satisfy the following principles: the 3' end of each forward primer sequence has a Tm enhancement sequence less than 8 nt in length. The 5' end of each forward primer sequence has a sequence that is the same as the DNA equivalent of the 5' end sequence of the target miRNA (replacing any U of the target miRNA with T) and is between 9-13 nt in length. The 5' end of each probe sequence has a sequence that is the same as the DNA equivalent of the 3' end sequence of the target miRNA and is between 9-12 nt in length. The 3' end of each probe sequence has a sequence that is the same as the 5' end sequence of the stem-loop sequence and is greater than 6 nt in length.

[0122] The forward / reverse primers and probes were synthesized by Hippobio Ltd., Huzhou, Zhejiang, China.

[0123] Extraction of miRNA samples from biological samples

[0124] In one embodiment, peripheral blood is used as the biological sample for miRNA extraction. It should be noted that other biological samples, such as cerebrospinal fluid, epithelial cells or bones can be used as the biological sample for miRNA extraction.

[0125] Serum processing: With informed consent from all subjects, 5 mL of peripheral blood was collected from each participant using a coagulant separation gel vacuum blood collection tube. After standing for 30 minutes, the sample was centrifuged at 3000×g for 10 minutes at room temperature, and the upper serum was collected and stored at −80°C for later use.

[0126] Extraction of serum miRNA: Qiagen serum / plasma miRNA extraction kit with column-based nucleic acid extraction method was used. 200 μL serum was used according to the instructions of the kit, and then 20 μL ddH2O was added for dissolution. The extracted miRNA was stored at -80 °C for later use.

[0127] Reverse transcription system and process

[0128] 1 μL of mixed target miRNA template or water was mixed with 2 μL of 10X RT mixture, 2 μL of HiScript II enzyme mixture and 1 μL of one of the stem-loop reverse transcription primers. The reaction volume was adjusted to 20 μL with ddH2O and then centrifuged briefly before performing reverse transcription. Table 6 shows the system used for reverse transcription.

[0129] Table 6. Systems for reverse transcription of target miRNA using each of the stem-loop reverse transcription primers

[0130]

[0131] The reverse transcription process was performed according to the thermal cycle in Table 7.

[0132] Table 7. Thermal cycles for reverse transcription.

[0133]

[0134] The reverse transcription products were immediately subjected to qPCR reactions or stored at -20°C.

[0135] When performing single reverse transcription, the specific stem-loop reverse transcription primer for reverse transcription of specific target miRNA is added to the above-mentioned reverse transcription system at a concentration of 2 μM and an amount of 0.5 μL. For multiple reverse transcription, all stem-loop reverse transcription primers (each of the stem-loop reverse transcription primers is selected for reverse transcription of one of multiple target miRNAs) should be added to the reverse transcription system at a concentration of 2 μM and an amount of 0.5 μL for each of the stem-loop reverse transcription primers. The stem-loop sequences of the stem-loop reverse transcription primers used to participate in the following experiments are shown in Table 2.

[0136] qPCR systems and procedures

[0137] Take 2 μL of the reverse transcription product and perform qPCR quantification according to the following qPCR system and program in Tables 8 and 9.

[0138] Table 8. Systems used for qPCR of reverse transcription products

[0139]

[0140] Table 9. Thermal cycling of qPCR

[0141]

[0142] When performing single-plex qPCR for only one specific target miRNA, the forward primer, reverse primer, and probe designed for the specific target miRNA are used in the qPCR system to quantify the specific target miRNA. In contrast, during multiplex qPCR, forward primer combinations, reverse primer combinations, and probe combinations designed for each and all target miRNAs are included to quantify multiple target miRNAs simultaneously. The forward primers, reverse primers, and probes involved in the experiment are shown in Tables 3-5.

[0143] Specificity of stem-loop reverse transcription primers based on anchor sequence length

[0144] Each of the stem-loop reverse transcription primers selected for one of the target miRNAs was formed into three variants; and each of the variants had a selected stem-loop structure connected to the following anchor sequences of different lengths: 6nt, 8nt, 11nt. Therefore, for each target miRNA, three stem-loop reverse transcription primer variants were formed, and each of the variants was used individually for single reverse transcription of a single target miRNA template, each template containing one of miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p. The single reverse transcription product was then subjected to single qPCR quantification.

[0145] Table 10. Stem-loop reverse transcription primers with anchor sequences of different lengths

[0146]

[0147] like Figure 1A As shown in Table 11, each of the stem-loop reverse transcription primer variants specifically reverse transcribes the target miRNA for which it is designed to reverse transcribe, and each of the stem-loop reverse transcription primer variants exhibits a negative result for the negative control group using ddH2O, indicating good specificity. The results are shown in Table 11.

[0148] Table 11. Singleplex RT-singleplex qPCR using each of the stem-loop reverse transcription primer variants

[0149]

[0150] Screening for anchor sequence lengths that achieve multi-specificity in stem-loop reverse transcription primer combinations

[0151] The following paragraphs describe an example in which the multi-specificity of the stem-loop reverse transcription primer is achieved by adjusting and / or screening the length of the anchor sequence of each stem-loop reverse transcription primer in the stem-loop reverse transcription primer. During the screening process, the present invention separately realizes a dual stem-loop reverse transcription primer combination and its use in dual RT-qPCR, a triple stem-loop reverse transcription primer combination and its use in triple RT-qPCR, and a final quadruple stem-loop reverse transcription primer combination and its use in quadruple RT-qPCR. Specifically, the dual stem-loop reverse transcription primer combination comprises two kinds of stem-loop reverse transcription primers, and enables two kinds of target miRNAs in the target miRNA to be quantified simultaneously in dual RT-qPCR. The triple stem-loop reverse transcription primer combination comprises three kinds of stem-loop reverse transcription primers, and enables three kinds of target miRNAs in the target miRNA to be quantified simultaneously in triple RT-qPCR. Therefore, a quadruple stem-loop reverse transcription primer combination containing four stem-loop reverse transcription primers was constructed, and the combination effectively quantified miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p in quadruple RT-qPCR. Based on homology restriction, the screening method used in this example was extended to multiplex quantitative detection of other target miRNAs.

[0152] Table 10 shows the stem-loop reverse transcription primers used in the experiments, while Tables 3-5 show the forward and reverse primers and probes used in the qPCR process.

[0153] In one embodiment of the present invention, the multiplex system comprises at least a multiplex stem-loop reverse transcription primer combination having multiple stem-loop reverse transcription primers, each of the multiple stem-loop reverse transcription primers corresponds to a target miRNA, and reverse transcribes the target miRNA in a single or multiplex RT reaction. In one embodiment, the multiplex system further comprises multiple forward primers, multiple reverse primers, and multiple probes, each of the multiple forward primers, each of the multiple reverse primers, and each of the probes corresponds to a target miRNA, and specifically participates in a single or multiplex qPCR of a reverse transcription product of the target miRNA.

[0154] In one embodiment, the multiplex system refers to a duplex system, wherein two stem-loop reverse transcription primers are used to quantify two target miRNAs in a duplex RT-qPCR. In one embodiment, the multiplex system refers to a triplex system, wherein three stem-loop reverse transcription primers are used to quantify three target miRNAs in a triplex RT-qPCR. In one embodiment, the multiplex system refers to a quadruple system, wherein four stem-loop reverse transcription primers are used to quantify four target miRNAs in a quadruple RT-qPCR. In one embodiment, the multiplex system includes more than four stem-loop reverse transcription primers, and the more than four stem-loop reverse transcription primers are used to quantify more than four target miRNAs in a multiplex RT-qPCR.

[0155] Dual system for quantification of two target miRNAs

[0156] The dual system comprises: a stem-loop reverse transcription primer combination, the stem-loop reverse transcription primer combination comprising any two stem-loop reverse transcription primers each showing specificity for one of the target miRNAs; their corresponding forward / reverse primers; and a probe. Dual RT-qPCR is performed to verify the sensitivity and specificity of the dual stem-loop reverse transcription primer combination. In one embodiment, two target miRNAs in the target miRNA are randomly selected to form one or more combinations.

[0157] In one embodiment, two combinations are formed:

[0158] Target miRNA combination 1: miR-34c-5p and miR-9-3p;

[0159] Target miRNA combination 2: miR-34c-5p and miR-16-5p.

[0160] For mixed RNA templates with target miRNAs in combination 1 or combination 2 above, dual RT was performed using corresponding stem-loop reverse transcription primers with anchor sequences of different lengths with mixed RNA templates and ddH2O as a negative control. Dual RT-qPCR quantification was performed using dual reverse transcription primers for mixed RNA template / negative control. Amplification results were observed to quantify target miRNAs in mixed RNA templates. The specificity of each stem-loop reverse transcription primer in the stem-loop reverse transcription primers with anchor sequences of different lengths was observed. The anchor length of each stem-loop reverse transcription primer in the two stem-loop reverse transcription primers in the dual system was determined based on the observed specificity.

[0161] Based on the above steps, it was finally determined that for miR-34c-5p, a stem-loop reverse transcription primer with an anchor sequence length of 6 nt was selected, and the stem-loop reverse transcription primer had SEQ ID No. 6. For miR-16-5p, a stem-loop reverse transcription primer with an anchor sequence length of 8 nt was selected, and the stem-loop reverse transcription primer had SEQ ID No. 5. For miR-9-3p, a stem-loop reverse transcription primer with an anchor sequence length of 6 nt was selected, and the stem-loop reverse transcription primer had SEQ ID No. 7.

[0162] Specificity of dual stem-loop reverse transcription primer combinations

[0163] Target miRNA combination 1 of miR-34c-5p and miR-9-3p was used to form mixed target miRNA template 1, and target miRNA combination 2 of miR-34c-5p and miR-16-5p was used to form mixed target miRNA template 2.

[0164] Each of the mixed target miRNA templates 1 and 2 was individually subjected to singleplex RT-qPCR, wherein each of the two corresponding stem-loop reverse transcription primers selected for the two target miRNAs was used for each of the singleplex RT-qPCR.

[0165] Figure 2A -F shows the results of four single-plex RT-qPCR of two target miRNA combinations 1 and 2. Specifically, Figure 2A -C shows that the stem-loop reverse transcription primer (SEQ ID No.7) of miR-9-3p specifically reverses miR-9-3p, and the stem-loop reverse transcription primer (SEQ ID No.6) of miR-34c-5p specifically reverses miR-34c-5p. Both stem-loop reverse transcription primers showed no signal in the negative control group using ddH2O, as shown in FIG. Figure 2C As shown. Figure 2D -F shows that the stem-loop reverse transcription primer (SEQ ID No. 5) of miR-16-5p specifically reverses miR-16-5p, and the stem-loop reverse transcription primer (SEQ ID No. 6) of miR-34c-5p specifically reverses miR-34c-5p. Both stem-loop reverse transcription primers showed no signal in the negative control group using ddH2O, as shown in FIG. Figure 2F shown.

[0166] Once the singleplex RT-qPCR showed good specificity for each selected corresponding stem-loop reverse transcription primer, duplex RT-qPCR was performed to determine whether there was any undesired cross-reaction between the two corresponding stem-loop reverse transcription primers selected for target miRNA combination 1 and 2.

[0167] Each of the mixed target miRNA templates 1 and 2 was individually subjected to dual RT-qPCR using a dual stem-loop reverse transcription primer combination comprising two corresponding stem-loop reverse transcription primers selected for target miRNA combination 1 and 2. Figure 3A -D shows the results of two duplex RT-qPCRs for two target miRNA combinations. Figure 3A and 3C shows the normal amplification curve of the target miRNA, and Figure 3B and 3D It shows that there is no signal in the negative control group using ddH 2 O. This indicates that there is no cross reaction between the stem-loop reverse transcription primer (SEQ ID No. 7) of miR-9-3p and the stem-loop reverse transcription primer (SEQ ID No. 6) of miR-34c-5p, and between the stem-loop reverse transcription primer (SEQ ID No. 5) of miR-16-5p and the stem-loop reverse transcription primer (SEQ ID No. 6) of miR-34c-5p.

[0168] Sensitivity test of dual system

[0169] Two mixed RNA templates 1 and 2 were used for two sets of experiments targeting miR-34c-5p / miR-9-3p and miR-34c-5p / miR-16-5p, respectively. Specifically, dual RT-qPCR was performed to determine the lowest detection limit, i.e., sensitivity, of each target miRNA in the target miRNA. Specifically, 1 pg of the mixed target miRNA template was added, dual reverse transcription was performed, and the resulting product was diluted 10 times in a gradient to a single copy number of the reverse transcription product.

[0170] All reverse transcription products obtained were then subjected to duplex qPCR. Figure 4A -D (Note: the amplification curves from left to right in each figure are the results at concentrations of 1 pg / μL, 0.1 pg / μL, 0.01 pg / μL, 1 fg / μL and 0.1 fg / μL, respectively).

[0171] The results showed that in the combination of miR-34c-5p and miR-9-3p, the detection limit of miR-34c-5p was 0.1 fg / μL, and the detection limit of miR-9-3p was 1 fg / μL. In the combination of miR-34c-5p and miR-16-5p, the detection limit of miR-34c-5p was 0.1 fg / μL, and the detection limit of miR-16-5p was 0.1 fg / μL.

[0172] Comparison of singleplex and duplex quantification of target miRNA

[0173] Serum samples were collected for single and dual RT-qPCR quantification of target miRNA samples, and dual RT-qPCR validation was performed using two target miRNA combinations: miR-34c-5p and miR-9-3p as combination 1, and miR-34c-5p and miR-16-5p as combination 2. A stem-loop reverse transcription primer (SEQ ID No.5) with an 8nt anchor sequence was selected for miR-16-5p, and its corresponding probe was labeled with VIC. A stem-loop reverse transcription primer (SEQ ID No.7) with a 6nt anchor sequence was selected for miR-9-3p, and its corresponding probe was labeled with ROX. A stem-loop reverse transcription primer (SEQID No.6) with a 6nt anchor sequence was selected for miR-34c-5p, and its corresponding probe was labeled with CY5. The results were shown in Figure 5A -F and shown in Table 12. The results showed that there was no significant difference in amplification curves and Ct values ​​between singleplex and duplex quantification of target miRNA.

[0174] Table 12. Singleplex and Duplex Quantification of Target miRNAs

[0175]

[0176] Triplex system for quantification of three target miRNAs

[0177] The triple system comprises: a stem-loop reverse transcription primer combination, the stem-loop reverse transcription primer combination comprising any three stem-loop reverse transcription primers each showing specificity for one of the target miRNAs; its corresponding forward / reverse primers; and its corresponding probe. Triple RT-qPCR is performed to verify the sensitivity and specificity of the triple stem-loop reverse transcription primer combination. In one embodiment, three target miRNAs in the target miRNA are randomly selected to form one or more combinations.

[0178] In one embodiment, two combinations are formed:

[0179] Target miRNA combination 3: miR-34c-5p, miR-9-3p, and miR-16-5p;

[0180] Target miRNA combination 4: miR-34c-5p, miR-16-5p and miR-9-5p.

[0181] For mixed RNA templates with target miRNAs in combination 3 or combination 4 above, triple RT was performed using corresponding stem-loop reverse transcription primers with anchor sequences of different lengths with mixed RNA templates and ddH2O as a negative control. Amplification results were observed to quantify the target miRNAs in the mixed RNA templates. The specificity of each of the stem-loop reverse transcription primers with anchor sequences of different lengths was observed. The anchor sequence length of each of the three stem-loop reverse transcription primers in the triple system was determined based on the observed specificity.

[0182] Based on the above steps, it was finally determined that in target miRNA combination 3, for miR-34c-5p, a stem-loop reverse transcription primer with an anchor sequence of 6 nt was selected, and the stem-loop reverse transcription primer had SEQ ID No. 6. For miR-16-5p, a stem-loop reverse transcription primer with an anchor sequence of 8 nt was selected, and the stem-loop reverse transcription primer had SEQ ID No. 5. For miR-9-3p, a stem-loop reverse transcription primer with an anchor sequence of 6 nt was selected, and the stem-loop reverse transcription primer had SEQ ID No. 7.

[0183] In target miRNA combination 4, for miR-34c-5p, a stem-loop reverse transcription primer with an anchor sequence of 6 nt was selected, and the stem-loop reverse transcription primer had SEQ ID No. 6. For miR-16-5p, a stem-loop reverse transcription primer with an anchor sequence of 8 nt was selected, and the stem-loop reverse transcription primer had SEQ ID No. 5. For miR-9-5p, a stem-loop reverse transcription primer with an anchor sequence of 11 nt was selected, and the stem-loop reverse transcription primer had SEQ ID No. 8.

[0184] Specificity of the triple stem-loop reverse transcription primer combination

[0185] Each of the target miRNA combinations 3 and 4 is used to form mixed target miRNA templates 3 and 4, respectively.

[0186] Each of the mixed target miRNA templates 3 and 4 was subjected to singleplex RT individually using each of the three corresponding stem-loop reverse transcription primers selected for the three target miRNAs in each combination. After singleplex RT, the reverse transcription products from each process of the singleplex RT process were subjected to multiplex qPCR. Fig. 6A -H shows the results of six singleplex RT-multiplex qPCR of two target miRNA combinations 3 and 4. Specifically, Fig. 6A-C shows that the stem-loop reverse transcription primer (SEQ ID No.7) of miR-9-3p specifically reverses transcribes miR-9-3p, the stem-loop reverse transcription primer (SEQ ID No.6) of miR-34c-5p specifically reverses transcribes miR-34c-5p, and the stem-loop reverse transcription primer (SEQ ID No.5) of miR-16-5p specifically reverses transcribes miR-16-5p. Fig.6D In the negative control group using ddH2O, all three stem-loop reverse transcription primers showed no signal.

[0187] exist Fig. 6E -G shows that the stem-loop reverse transcription primer (SEQ ID No.5) of miR-16-5p specifically reverses transcribes miR-16-5p, the stem-loop reverse transcription primer (SEQ ID No.6) of miR-34c-5p specifically reverses transcribes miR-34c-5p, and the stem-loop reverse transcription primer (SEQ ID No.8) of miR-9-5p specifically reverses transcribes miR-9-5p. Figure 6H In the negative control group using ddH2O, all three stem-loop reverse transcription primers showed no signal.

[0188] Once the singleplex RT-multiplex qPCR showed good specificity for each selected corresponding stem-loop reverse transcription primer, a triplex RT-qPCR was performed to determine whether there was any undesired cross-reaction between the three corresponding stem-loop reverse transcription primers selected for target miRNA combinations 3 and 4.

[0189] Each of the resulting mixed target miRNA templates 3 and 4 was individually subjected to triple RT-qPCR using its corresponding triple stem-loop reverse transcription primer combination. Fig. 7A -D shows the results of two triple RT-qPCRs for two target miRNA combinations 3 and 4, where Fig. 7A and 7C The amplification curve of the target miRNA is normal, and Figure 7B and 7D As shown, there is no signal in the negative control group using ddH2O. This indicates that there is no cross-reaction between the stem-loop reverse transcription primer (SEQ ID No.7) of miR-9-3p, the stem-loop reverse transcription primer (SEQ ID No.6) of miR-34c-5p, and the stem-loop reverse transcription primer (SEQ ID No.5) of miR-16-5p; and there is no cross-reaction between the stem-loop reverse transcription primer (SEQ ID No.5) of miR-16-5p, the stem-loop reverse transcription primer (SEQ ID No.6) of miR-34c-5p, and the stem-loop reverse transcription primer (SEQ ID No.8) of miR-9-5p.

[0190] Sensitivity test of dual system

[0191] Mixed RNA templates with two target miRNA combinations 3 and 4 were used for two sets of experiments, respectively. Triple RT-qPCR was performed to determine the lowest detection limit, i.e., sensitivity, of each target miRNA. Specifically, 1 pg of mixed target miRNA template was added, triple reverse transcription was performed, and the resulting product was diluted 10 times in a gradient to a single copy number of the reverse transcription product.

[0192] All reverse transcription products obtained were then subjected to triple qPCR. Fig. 8A -F (Note: the amplification curves from left to right in each figure are the results at concentrations of 1 pg / μL, 0.1 pg / μL, 0.01 pg / μL, 1 fg / μL and 0.1 fg / μL, respectively).

[0193] The results showed that in combination 3, the detection limit of miR-16-5p was 0.1 fg / μL, the detection limit of miR-34c-5p was 0.1 fg / μL, and the detection limit of miR-9-3p was 1 fg / μL. In combination 4, the detection limit of miR-9-5p was 0.1 fg / μL, the detection limit of miR-34c-5p was 0.1 fg / μL, and the detection limit of miR-16-5p was 0.1 fg / μL.

[0194] Comparison of singleplex and tripleplex quantification of target miRNA

[0195] Serum samples were collected for single and triple RT-qPCR quantification of target miRNA samples, and triple RT-qPCR validation was performed using two target miRNA combinations 3 and 4. A stem-loop reverse transcription primer (SEQ ID No.5) with an 8nt anchor sequence was selected for miR-16-5p, and its corresponding probe was labeled with VIC (SEQ ID No.21). A stem-loop reverse transcription primer (SEQ ID No.7) with a 6nt anchor sequence was selected for miR-9-3p, and its corresponding probe was labeled with ROX (SEQ ID No.23). A stem-loop reverse transcription primer (SEQ ID No.6) with a 6nt anchor sequence was selected for miR-34c-5p, and its corresponding probe was labeled with CY5 (SEQ ID No.22). A stem-loop reverse transcription primer (SEQ ID No.8) with an 11nt anchor sequence was selected for miR-9-5p, and its corresponding probe was labeled with FAM (SEQ ID No.24). The results were as follows. Fig. 9A-H and are shown in Table 13. The results showed that there was no significant difference in amplification curves and Ct values ​​between singleplex and tripleplex quantification of target miRNA.

[0196] Table 13. Singleplex and triplex quantification of target miRNAs

[0197]

[0198] Quadruple system for quantification of four target miRNAs

[0199] The quadruple system comprises: a stem-loop reverse transcription primer combination, wherein the stem-loop reverse transcription primer combination has four stem-loop reverse transcription primers each showing specificity for one of the target miRNAs; its corresponding forward / reverse primers; and its corresponding probe. Quadruple RT-qPCR is performed to verify the sensitivity and specificity of the quadruple stem-loop reverse transcription primer combination. In one embodiment, the target miRNA combination 5 comprises miR-34c-5p, miR-16-5p, miR-9-3p, and miR-9-5p.

[0200] For mixed RNA template 5 having all of miR-34c-5p, miR-16-5p, miR-9-3p, and miR-9-5p, quadruple RT was performed using corresponding stem-loop reverse transcription primers with different lengths of anchor sequences with mixed RNA template 5 and ddH2O as a negative control. Amplification results were observed to quantify target miRNAs in mixed RNA template 5. The specificity of each of the stem-loop reverse transcription primers with different lengths of anchor sequences was observed. The anchor sequence length of each of the four stem-loop reverse transcription primers in the quadruple system was determined based on the observed specificity.

[0201] Based on the above steps, it was finally determined that for miR-34c-5p, a stem-loop reverse transcription primer with an anchor sequence of 6 nt was selected, and the stem-loop reverse transcription primer had SEQ ID No. 6. For miR-16-5p, a stem-loop reverse transcription primer with an anchor sequence of 8 nt was selected, and the stem-loop reverse transcription primer had SEQ ID No. 5. For miR-9-3p, a stem-loop reverse transcription primer with an anchor sequence of 6 nt was selected, and the stem-loop reverse transcription primer had SEQ ID No. 7. For miR-9-5p, a stem-loop reverse transcription primer with an anchor sequence of 11 nt was selected, and the stem-loop reverse transcription primer had SEQ ID No. 8.

[0202] Specificity of the quadruple stem-loop reverse transcription primer combination

[0203] Mixed target miRNA template 5 was subjected to singleplex RT using each of the four corresponding stem-loop reverse transcription primers selected for the four target miRNAs, and then quadruplex qPCR was formed on each of the singleplex reverse transcription products. Fig. 10A -F shows the results of four single-plex RT-quadruple qPCRs, each using a corresponding stem-loop reverse transcription primer. Fig. 10A -D shows that the stem-loop reverse transcription primer (SEQ ID No.7) of miR-9-3p specifically reverses miR-9-3p, the stem-loop reverse transcription primer (SEQ ID No.6) of miR-34c-5p specifically reverses miR-34c-5p, and the stem-loop reverse transcription primer (SEQ ID No.5) of miR-16-5p specifically reverses miR-16-5p, and the stem-loop reverse transcription primer (SEQ ID No.8) of miR-9-5p specifically reverses miR-9-5p. Fig.10E In the negative control group using ddH2O in -F, all four stem-loop reverse transcription primers showed no signal.

[0204] Once the singleplex RT-quadruplex qPCR showed good specificity for each selected corresponding stem-loop reverse transcription primer, quadruplex RT-qPCR was performed to determine whether there was any undesired cross-reaction between the four corresponding stem-loop reverse transcription primers selected for the target miRNA.

[0205] Mixed Target miRNA Template 5 Quadruple RT-qPCR was performed using a quadruple stem-loop reverse transcription primer combination containing all four corresponding stem-loop reverse transcription primers selected for the target miRNAs. Fig.11A -B shows the results of quadruple RT-qPCR of mixed target miRNA combination 5, in which Fig.11A The amplification curve of the target miRNA is normal, and Fig. 11B As shown, there was no signal in the negative control group using ddH2O. This indicates that there is no cross-reaction between the stem-loop reverse transcription primer of miR-9-3p (SEQ ID No.7), the stem-loop reverse transcription primer of miR-34c-5p (SEQ ID No.6), the stem-loop reverse transcription primer of miR-16-5p (SEQ ID No.5), and the stem-loop reverse transcription primer of miR-9-5p (SEQ ID No.8).

[0206] Sensitivity test of dual system

[0207] Quadruple RT-qPCR was performed to determine the lowest detection limit, i.e., sensitivity, of each target miRNA in the mixed target miRNA template combination 5. Specifically, 1 pg of the mixed target miRNA template 5 was added, quadruple RT was performed, and the resulting product was serially diluted 10-fold to a single copy number of the reverse transcription product.

[0208] All reverse transcription products obtained were then subjected to quadruple qPCR. Fig. 12A -D (Note: the amplification curves from left to right in each figure are the results at concentrations of 1 pg / μL, 0.1 pg / μL, 0.01 pg / μL, 1 fg / μL and 0.1 fg / μL, respectively).

[0209] The results showed that the detection limit of miR-16-5p was 0.1 fg / μL, the detection limit of miR-34c-5p was 0.1 fg / μL, and the detection limit of miR-9-3p was 0.1 fg / μL. In combination 4, the detection limit of miR-9-5p was 1 fg / μL, the detection limit of miR-34c-5p was 1 fg, and the detection limit of miR-16-5p was 1 fg / μL.

[0210] Comparison of singleplex and quadruplex quantification of target miRNA

[0211] Serum samples were collected for single and quadruple RT-qPCR quantification of target miRNAs, and quadruple RT-qPCR validation was performed using mixed target miRNA template combination 5. A stem-loop reverse transcription primer with an 8nt anchor sequence (SEQ ID No.5) was selected for miR-16-5p, and its corresponding probe was labeled with VIC (SEQ ID No.21). A stem-loop reverse transcription primer with a 6nt anchor sequence (SEQ ID No.7) was selected for miR-9-3p, and its corresponding probe was labeled with ROX (SEQ IDNo.23). A stem-loop reverse transcription primer with a 6nt anchor sequence (SEQ ID No.6) was selected for miR-34c-5p, and its corresponding probe was labeled with CY5 (SEQ ID No.22). A stem-loop reverse transcription primer with an 11nt anchor sequence (SEQ IDNo.8) was selected for miR-9-5p, and its corresponding probe was labeled with FAM (SEQ ID No.24). The results were as follows. Fig.13A The results show that there is no significant difference in amplification curves and Ct values ​​between singleplex and quadruplex quantification of target miRNA.

[0212] Robustness of quadruple RT-qPCR quantification

[0213] The test size was expanded to 20 subjects to determine the robustness of the quadruple RT-qPCR quantification. Fig.14A -F and shown in Table 14. Specifically, as reflected, the quadruple stem-loop reverse transcription primer combination (SEQ ID No. 5-8) can accurately quantify the target miRNAs of miR-34c-5p, miR-9-3p, miR-16-5p and miR-9-5p using quadruple RT-qPCR.

[0214] Table 14. Singleplex and quadruplex quantification of target miRNAs

[0215]

[0216]

[0217] Specificity of multiple stem-loop reverse transcription primer combinations with different anchor sequence lengths

[0218] If the stem-loop reverse transcription primer is modified, the accuracy of multiplex RT-qPCR quantification will be compromised. In one embodiment, a single reverse transcription is performed using a mixed RNA template comprising miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p. An alternative stem-loop reverse transcription primer combination is selected. Among them, miR-16-5p selects a reverse transcription primer with an anchor sequence length of 11nt and stem loop 1 (SEQ ID No.62), miR-34c-5p selects a reverse transcription primer with an anchor sequence length of 8nt and stem loop 2 (SEQ ID No.65), miR-9-3p selects a reverse transcription primer with an anchor sequence length of 8nt and stem loop 3 (SEQ ID No.67), and miR-9-5p selects a reverse transcription primer with an anchor sequence length of 6nt and stem loop 4 (SEQ ID No.69).

[0219] Then, multiplex qPCR was performed to determine if there was any cross-reaction between the quantification of the four target miRNAs, to verify the specificity of the alternative stem-loop reverse transcription primer combination, and to compare the Ct values ​​of each of the target miRNAs. Fig.15A -D and shown in Table 15.

[0220] Table 15. Singleplex and quadruplex quantitation by alternative stem-loop reverse transcription primers

[0221]

[0222] By observing the amplification curves of the target miRNA, it was found that when miR-9-3p was reverse transcribed alone, there was signal interference from miR-16-5p and miR-9-5p; when miR-9-5p was reverse transcribed alone, there was signal interference from miR-16-5p; when miR-16-5p was reverse transcribed alone, there was signal interference from miR-9-5p; when miR-34c-5p was reverse transcribed alone, there was signal interference from miR-16-5p and miR-9-5p; and there were significant differences in Ct values ​​between single and quadruple quantitative detection.

[0223] The multiple target miRNA quantitative method of the present invention is also applicable to other miRNA targets, as long as the homology of the miRNA sequence is low. The 3' end of the miRNA sequence cannot contain two or more continuous identical bases to avoid the non-specific binding of the stem-loop reverse transcription primer and affect the experimental results. In the construction of the multiple target miRNA quantitative system, stem-loop reverse transcription primers with different stem-loop sequences are used to obtain cDNA products with significantly different sequences. Specific primers and probes are designed for cDNA to verify the sensitivity and specificity of the multiple system. Once the multiple specificity and sensitivity of the multiple RT-qPCR using the multiple stem-loop reverse transcription primer combination are verified, sample quantification can be performed in a desired environment (e.g., clinical diagnosis of a disease).

[0224] In one embodiment, miRNAs other than miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p can be used as target miRNAs for determining Alzheimer's disease. Each target miRNA has a corresponding stem-loop reverse transcription primer for its reverse transcription, and a corresponding forward primer, a corresponding reverse primer and a corresponding probe with a fluorescent reporter group and a quencher group for qPCR of the reverse transcription product. Table 16 shows the target miRNA and its corresponding forward / reverse primer and probe for qPCR.

[0225] Table 16. Target miRNAs of Alzheimer's disease and their corresponding stem-loop reverse transcription primers, forward / reverse primers and probes for qPCR

[0226]

[0227]

[0228]

[0229]

[0230]

[0231] To summarize the present invention, the sequences of stem-loop reverse transcription primers, forward / reverse primers and probes are listed below in Tables 17 and 18.

[0232] Table 17. Primers and probes used for multiplex RT-qPCR

[0233]

[0234]

[0235] Table 17. Stem-loop primer variants with different anchor lengths

[0236]

[0237] The above description of the illustrative embodiments of the present invention has been presented for the purpose of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and modifications and variations may be made in view of the above teachings or may be obtained from the practice of the present invention. The embodiments are selected and described in order to explain the principles of the present invention and as a practical application of the present invention, so that those skilled in the art can utilize the present invention in various embodiments with various modifications suitable for the specific purpose contemplated. It is intended that the scope of the present invention should be limited by the appended claims and their equivalents.

Claims

1. A multiple reverse transcription primer combination for simultaneously quantifying miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p, the multiple reverse transcription primer combination comprising: A first stem-loop reverse transcription primer, wherein the first stem-loop reverse transcription primer comprises a nucleic acid sequence of SEQ ID No. 5; a second stem-loop reverse transcription primer, wherein the second stem-loop reverse transcription primer comprises a nucleic acid sequence of SEQ ID No.6; a third stem-loop reverse transcription primer, wherein the third stem-loop reverse transcription primer comprises the nucleic acid sequence of SEQ ID No.7; as well as A fourth stem-loop reverse transcription primer, wherein the fourth stem-loop reverse transcription primer comprises the nucleic acid sequence of SEQ ID No.

8.

2. A multiple reverse transcription primer combination for simultaneously quantifying multiple target miRNAs, the multiple reverse transcription primer combination comprising: Various stem-loop reverse transcription primers; wherein each of the plurality of stem-loop reverse transcription primers has a stem-loop sequence forming a stem-loop structure and an anchor sequence complementary to a unique 3' sequence of one of the plurality of target miRNAs; The lengths of the anchor sequences of at least two of the stem-loop reverse transcription primers are different from each other. 3 . The multiplex reverse transcription primer combination according to claim 2 , wherein the length of each stem-loop reverse transcription primer in the plurality of stem-loop reverse transcription primers is about 40-65 nt. 4 . The multiplex reverse transcription primer combination according to claim 2 , wherein the length of the anchor sequence of each stem-loop reverse transcription primer in the plurality of stem-loop reverse transcription primers is about 3-12 nt.

5. The multiple reverse transcription primer combination according to claim 2, wherein the plurality of stem-loop reverse transcription primers comprises a first stem-loop reverse transcription primer having a first stem-loop sequence and a first anchor sequence; as well as a second stem-loop reverse transcription primer having a second stem-loop sequence and a second anchor sequence, wherein each of the first stem-loop sequence and the second stem-loop sequence is selected from the group consisting of SEQ ID No. 9-12; and the first stem-loop sequence and the second stem-loop sequence are different from each other; and The length of each of the first anchor sequence and the second anchor sequence is selected from one of 4 nt, 6 nt, 8 nt and 11 nt.

6. The multiple reverse transcription primer combination of claim 5, wherein the first anchor sequence of the first stem-loop reverse transcription primer is complementary to a unique 3' sequence of a first target miRNA among the multiple target miRNAs, and the second anchor sequence of the second stem-loop reverse transcription primer is complementary to a unique 3' sequence of a second target miRNA among the multiple target miRNAs.

7. The multiple reverse transcription primer combination according to claim 5, wherein the plurality of stem-loop reverse transcription primers further comprises a third stem-loop reverse transcription primer having a third stem-loop sequence and a third anchor sequence, wherein the third stem-loop sequence is selected from the group consisting of SEQ ID No.9-12; and the third stem-loop sequence is different from the first stem-loop sequence and the second stem-loop sequence; wherein the length of the third anchor sequence is selected from one of 4nt, 6nt, 8nt and 11nt; and The third anchor sequence is complementary to a unique 3' sequence of a third target miRNA among the plurality of target miRNAs.

8. The multiple reverse transcription primer combination according to claim 7, wherein the plurality of stem-loop reverse transcription primers further comprises a fourth stem-loop reverse transcription primer having a fourth stem-loop sequence and a fourth anchor sequence, wherein the fourth stem-loop sequence is selected from the group consisting of SEQ ID No. 9-12; and the fourth stem-loop sequence is different from the first stem-loop sequence, the second stem-loop sequence and the third stem-loop sequence; wherein the length of the fourth anchor sequence is selected from one of 4nt, 6nt, 8nt and 11nt; and The fourth anchor sequence is complementary to a unique 3' sequence of a fourth target miRNA among the plurality of target miRNAs.

9. The multiple reverse transcription primer combination of claim 8, wherein the multiple reverse transcription primer combination has multi-specificity, such that the first stem-loop reverse transcription primer effectively and only reverse transcribes the first target miRNA, the second stem-loop reverse transcription primer effectively and only reverse transcribes the second target miRNA, the third stem-loop reverse transcription primer effectively and only reverse transcribes the third target miRNA, and the fourth stem-loop reverse transcription primer effectively and only reverse transcribes the fourth target miRNA.

10. The multiple reverse transcription primer combination according to claim 9, wherein each of the first target miRNA, the second target miRNA, the third target miRNA and the fourth target miRNA is selected from the group consisting of miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p, wherein each of the first target miRNA, the second target miRNA, the third target miRNA and the fourth target miRNA are different from each other.

11. A multiple reverse transcription primer combination according to claim 8, wherein the first stem-loop reverse transcription primer comprises a nucleic acid sequence of SEQ ID No.5, the second stem-loop reverse transcription primer comprises a nucleic acid sequence of SEQ ID No.6, the third stem-loop reverse transcription primer comprises a nucleic acid sequence of SEQ ID No.7, and the fourth stem-loop reverse transcription primer comprises a nucleic acid sequence of SEQ ID No.

8.

12. A kit for simultaneously quantifying the expression levels of multiple target miRNAs, the kit comprising: a first stem-loop reverse transcription primer and a second stem-loop reverse transcription primer, wherein the first stem-loop reverse transcription primer has a first stem-loop sequence and a first anchor sequence, and the second stem-loop reverse transcription primer has a second stem-loop sequence and a second anchor sequence, wherein each of the first stem-loop sequence and the second stem-loop sequence is selected from the group consisting of SEQ ID No. 9-12; and the first stem-loop sequence and the second stem-loop sequence are different from each other, wherein the length of each anchor sequence in the first anchor sequence and the second anchor sequence is selected from one of 4 nt, 6 nt, 8 nt and 11 nt; a first forward primer and a second forward primer, wherein each of the first forward primer and the second forward primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 17-20, and the first forward primer is different from the second forward primer; a first reverse primer and a second reverse primer, wherein each of the first reverse primer and the second reverse primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 25-28, and the first reverse primer is different from the second reverse primer; as well as a first probe and a second probe, wherein the first probe comprises a first probe sequence, a first fluorescent reporter group and a first quencher group; and the second probe comprises a second probe sequence, a second fluorescent reporter group and a second quencher group, Each of the first probe sequence and the second probe sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 21-24.

13. The kit according to claim 12, wherein Each of the first fluorescent reporter group and the second fluorescent reporter group is selected from the group consisting of VIC, CY5, ROX and FAM; and the first fluorescent reporter group is different from the second fluorescent reporter group.

14. The kit of claim 13, wherein the first anchor sequence is complementary to a 3' sequence of a first target miRNA among the plurality of target miRNAs, and the second anchor sequence is complementary to a unique 3' sequence of a second target miRNA among the plurality of target miRNAs.

15. The kit according to claim 12, further comprising: a third stem-loop reverse transcription primer, wherein the third stem-loop reverse transcription primer has a third stem-loop sequence and a third anchor sequence, wherein the third stem-loop sequence is selected from the group consisting of SEQ ID No. 9-12; and the third stem-loop sequence is different from the first stem-loop sequence and the second stem-loop sequence, and wherein the length of the third anchor sequence is selected from one of 4nt, 6nt, 8nt and 11nt; a third forward primer, wherein the third forward primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. 17-20; and the third forward primer is different from the first forward primer and the second forward primer; a third reverse primer, wherein the third reverse primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 25-28; and the third reverse primer is different from the first reverse primer and the second reverse primer; as well as The third probe, wherein the third probe comprises a third probe sequence, a third fluorescent reporter group and a third quencher group; wherein the third probe sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. 21-24.

16. The kit according to claim 15, wherein The third fluorescent reporter group is selected from the group consisting of VIC, CY5, ROX and FAM; and the third fluorescent reporter group is different from the first fluorescent reporter group and the second fluorescent reporter group.

17. The kit according to claim 16, further comprising: a fourth stem-loop reverse transcription primer, wherein the fourth stem-loop reverse transcription primer has a fourth stem-loop sequence and a fourth anchor sequence, wherein the fourth stem-loop sequence is selected from the group consisting of SEQ ID No. 9-12; and the fourth stem-loop sequence is different from the first stem-loop sequence, the second stem-loop sequence and the third stem-loop sequence, wherein the length of the fourth anchor sequence is selected from one of 4nt, 6nt, 8nt and 11nt; a fourth forward primer, wherein the fourth forward primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 17-20; and the fourth forward primer is different from the first forward primer, the second forward primer, and the third forward primer; a fourth reverse primer, wherein the fourth reverse primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 25-28; and the fourth reverse primer is different from the first reverse primer, the second reverse primer, and the third reverse primer; as well as The fourth probe comprises a fourth probe sequence, a fourth fluorescent reporter group and a fourth quencher group; wherein the fourth probe sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. 21-24.

18. The kit according to claim 17, wherein The fourth fluorescent reporter group is selected from the group consisting of VIC, CY5, ROX and FAM; and the fourth fluorescent reporter group is different from the first fluorescent reporter group, the second fluorescent reporter group and the third fluorescent reporter group.

19. A kit for simultaneously quantifying the expression levels of multiple target miRNAs, the kit comprising: A multiple reverse transcription primer combination, the multiple reverse transcription primer combination comprising a first stem-loop reverse transcription primer comprising a nucleic acid sequence of SEQ ID No.5, a second stem-loop reverse transcription primer comprising a nucleic acid sequence of SEQ ID No.6, a third stem-loop reverse transcription primer comprising a nucleic acid sequence of SEQ ID No.7, and a fourth stem-loop reverse transcription primer comprising a nucleic acid sequence of SEQ ID No.8; A forward primer combination, the forward primer combination comprising a first forward primer comprising a nucleic acid sequence of SEQ ID No.17, a second forward primer comprising a nucleic acid sequence of SEQ ID No.18, a third forward primer comprising a nucleic acid sequence of SEQ ID No.19, and a fourth forward primer comprising a nucleic acid sequence of SEQ ID No.20; A reverse primer combination, the reverse primer combination comprising a first reverse primer comprising a nucleic acid sequence of SEQ ID No.25, a second reverse primer comprising a nucleic acid sequence of SEQ ID No.26, a third reverse primer comprising a nucleic acid sequence of SEQ ID No.27, and a fourth reverse primer comprising a nucleic acid sequence of SEQ ID No.28; as well as A probe combination, the probe combination comprising a first probe comprising a nucleic acid sequence of SEQ ID No.21 and a first fluorescent reporter group of VIC, a second probe comprising a nucleic acid sequence of SEQ ID No.22 and a second fluorescent reporter group of CY5, a third probe comprising a nucleic acid sequence of SEQ ID No.23 and a third fluorescent reporter group of ROX, and a fourth probe comprising a nucleic acid sequence of SEQ ID No.24 and a fourth fluorescent reporter group of FAM.

20. A method for simultaneously quantifying miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p, the method comprising performing multiplex RT-qPCR using the multiplex reverse transcription primer combination according to claim 2.

21. A method for determining a medical condition of a living subject using the quantitative results of miR-16-5p, miR-34c-5p, miR-9-3p, and miR-9-5p, the method comprising: Performing multiplex RT-qPCR using the kit according to claim 19; Obtaining quantitative results of miR-16-5p, miR-34c-5p, miR-9-3p, and miR-9-5p from the multiplex RT-qPCR; and using the quantitative results to determine a medical condition of the living subject, wherein said medical condition of said living subject is Alzheimer's Disease.

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