Multiple reverse transcription primer combination for multiple reverse transcription-quantitative PCR reaction

Abstract

The invention discloses a multiple reverse transcription primer combination for simultaneously quantifying multiple target miRNAs. The multiple reverse transcription primer combination comprises multiple 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.

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 entitled “Method for Screening Multiplex Reverse Transcription Primer Combinations for Quantifying Multiple miRNAs” with attorney docket number 1010469.100WO1; co-pending PCT patent application entitled “Method and Kit for Multiplex Quantitative Detection of miRNAs for Alzheimer's Disease” with attorney docket number 1010469.101WO1; and co-pending PCT patent application entitled “Method and Kit for Multiplex Quantitative Detection of miRNAs for Lung Cancer” with attorney docket number 1010469.102WO1. and a co-pending PCT patent application with attorney docket number 1010469.104WO1, entitled “Method for Clinical Detection of Multiple Target miRNAs,” filed on the same day as the present application and with the same applicant as the present application, any and all of which are hereby incorporated 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 stem-loop reverse transcription primer combination for simultaneously quantifying multiple target miRNAs. 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 tiny non-coding single-stranded RNA. According to miRBase (version 22), the human genome encodes approximately 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 various life processes, such as the occurrence and development of diseases and cell aging. Existing studies have shown that changes in the expression levels of multiple miRNAs in specific combinations can be used for the screening, diagnosis and prognosis of many 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] Currently, single-plex detection for specific target miRNA can be achieved by single-plex reverse transcription-single-plex quantitative PCR (RT-qPCR). Specifically, the target miRNA is reverse transcribed into cDNA using a reverse transcription primer in reverse transcription (RT), and then the reverse transcription product containing cDNA is subjected to fluorescent quantitative PCR (qPCR) to quantify the target miRNA.

[0012] Since the length of miRNA is generally short and generally in the range of 18nt to 25nt, it is difficult to obtain a reverse transcription product, such as cDNA, with a length suitable for qPCR. In order to solve this problem, various miRNA reverse transcription technologies are intended to extend the length of the reverse transcription product (such as cDNA). Among them, stem-loop reverse transcription is a preferred reverse transcription method, which effectively extends the length of the reverse transcription product. Specifically, the reverse transcription primer used in the method has a stem-loop structure sequence and an anchor sequence that is complementary to the 3' sequence of the target miRNA. The stem-loop structure can effectively extend the sequence length of the reverse transcription product, and can prevent itself from pairing with other sequence structures due to its own complementary structure, and thus reduce non-specific binding. Once the reverse transcription is completed, the reverse transcription product obtained by reverse transcription is subjected to qPCR, in which forward / reverse primers and specific fluorescent probes are added. Common probes include The target miRNA is quantified in qPCR and the quantitative results in the form of Ct values ​​will be provided.

[0013] In 2005, 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 purified by MultiScribe. TMReverse 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.

[0014] 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.

[0015] Therefore, there is a major drawback in this technical field, that is, it is difficult to achieve multiple quantification using RT-qPCR - quantifying multiple target miRNAs at the same time. Moreover, it is difficult to achieve stable standardized control. Therefore, in order to quantify multiple target miRNAs whose expression level changes are associated with a specific disease, a single RT-qPCR of each target miRNA must be completed separately, and thus significantly increases the cost and time consumption. This defect is the biggest obstacle to the clinical use of miRNA detection / quantification (i.e., disease diagnostic methods).

[0016] Therefore, there is still an urgent need for a multiplexed quantitative method that can simultaneously quantify a combination of multiple target miRNAs. Summary of the invention

[0017] In view of the above, the present invention discloses a method for selecting and screening a combination of stem-loop reverse transcription primers capable of simultaneously quantifying a plurality of target miRNAs.

[0018] In one aspect of the present invention, the present invention discloses a method for screening a multiple stem-loop reverse transcription primer combination showing multi-specificity for simultaneously quantifying multiple target miRNAs, the method comprising (A) providing the multiple target miRNAs, wherein each of the multiple target miRNAs has a unique 3' sequence; (B) providing 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 the unique 3' sequence of one of the multiple target miRNAs, wherein the multiple stem-loop reverse transcription primers The primers are selected by a selection process, the selection process comprising a) selecting the stem-loop sequence for each of the plurality of stem-loop reverse transcription primers; and b) selecting a certain length for the anchor sequence of each of the plurality of stem-loop reverse transcription primers, wherein the lengths of the anchor sequences of at least two of the plurality of stem-loop reverse transcription primers are different from each other; (c) screening the plurality of stem-loop reverse transcription primers by a screening process, or determining the multi-specificity of the plurality of stem-loop reverse transcription primers, wherein the screening process comprises i) using the plurality of stem-loop reverse transcription primers to be quantified; The method comprises the steps of: i) performing a mixed target miRNA template of a target miRNA, performing a single reverse transcription process for each of the plurality of stem-loop reverse transcription primers; ii) collecting a set of reverse transcription products from each of the single reverse transcription processes of the single reverse transcription processes of step i); iii) performing a multiplex qPCR amplification process for each set of the sets of reverse transcription products from step ii) to form a set of multiplex qPCR products for each set of the sets of reverse transcription products; and iv) obtaining a set of multiplex qPCR products based on each set of the sets of the multiplex qPCR products. obtaining a quantitative result for each of the plurality of target miRNAs, and thereby obtaining a set of the quantitative results for the plurality of target miRNAs; and v) determining the multi-specificity of the plurality of stem-loop reverse transcription primers based on the set of quantitative results formed in step iv), wherein the multi-specificity of the plurality of stem-loop reverse transcription primers is confirmed when the set of quantitative results shows that each of the plurality of stem-loop reverse transcription primers effectively and only reverse transcribes a target miRNA complementary to the anchor sequence of each of the plurality of stem-loop reverse transcription primers;and (D) if the multi-specificity of the plurality of stem-loop reverse transcription primers is not confirmed by the set of quantitative results, modifying the plurality of stem-loop reverse transcription primers by an iterative process, the iterative process comprising: providing a modified plurality of stem-loop reverse transcription primers by repeating step b) of the selection process to select a new length for the anchor sequence of each of those stem-loop reverse transcription primers of the plurality of stem-loop reverse transcription primers that fail to effectively and only reverse transcribe the target miRNA complementary to the anchor sequence of the stem-loop reverse transcription primer, wherein the length of the anchor sequence is selected between 3 nt and 12 nt; and repeating steps i)-v) of the screening process to determine the multi-specificity of the modified plurality of stem-loop reverse transcription primers until the modified plurality of stem-loop reverse transcription primers exhibit the multi-specificity and are simultaneously operative to quantify the plurality of target miRNAs. ;

[0019] In one embodiment, step i) of the screening process includes a hybridization reaction, wherein each of the multiple stem-loop reverse transcription primers hybridizes with at least one target miRNA of the multiple target miRNAs to form a collection of at least one type of miRNA-stem-loop reverse transcription primer complexes; and an extension reaction, wherein at least one type of cDNA is formed by extending the 3' of the stem-loop reverse transcription primer in the collection of the at least one type of miRNA-stem-loop reverse transcription primer complexes.

[0020] In one embodiment, step iii) of the screening process includes adding a combination of forward primers and at least one reverse primer to each of the sets of reverse transcription products from step ii), wherein each of the forward primers has a sequence identical to the 5' sequence of one of the multiple target miRNAs; adding a combination of probes to each of the sets of reverse transcription products from step ii), wherein each of the probes has a sequence identical to a portion of one of the target miRNAs, and each of the probes has a fluorescent reporter group different from the fluorescent reporter groups of the other probes, and each of the probes having a quenching group that inhibits the fluorescence generated by the fluorescent reporter group; individually amplifying each of the sets of the reverse transcription products from step ii) with the combination of the forward primer, the at least one reverse primer and the combination of the probes to individually form a set of multiplex qPCR products for each of the sets of the reverse transcription products; detecting the intensity of the fluorescence generated by the fluorescent reporter group of the combination of the probes in each of the sets in the set of the multiplex qPCR products; and quantifying each of the multiple target miRNAs based on the intensity of the fluorescence detected in each of the sets in the set of the multiplex qPCR products.

[0021] In one embodiment, the quantification result for each target miRNA in the plurality of target miRNAs comprises a Ct value.

[0022] In one embodiment, the stem-loop sequences of at least two stem-loop reverse transcription primers among the plurality of stem-loop reverse transcription primers selected in step a) are identical.

[0023] In one embodiment, each of the unique 3' sequences of the plurality of target miRNAs differs from the other unique 3' sequences of the plurality of target miRNAs by at least 1 nt.

[0024] In one embodiment, the method further includes a sensitivity test of the multiple stem-loop reverse transcription primers, wherein the sensitivity test includes 1) preparing multiple mixed target miRNA templates with a concentration gradient, wherein the concentration of each template in the multiple mixed target miRNA templates is different from the concentration of other mixed target miRNA templates; 2) using each stem-loop reverse transcription primer in the multiple stem-loop reverse transcription primers to perform a single reverse transcription process on each template in the mixed target miRNA template separately, and collecting a collection of reverse transcription products from each process in the single reverse transcription process; 3) using each collection of the reverse transcription products from step 2) to perform a multiple qPCR process separately, and obtaining a collection of quantitative results of each target miRNA in the target miRNA from each process in the multiple qPCR process; 4) arranging the collection of quantitative results according to each stem-loop reverse transcription primer in the multiple stem-loop reverse transcription primers; 5) determining the minimum concentration of the mixed target miRNA template for each stem-loop reverse transcription primer in the multiple stem-loop reverse transcription primers based on the arranged quantitative results.

[0025] In one embodiment, the stem-loop sequences of at least two stem-loop reverse transcription primers among the plurality of stem-loop reverse transcription primers are identical.

[0026] In one embodiment, the stem-loop sequences of all of the plurality of stem-loop reverse transcription primers are identical.

[0027] In one embodiment, the multiple target miRNAs include a combination of any two or more of hsa-miR-210-3p, hsa-miR-126-3p, hsa-miR-205-5p and hsa-miR-486-5p.

[0028] In one embodiment, the plurality of target miRNAs include a combination of any two or more of miR-16-5p, miR-34c-5p, miR-9-3p, and miR-9-5p.

[0029] In another aspect of the present invention, a method for screening a multiple stem-loop reverse transcription primer combination showing multi-specificity for simultaneously quantifying multiple target miRNAs, the method comprising the following steps: (A) providing the multiple target miRNAs, wherein each of the multiple target miRNAs has a unique 3' sequence; (B) providing 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 the unique 3' sequence of one of the multiple target miRNAs, and wherein the lengths of the anchor sequences of at least two of the multiple stem-loop reverse transcription primers are identical to each other. different; (C) screening the plurality of stem-loop reverse transcription primers to determine the multi-specificity of the plurality of stem-loop reverse transcription primers, wherein the multi-specificity of the plurality of stem-loop reverse transcription primers is confirmed when each of the plurality of stem-loop reverse transcription primers effectively and only reverse transcribes a target miRNA that is complementary to the anchor sequence of each of the plurality of stem-loop reverse transcription primers, and (D) if the multi-specificity of the plurality of stem-loop reverse transcription primers is not confirmed, modifying the plurality of stem-loop reverse transcription primers by repeating the process until the modified plurality of stem-loop reverse transcription primers exhibit the multi-specificity and simultaneously operably quantify the plurality of target miRNAs.

[0030] 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 multiple stem-loop reverse transcription primers are different from each other.

[0031] 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.

[0032] 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.

[0033] In one embodiment, the plurality of 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; and the plurality of target miRNAs include a first target miRNA and a second target miRNA.

[0034] In one embodiment, the first anchor sequence of the first stem-loop reverse transcription primer is complementary to the 3' sequence of the first target miRNA, and the second anchor sequence of the second stem-loop reverse transcription primer is complementary to the 3' sequence of the second target miRNA.

[0035] In one embodiment, when multiple reverse transcription is performed using a mixed target miRNA template comprising the first target miRNA and the second target miRNA and the multiple reverse transcription primer combination, the first stem-loop reverse transcription primer effectively and only reverse transcribes the first target miRNA; and the second stem-loop reverse transcription primer effectively and only reverse transcribes the second target miRNA.

[0036] In one embodiment, the first stem-loop sequence of the first stem-loop reverse transcription primer and the second stem-loop sequence of the second stem-loop reverse transcription primer are the same.

[0037] In one embodiment, 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 anchor sequence is complementary to the 3' sequence of a third target miRNA in the plurality of target miRNAs.

[0038] In one embodiment, 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 anchor sequence is complementary to the 3' sequence of a fourth target miRNA in the plurality of target miRNAs.

[0039] 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.

[0040] In one embodiment, at least two of the first stem-loop structure, the second stem-loop structure, the third stem-loop structure, and the fourth stem-loop structure are identical.

[0041] In one embodiment, all of the first stem-loop structures, the second stem-loop structures, the third stem-loop structures, and the fourth stem-loop structures are identical.

[0042] In one embodiment, each of the first fluorescent reporter group, the second fluorescent reporter group, the third fluorescent reporter group and the fourth fluorescent reporter group includes one of VIC, CY5, ROX, and FAM, and each of the first quencher group, the second quencher group, the third quencher group and the fourth quencher group includes one of BHQ1, BHQ2, BHQ3 and MGB.

[0043] 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 having at least 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; a first forward primer and a second forward primer, wherein the first forward primer comprises a Tm enhancement tail and a first forward primer sequence, and the second forward primer comprises a second Tm enhancement tail and a second forward primer sequence; at least one reverse primer, wherein the at least one reverse primer is complementary to the first stem-loop sequence and the second stem-loop sequence; and a first probe and a second probe, wherein the first probe comprises a first probe sequence identical to a portion of the first stem-loop reverse transcription primer, a first fluorescent reporter group and a first quencher group, and the second probe comprises a second probe sequence identical to a portion of the second stem-loop reverse transcription primer, a second fluorescent reporter group and a second quencher group, wherein the first fluorescent reporter group is different from the second fluorescent reporter group.

[0044] In one embodiment, each of the first stem-loop reverse transcription primer and the second stem-loop reverse transcription primer has a length of about 40-60 nt.

[0045] In one embodiment, each of the first anchor sequence and the second anchor sequence is about 3-12 nt in length.

[0046] In one embodiment, the first anchor sequence is complementary to a 3' sequence of a first target miRNA in the plurality of target miRNAs, and the second anchor sequence is complementary to a 3' sequence of a second target miRNA in the plurality of target miRNAs.

[0047] In one embodiment, the first stem-loop sequence of the first stem-loop reverse transcription primer and the second stem-loop sequence of the second stem-loop reverse transcription primer are the same.

[0048] In one embodiment, the length of the first anchor sequence of the first stem-loop reverse transcription primer is different from the length of the second anchor sequence of the second stem-loop reverse transcription primer.

[0049] In one embodiment, the multiple reverse transcription primer combination further includes a third stem-loop reverse transcription primer and a fourth stem-loop reverse transcription primer, wherein the third stem-loop reverse transcription primer has a third stem-loop sequence and a third anchor sequence, and the fourth stem-loop reverse transcription primer has a fourth stem-loop sequence and a fourth anchor sequence; a third forward primer and a fourth forward primer, wherein the third forward primer includes a third Tm enhancement tail and a third forward primer sequence, and the fourth forward primer includes a fourth Tm enhancement tail and a fourth forward primer sequence; and a third probe and a fourth probe, wherein the third probe includes a third probe sequence identical to a portion of the third stem-loop reverse transcription primer, a third fluorescent reporter group, and a third quencher group, and the fourth probe includes a fourth probe sequence identical to a portion of the fourth stem-loop reverse transcription primer, a fourth fluorescent reporter group, and a fourth quencher group, wherein each fluorescent reporter group of the first fluorescent reporter group, the second fluorescent reporter group, the third fluorescent reporter group, and the fourth fluorescent reporter group is different from the other fluorescent reporter groups.

[0050] In one embodiment, each of the first probe, the second probe, the third probe, and the fourth probe is probe or MGB probe.

[0051] In one aspect of the present invention, a method for determining the physiological or mental condition of a living subject using quantitative results of multiple target miRNAs in the living subject, the method comprising collecting a biological sample from the living subject; extracting a miRNA sample having the multiple target miRNAs from the biological sample, wherein the multiple target miRNAs include a first target miRNA and a second target miRNA; performing a multiple reverse transcription process on the miRNA sample using a multiple stem-loop reverse transcription primer combination, wherein the multiple stem-loop reverse transcription primer combination includes 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 the first anchor sequence is complementary to the 3' sequence of the first target miRNA, and the second anchor sequence is complementary to the 3' sequence of the first target miRNA. wherein the length of the first anchor sequence of the first stem-loop reverse transcription primer is different from the length of the second anchor sequence of the second stem-loop reverse transcription primer; collecting a collection of multiple reverse transcription products from the multiple reverse transcription process; performing a multiple qPCR process on the collection of reverse transcription products to obtain a quantitative result for each of the multiple target miRNAs; obtaining a collection of quantitative results for the multiple target miRNAs; and using the collection of quantitative results for the multiple target miRNAs to determine the physiological or mental condition of the living subject, wherein the multiple target miRNAs are selected based on the physiological or mental condition to be determined, and wherein the multiple stem-loop reverse transcription primer combination has multiple specificities.

[0052] In one embodiment, the plurality of target miRNAs are selected based on the physiological or psychiatric condition to be determined.

[0053] In one embodiment, the plurality of target miRNAs comprises at least a first target miRNA and a second target miRNA.

[0054] In one embodiment, the multiple reverse transcription primer combination includes 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.

[0055] In one embodiment, the first anchor sequence of the first stem-loop reverse transcription primer is complementary to the 3' sequence of the first target miRNA, and the second anchor sequence of the second stem-loop reverse transcription primer is complementary to the 3' sequence of the second target miRNA, wherein the length of the first anchor sequence of the first stem-loop reverse transcription primer is different from the length of the second anchor sequence of the second stem-loop reverse transcription primer.

[0056] In one embodiment, each of the first stem-loop reverse transcription primer and the second stem-loop reverse transcription primer has a length of about 40-60 nt.

[0057] In one embodiment, each of the first anchor sequence and the second anchor sequence is about 3-12 nt in length.

[0058] In one embodiment, during the reverse transcription of the plurality of target miRNAs, the first stem-loop reverse transcription primer efficiently and only reverse transcribes the first target miRNA; and the second stem-loop reverse transcription primer efficiently and only reverse transcribes the second target miRNA.

[0059] In one embodiment, the first stem-loop sequence of the first stem-loop reverse transcription primer and the second stem-loop sequence of the second stem-loop reverse transcription primer are the same.

[0060] In one embodiment, the first stem-loop sequence of the first stem-loop reverse transcription primer is different from the second stem-loop sequence of the second stem-loop reverse transcription primer.

[0061] In one embodiment, the multiple reverse transcription primer combination further comprises a third stem-loop reverse transcription primer having a third stem-loop sequence and a third anchor sequence, wherein the third anchor sequence is complementary to the 3' sequence of a third target miRNA among the plurality of target miRNAs.

[0062] In one embodiment, the multiple reverse transcription primer combination further comprises a fourth stem-loop reverse transcription primer having a fourth stem-loop sequence and a fourth anchor sequence, wherein the fourth anchor sequence is complementary to the 3' sequence of a fourth target miRNA among the plurality of target miRNAs.

[0063] In one embodiment, the multiplex qPCR process is performed using a combination of forward primers, at least one reverse primer, and a combination of probes.

[0064] In one embodiment, each of the forward primers is complementary to the set of reverse transcription products of only one of the multiple target miRNAs, and each of the probes is complementary to the set of reverse transcription products of only one of the target miRNAs.

[0065] In one embodiment, each of the probes comprises a fluorescent reporter and a quencher, and the fluorescent reporter of each of the probes is different from the fluorescent reporters of other probes.

[0066] In one embodiment, each of the probes is probe or MGB probe. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] 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.

[0068] Figure 1A -B is a diagram showing a selection process for selecting a stem-loop reverse transcription primer combination and a screening process for testing the multi-specificity of the selected stem-loop reverse transcription primer combination. Figure 1A The selection and screening process for a combination of four stem-loop reverse transcription primers is shown. Figure 1B The selection and screening process for various stem-loop reverse transcription primer combinations is shown.

[0069] Figure 2A -D shows the amplification plot of single-plex RT-qPCR for each of the target miRNAs using each of the stem-loop reverse transcription primers with different anchor sequence lengths (6 / 8 / 11 nt). Figure 2A Amplification plots of miR-16-5p / ddH2O using corresponding stem-loop reverse transcription primers with different anchor sequence lengths (6 / 8 / 11 nt) are shown. Figure 2B Amplification plots of miR-34c-5p / ddH2O using corresponding stem-loop reverse transcription primers with different anchor sequence lengths (6 / 8 / 11 nt) are shown. Figure 2C Amplification plots of miR-9-3p / ddH2O using corresponding stem-loop reverse transcription primers with different anchor sequence lengths (6 / 8 / 11 nt) are shown. Figure 2D Amplification plots of miR-9-5p / ddH2O using corresponding stem-loop reverse transcription primers with different anchor sequence lengths (6 / 8 / 11 nt) are shown.

[0070] Figure 3A -E show the amplification plots of singleplex reverse transcription using each of the screened stem-loop reverse transcription primers and a mixed miRNA template with miR-9-3p, miR-34c-5p, miR-9-5p and miR-16-5p, followed by multiplex qPCR, as well as the amplification plots of ddH2O as a negative control. Figure 3A Shown is an amplification plot of singleplex RT-multiplex qPCR using stem-loop reverse transcription primers and mixed miRNA templates screened against miR-9-3p. Figure 3B Shown is an amplification plot of singleplex RT-multiplex qPCR using stem-loop reverse transcription primers and mixed miRNA templates screened against miR-34c-5p. Figure 3CShown is an amplification plot of singleplex RT-multiplex qPCR using stem-loop reverse transcription primers and mixed miRNA templates screened against miR-9-5p. Figure 3D Shown is an amplification plot of singleplex RT-multiplex qPCR using stem-loop reverse transcription primers and mixed miRNA templates screened against miR-16-5p. Figure 3E Shown is the amplification plot of singleplex RT-multiplex qPCR using a combination of stem-loop reverse transcription primers and ddH2O as a negative control.

[0071] Figure 4A -D shows the sensitivity test results of each of the stem-loop reverse transcription primers to each of the target miRNAs. Figure 4A Shown are the results of sensitivity testing of stem-loop reverse transcription primers screened for miR-9-5p. Figure 4B Shown are the results of sensitivity testing of stem-loop reverse transcription primers screened for miR-9-3p. Figure 4C The sensitivity test results of the stem-loop reverse transcription primers screened for miR-16-5p are shown. Figure 4D Shown are the results of sensitivity testing of stem-loop reverse transcription primers screened for miR-34c-5p.

[0072] Figure 5A -F show the comparison between singleplex reverse transcription and multiplex reverse transcription of multiple target miRNAs, respectively. Figure 5A Amplification plot of miR-34c-5p using singleplex reverse transcription is shown. Figure 5B Amplification plots of miR-9-5p using singleplex reverse transcription are shown. Figure 5C Amplification plots of miR-9-3p using singleplex reverse transcription are shown. Figure 5D Amplification plot of miR-16-5p using singleplex reverse transcription is shown. Figure 5E Amplification plots of multiplex reverse transcription using a stem-loop reverse transcription combination are shown. Fig. 5F is a schematic diagram showing a comparison of Ct values ​​of target miRNAs using singleplex reverse transcription and multiplex reverse transcription.

[0073] Fig. 6A -H show amplification plots of single-plex RT-qPCR using each of the stem-loop reverse transcription primers with different anchor sequence lengths (4 / 6 / 8 / 11 nt) and a mixed target miRNA template or a negative control, respectively. Fig. 6A Amplification plots of singleplex RT-qPCR of miR-210-3p using stem-loop reverse transcription primers with different anchor sequence lengths (4 / 6 / 8 / 11 nt) are shown. Figure 6BAmplification plots of singleplex RT-qPCR of negative control using miR-210-3p stem-loop reverse transcription primers with different anchor sequence lengths (4 / 6 / 8 / 11 nt) are shown. Figure 6C Amplification plots of singleplex RT-qPCR of miR-126-3p using stem-loop reverse transcription primers with different anchor sequence lengths (4 / 6 / 8 / 11 nt) are shown. Fig.6D Amplification plots of singleplex RT-qPCR of negative control using miR-126-3p stem-loop reverse transcription primers with different anchor sequence lengths (4 / 6 / 8 / 11 nt) are shown. Fig. 6E Amplification plots of singleplex RT-qPCR of miR-205-5p using stem-loop reverse transcription primers with different anchor sequence lengths (4 / 6 / 8 / 11 nt) are shown. Fig. 6F Amplification plots of singleplex RT-qPCR of negative control using miR-205-5p stem-loop reverse transcription primers with different anchor sequence lengths (4 / 6 / 8 / 11 nt) are shown. Figure 6G Amplification plots of singleplex RT-qPCR of miR-486-5p using stem-loop reverse transcription primers with different anchor sequence lengths (4 / 6 / 8 / 11 nt) are shown. Figure 6H Amplification plots of singleplex RT-qPCR of negative control using miR-486-3p stem-loop reverse transcription primers with different anchor sequence lengths (4 / 6 / 8 / 11 nt) are shown.

[0074] Fig. 7A -D show amplification plots of singleplex RT-multiplex qPCR using each of the screened stem-loop RT primers for mixed target miRNA templates, and amplification plots of singleplex RT-multiplex qPCR using ddH2O and a negative control, respectively. Fig. 7A Amplification plots of singleplex RT-multiplex qPCR of hsa-miR-210-3p using stem-loop reverse transcription primers are shown. Figure 7B Amplification plots of singleplex RT-multiplex qPCR of hsa-miR-126-3p using stem-loop reverse transcription primers are shown. Figure 7C Amplification plots of singleplex RT-multiplex qPCR of hsa-miR-205-5p using stem-loop reverse transcription primers are shown. Fig.7D Amplification plots of singleplex RT-multiplex qPCR of hsa-miR-486-5p using stem-loop reverse transcription primers are shown.

[0075] Fig. 8A -D shows the sensitivity test results of each of the target miRNAs. Fig. 8A The sensitivity test results of hsa-miR-210-3p are shown. Figure 8B The sensitivity test results of hsa-miR-126-3p are shown. Figure 8C The sensitivity test results of hsa-miR-205-5p are shown. Fig.8D Sensitivity test results of hsa-miR-486-5p are shown.

[0076] Fig. 9 is a schematic diagram showing multiplex detection of target miRNA combinations of hsa-miR-210-3p, hsa-miR-126-3p, hsa-miR-205-5p and hsa-miR-486-5p by multiplex RT-qPCR using the screened multiple stem-loop reverse transcription primer pairs.

[0077] Fig. 10A -D shows the amplification plot of quadruple RT-qPCR of mixed target miRNA templates using the stem-loop reverse transcription primer combination according to Table 23. Fig. 10A Shown are the results of qPCR using the fluorescent reporter VIC. Fig. 10B Shown are the results of qPCR using the fluorescent reporter ROX. Fig. 10C Shown are the results of qPCR using the fluorescent reporter CY5. Fig. 10D Shown are the results of qPCR using the fluorescent reporter FAM.

[0078] Fig.11 A diagram showing a multiplex RT-qPCR process for determining physiological or mental conditions in living subjects. DETAILED DESCRIPTION

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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 and its substitutes, such as amplification products and native sequences. The target miRNA of the present teachings can be derived from any source in 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 (such as 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. Generally, the target miRNA of the present teachings will be single-stranded.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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 TMand 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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)).

[0109] 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.

[0110] 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.

[0111] "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.

[0112] “T m " 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 chosen to be equal to the T 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 (for a description of SSC buffer, see Sambrook, below). 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 salt concentrations below 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 temperatures 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.

[0113] In view of the shortcomings of the prior art, namely reverse transcription-quantitative PCR (RT-qPCR) can only be used to quantify the expression level of a single target miRNA, the present invention provides a method for screening a multiple reverse transcription primer combination for quantifying multiple target miRNAs. Therefore, the present invention enables multiple target miRNAs to be quantified simultaneously by a multiple reverse transcription primer combination.

[0114] Multiple reverse transcription primer combination of the present invention comprises multiple stem-loop reverse transcription primers, and every kind of stem-loop reverse transcription primer comprises 5' end, stem-loop sequence, anchor base sequence and 3' end successively from its 5' to 3' end.The number of nucleotide bases 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 that can not form double-stranded structure between complementary fragment pairs protrudes to form "ring", and described ring is usually the sequence of 16-36 nucleotide bases.In one embodiment, design has the stem-loop reverse transcription primer with different stem-loop sequences.In one embodiment, design all has the stem-loop reverse transcription primer with identical stem-loop sequence.

[0115] 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.

[0116] The method for selecting one or more stem-loop reverse transcription primers for a multiplex reverse transcription primer combination mainly comprises the following steps: a) selecting a stem-loop sequence for each of the stem-loop reverse transcription primers; b) selecting the length of the anchor sequence of each of the stem-loop reverse transcription primers; and c) determining at least one combination of stem-loop reverse transcription primers that has multi-specificity and thus achieves multiplex RT-qPCR for simultaneously quantifying multiple target miRNAs.

[0117] The present application discloses that when the length of the anchor sequence is used as a variable for screening each stem-loop reverse transcription primer in the stem-loop reverse transcription primer for multiple RT, a combination of stem-loop reverse transcription primers that can be used for multiple RT-qPCR can be screened out. When this stem-loop reverse transcription primer combination is used for the multiple RT of multiple target miRNAs, cross-reactions will not occur, and therefore it will show multiple specificities to subsequent multiple qPCR. Therefore, when using the combination of stem-loop reverse transcription primers to reverse transcribe multiple target miRNAs in multiple RT, the efficiency of detection / quantification is greatly improved, the operating process is simplified, and the detection cost is reduced.

[0118] In order to quantify multiple target miRNAs by multiplex RT-qPCR, the above screening method can be used to screen stem-loop reverse transcription primer combinations for multiplex RT. Thus, miRNA samples with multiple target miRNAs are subjected to multiplex RT using the screened stem-loop reverse transcription primer combination before their reverse transcription products are subjected to multiplex qPCR.

[0119] During multiplex qPCR, a combination of forward primers, a universal reverse primer or a combination of reverse primers, and a combination of probes are added to the reverse transcription products for amplification during multiplex qPCR.

[0120] The principle for designing the forward primer used in qPCR is as follows. Specifically, the 5' end to the 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 generally 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 universal reverse primer should be within 1°C, and the Tm values ​​of the forward primer and the universal reverse primer should both be about 55-60°C.

[0121] The principles for designing the reverse primer used in qPCR are as follows. Specifically, 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.

[0122] The principles for designing probes used in qPCR are as follows. In one embodiment, The probe is used as a probe in a qPCR reaction. The length of each probe is usually in the range of 25 to 32 nt, and the Tm value is between 65-75°C, which is 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 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.

[0123] Figure 1A -B shows a diagram of a method for screening a multiple stem-loop reverse transcription primer combination 110 showing multi-specificity for simultaneously quantifying multiple target miRNAs, the method comprising a selection process, a screening process, and an optional repetition process. Specifically, Figure 1A A diagram showing a method for screening a multiplex stem-loop reverse transcription primer combination 110 having four stem-loop reverse transcription primers is shown. Figure 1B A diagram showing a method for screening a multiplex stem-loop reverse transcription primer combination 110 having multiple stem-loop reverse transcription primers is shown. The screened multiplex stem-loop reverse transcription primer combination is capable of quantifying multiple target miRNAs simultaneously.

[0124] In one embodiment, the number of stem-loop reverse transcription primers is selected according to the number of target miRNA to be quantified. In one embodiment, the combination has two kinds of stem-loop reverse transcription primers. In one embodiment, the combination has three kinds of stem-loop reverse transcription primers. In one embodiment, the combination has four kinds of stem-loop reverse transcription primers. In one embodiment, the combination has five kinds of stem-loop reverse transcription primers. In one embodiment, the combination has six kinds of stem-loop reverse transcription primers. In one embodiment, the combination has seven kinds of stem-loop reverse transcription primers. In one embodiment, the combination has eight kinds of stem-loop reverse transcription primers. In one embodiment, the combination has nine kinds of stem-loop reverse transcription primers. In one embodiment, the combination has ten kinds of stem-loop reverse transcription primers.

[0125] It should be noted that, although there are multiple stem-loop reverse transcription primers in the combination, the method for screening the combination with multi-specificity is the same. Once the combination of stem-loop reverse transcription primers shows multi-specificity, it is confirmed that any sub-combination of the screened combination with any number of the screened stem-loop reverse transcription primers in the screened combination also has multi-specificity.

[0126] In one embodiment, the selection process 100 starts with selecting a stem-loop reverse transcription primer combination 110 having more than one stem-loop reverse transcription primer 111 / 113 / 115 / 117. Each of the stem-loop reverse transcription primers 111 / 113 / 115 / 117 has a stem-loop sequence 1011 / 1013 / 1015 / 1017 that forms a stem-loop structure and an anchor sequence 1111 / 1113 / 1115 / 1117 that is complementary to the 3' sequence of one of the target miRNAs 121 / 123 / 125 / 127.

[0127] In one embodiment, the 3' sequence of each target miRNA in target miRNA 121 / 123 / 125 / 127 is different from the 3' sequence of other target miRNAs. In one embodiment, when the term "unique" is used for the 3' sequence of each target miRNA in target miRNA, it means that the 3' sequence of each target miRNA in target miRNA differs from the 3' sequence of other target miRNAs by at least 1nt.

[0128] When selecting each of the stem-loop reverse transcription primers 111 / 113 / 115 / 117, firstly a stem-loop sequence 1011 / 1013 / 1015 / 1017 is selected for each of the stem-loop reverse transcription primers, and then a certain length is selected for the anchor sequence of each of the stem-loop reverse transcription primers.

[0129] In one embodiment, a screening process is performed to test the multi-specificity of the selected stem-loop reverse transcription primer combination 110. The screening process starts with step i) of separately performing a single-plex reverse transcription process 201 / 203 using each of the stem-loop reverse transcription primers 111 / 113 / 115 / 117 with a mixed target miRNA template including a plurality of target miRNAs 121 / 123 / 125 / 127 to be quantified. In step ii), the reverse transcription products 141 / 143 / 145 / 147 of each single-plex reverse transcription process from the single-plex reverse transcription process of step i) are separately collected.

[0130] Then, each set of reverse transcription products 141 / 143 / 145 / 147 from step ii) is subjected to multiplex qPCR 205 / 207 individually, and each multiplex qPCR produces a set of multiplex qPCR products 161 / 163 / 165 / 167. During multiplex qPCR 205 / 207, quantitative results of each target miRNA in target miRNA 121 / 123 / 125 / 127 are obtained, thereby forming a set of quantitative results of multiple target miRNAs.

[0131] The multi-specificity of the stem-loop reverse transcription primer combination 110 can be determined based on the set of quantitative results. Specifically, when the set of quantitative results shows that each of the stem-loop reverse transcription primers 111 / 113 / 115 / 117 effectively and only reverse transcribes the target miRNA 111 / 113 / 115 / 117 complementary to its anchor sequence 1111 / 1113 / 1115 / 1117, the multi-specificity of the stem-loop reverse transcription primer combination 110 is confirmed.

[0132] However, when the selected stem-loop reverse transcription primer combination 110 failed to show multi-specificity according to the quantitative results 161 / 163 / 165 / 167, the stem-loop reverse transcription primer combination 110 was modified through a repeated process.

[0133] Specifically, the iterative process begins with selecting a new length for the anchor sequence 1111 / 1113 / 1115 / 1117 of any of the stem-loop reverse transcription primers 111 / 113 / 115 / 117 that failed the multi-specificity test during the screening process. That is, the stem-loop reverse transcription primer reverse transcribes more than one target miRNA, or cannot effectively reverse transcribe a target miRNA that is complementary to the anchor sequence of the stem-loop reverse transcription primer.

[0134] In one embodiment, the length of the anchor sequence 1111 / 1113 / 1115 / 1117 is selected between 3nt and 12nt. In one embodiment, the length of the anchor sequence 1111 / 1113 / 1115 / 1117 is selected between 4nt and 11nt. In one embodiment, the length of the anchor sequence 1111 / 1113 / 1115 / 1117 is selected between 4nt and 8nt. In one embodiment, the length of the anchor sequence 1111 / 1113 / 1115 / 1117 is selected between 6nt and 11nt. In one embodiment, the length of the anchor sequence 1111 / 1113 / 1115 / 1117 is selected between 6nt and 8nt.

[0135] Once a new length of the anchor sequence 1111 / 1113 / 1115 / 1117 of the stem-loop reverse transcription primer 111 / 113 / 115 / 117 that failed the multi-specificity test was reselected, a modified stem-loop reverse transcription primer combination was formed.

[0136] The next step in the iterative process is to repeat the screening process 200 with the modified stem-loop reverse transcription primer combination to determine the multi-specificity of the modified stem-loop reverse transcription primer combination.

[0137] The iterative process can generate one or more new modified stem-loop reverse transcription primer combinations by adjusting the length of the anchor sequence of any of the stem-loop reverse transcription primers. In one embodiment, only the length of the anchor sequence of one stem-loop reverse transcription primer is adjusted during one round of iterative process. In another embodiment, the length of the anchor sequence of more than one stem-loop reverse transcription primer is adjusted during one round of iterative process.

[0138] Once the stem-loop reverse transcription primer combination 110 is screened and confirmed to have multi-specificity, the method stops. In one embodiment, multiple RT-qPCR is then performed using the screened stem-loop reverse transcription primer combination 110 and multiple target miRNAs 121 / 123 / 125 / 127. Single RT performed using each stem-loop reverse transcription primer in stem-loop reverse transcription primers 111 / 113 / 115 / 117 can be compared with multiple RT performed using combination 110 to verify that the quantitative results of the multiple RT-qPCR are accurate.

[0139] In any single-plex RT process, a hybridization reaction 201 and an extension reaction 203 are included. In the hybridization reaction 201, each of the selected stem-loop reverse transcription primers is individually hybridized with the target miRNA 121 / 123 / 125 / 127 complementary to its anchor sequence to form a collection of miRNA-stem-loop reverse transcription primer complexes 131 / 133 / 135 / 137. In the following extension reaction 203, cDNA 141 / 143 / 145 / 147 is formed by extending the 3' of the stem-loop reverse transcription primer 111 / 113 / 115 / 117 for the target miRNA 121 / 123 / 125 / 127.

[0140] Unlike the single-plex RT process, in any of the multiplex RT processes, the hybridization reaction 201 is performed using more than one or all of the stem-loop reverse transcription primers that hybridize to a plurality of target miRNAs. When the selected stem-loop reverse transcription primer combination 110 has multiple specificities, each of the stem-loop reverse transcription primers only binds to the target miRNA that is complementary to its anchor sequence.

[0141] The multiplex qPCR 205 / 207 of the screening process 200 uses a combination of forward primers and a universal reverse primer or a combination of reverse primers. Each of the forward primers has a sequence complementary to only one type of cDNA 141 / 143 / 145 / 147 or only one stem-loop reverse transcription primer for reverse transcription.

[0142] The combination of probes 151 / 153 / 155 / 157 is also added to the reverse transcription of the reverse transcription product. In one embodiment, each of the probes is complementary to only one type of cDNA 141 / 143 / 145 / 147, and each of the probes has a fluorescent reporter group that is different from the fluorescent reporter groups of the other probes. In one embodiment, the probes are Probe. In one embodiment, the probe is a MGB probe.

[0143] Once the primers and probes are added to the reverse transcription products, multiplex qPCR amplifies the reverse transcription products. During the amplification process, the probes continue to bind to the generated cDNA, and whenever the cDNA extension forces each probe in the probe to leave a complementary cDNA sequence and the fluorescent reporter group is no longer inhibited by the quencher group, the fluorescent reporter group of each probe in the probe will produce fluorescence. Each target mRNA in the target mRNA is quantified by the fluorescence intensity detected in each set of the multiplex qPCR products.

[0144] In one embodiment, the quantitative result of each target miRNA in the target mRNA is a Ct value.

[0145] In one embodiment, the stem-loop sequences of at least two of the stem-loop reverse transcription primers are identical. In another embodiment, the stem-loop sequences of all stem-loop reverse transcription primers are identical.

[0146] In one embodiment, once a combination of stem-loop reverse transcription primers has been screened and confirmed to be multi-specific, the combination of stem-loop reverse transcription primers is subjected to a sensitivity test.

[0147] Sensitivity test is performed using multiple mixed target miRNA 121 / 123 / 125 / 127 templates with concentration gradient. That is, each template in the mixed target miRNA template has a concentration different from other mixed target miRNA templates. In one embodiment, each template in the mixed target miRNA template is diluted to 10% concentration with the previous template. In one embodiment, each template in the mixed target miRNA template is diluted to 50% concentration with the previous template. In one embodiment, each template in the mixed target miRNA template is diluted to 20% concentration with the previous template.

[0148] Once a plurality of mixed target miRNA 121 / 123 / 125 / 127 templates with a concentration gradient are prepared, a single RT process is performed individually on each mixed miRNA template in the mixed miRNA template using each of the stem-loop reverse transcription primers. That is, each of the stem-loop reverse transcription primers is individually used for single reverse transcription of a plurality of mixed target miRNA templates.

[0149] The reverse transcription products of each single reverse transcription process in the single reverse transcription process are collected. Multiple qPCR is performed using each set of the reverse transcription product sets. A set of quantitative results of each target miRNA in the target miRNA in the multiple mixed target miRNA templates is obtained. Thus, for each stem-loop reverse transcription primer in the stem-loop reverse transcription primer, its quantitative results are arranged according to the concentration of the multiple mixed target miRNA templates, and the lowest concentration of the mixed target miRNA template effectively transcribed by the stem-loop reverse transcription primer is considered to be the result of the sensitivity test.

[0150] Each of the stem-loop reverse transcription primers has a stem-loop sequence forming a stem-loop structure and an anchor sequence complementary to the 3′ sequence of one of the plurality of target miRNAs.

[0151] In one embodiment, the length of the anchor sequence of at least one of the stem-loop reverse transcription primers is different from the length of the anchor sequence of the other stem-loop reverse transcription primers. In one embodiment, the length of each of the stem-loop reverse transcription primers is about 40-60 nt.

[0152] In one embodiment, the stem-loop structure of some stem-loop reverse transcription primers in the stem-loop reverse transcription primer is identical. In one embodiment, the stem-loop structure of all stem-loop reverse transcription primers is identical. In one embodiment, the stem-loop structure of at least one stem-loop reverse transcription primer in the stem-loop reverse transcription primer is different from the stem-loop structure of other stem-loop reverse transcription primers. In one embodiment, every kind of stem-loop structure in the stem-loop structure is unique and is different from other stem-loop structures.

[0153] In one embodiment, a kit for simultaneously quantifying multiple target miRNAs is provided. The kit comprises: a multiple stem-loop reverse transcription primer combination, wherein the multiple stem-loop reverse transcription primer combination comprises two to ten stem-loop reverse transcription primers; a combination of two to ten forward primers, wherein each of the forward primers comprises a Tm enhancement tail and a forward primer sequence complementary to the reverse transcription product produced in RT; and a universal reverse primer or a combination of reverse primers.

[0154] The kit also includes a combination of probes. Each probe in the probe has a probe sequence identical to a portion of the sequence of a stem-loop reverse transcription primer. Each probe in the probe has a fluorescent reporter group and a quencher group. Importantly, each probe in the probe has a fluorescent reporter group that is different from the fluorescent reporter of other probes.

[0155] Example 1 - Multiple stem-loop reverse transcription primers for quantification of miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p

[0156] The following is an example of a screening method for screening a stem-loop reverse transcription primer combination in the present application, wherein the stem-loop reverse transcription primer combination is used to simultaneously detect a combination of multiple target miRNAs comprising miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p. The sequences of these miRNAs are reported in the microRNA database (www.mirbase.org). Specifically, the sequence of miR-16-5p is SEQ ID No.1, the sequence of miR-34c-5p is SEQ ID No.2, the sequence of miR-9-3p is SEQ ID No.3, and the sequence of miR-9-5p is SEQ ID No.4.

[0157] A variety of stem-loop reverse transcription primers with different anchor sequence lengths are designed for the above-mentioned target miRNA combination. The length of each stem-loop reverse transcription primer in the stem-loop reverse transcription primer is 40-60 nucleotides. Each stem-loop reverse transcription primer in the stem-loop reverse transcription primer comprises a stem-loop sequence and an anchor sequence. In Example 1, the stem-loop sequence of each stem-loop reverse transcription primer is shown in Table 1, and the anchor sequence of each stem-loop reverse transcription primer in the stem-loop reverse transcription primer is complementary to the 3' sequence of the target miRNA to be specifically detected by the stem-loop reverse transcription primer. In one embodiment, the length of the anchor sequence is selected in the range of 3-12nt.

[0158] Table 1. Sequences of the stem-loop structure of each stem-loop reverse transcription primer in Example 1

[0159]

[0160] Forward primers and reverse primers for multiplex qPCR were designed for the reverse transcription product (cDNA), as shown in Table 2:

[0161] Table 2. Forward and reverse primers for qPCR

[0162]

[0163]

[0164] about The probes were designed by modifying each probe with a different fluorescent group (FAM, VIC, CY5, ROX, etc.) to distinguish the detection results of different target miRNAs based on different fluorescent reporter groups, as shown in Table 3.

[0165] Table 3. Reporter and quencher groups for each probe in the probe set.

[0166]

[0167] Specificity of each stem-loop reverse transcription primer and probe

[0168] Each stem-loop reverse transcription primer was subjected to singleplex reverse transcription using a mixed target miRNA template or ddH2O. Each of the stem-loop reverse transcription primers had an anchor sequence of 4, 6, 8 or 11 nt in length and a stem-loop sequence shown in Table 1. The singleplex reverse transcription system is shown in Table 4:

[0169] Table 4. Reverse transcription system

[0170]

[0171] The reverse transcription thermal cycle is shown in Table 5.

[0172] Table 5. Thermal cycles for reverse transcription

[0173]

[0174] The reverse transcription products collected from each of the singleplex reverse transcriptions were subjected to multiplex qPCR, where the forward / reverse primers and probes were shown in Tables 2 and 3. The qPCR system is shown in Table 6.

[0175] Table 6. qPCR system

[0176]

[0177] The qPCR amplification thermal cycle is shown in Table 7:

[0178] Table 7. Thermal cycling of qPCR.

[0179]

[0180] The quantitative results generated by qPCR are shown in Table 8 and Figure 2A -D. The quantitative results of single-plex RT-multiplex qPCR of miR-16-5p using stem-loop reverse transcription primers (with 6, 8 or 11 nt as anchor sequence length) are shown in Figure 2A The quantitative results of single-plex RT-multiplex qPCR of miR-34c-5p (with 6, 8 or 11 nt as anchor sequence length) are shown in Figure 2B The quantitative results of single-plex RT-multiplex qPCR of miR-9-3p (with 6, 8 or 11 nt as anchor sequence length) are shown in Figure 2C The quantitative results of single-plex RT-multiplex qPCR of miR-9-5p (with 6, 8 or 11 nt as anchor sequence length) are shown in Figure 2D As shown in the figure, all stem-loop reverse transcription primers did not amplify in ddH2O, indicating good specificity.

[0181] Table 8. Results of single-plex RT-qPCR using each of the stem-loop reverse transcription primers with different anchor sequence lengths (6 / 8 / 11 nt)

[0182]

[0183]

[0184] Construction of multiplex RT-qPCR for quantification of multiple target miRNAs

[0185] Each stem-loop reverse transcription primer in the stem-loop reverse transcription primer is selected for each target miRNA in the target miRNA. The length of the anchor sequence of each stem-loop reverse transcription primer in the stem-loop reverse transcription primer can be adjusted to form more than one possible combination of stem-loop reverse transcription primers. In one embodiment, the stem-loop sequences of all stem-loop reverse transcription primers are the same. In another embodiment, the stem-loop sequence of each stem-loop reverse transcription primer in the stem-loop reverse transcription primer is different from the stem-loop sequence of other stem-loop reverse transcription primers. Each combination in the combination of stem-loop reverse transcription primers is subjected to multi-specificity and sensitivity tests. When a combination passes the specificity test, it is considered to be a potential combination for multiple RT-qPCR for quantifying multiple target miRNAs. Thereafter, a sensitivity test can be performed to determine the detection limit of the target miRNA. In order to improve the screening efficiency, once the multi-specificity of a particular combination is confirmed, the selection of the stem-loop reverse transcription primer can be stopped. The combination is then used for multiple RT-qPCR quantification of the target miRNA combination. In order to obtain an optimized stem-loop reverse transcription primer combination, the length of the anchor sequence of the stem-loop reverse transcription primer can be adjusted, and more combinations with different anchor sequence lengths can be formed. These combinations with different anchor sequences were tested for multiple specificities and sensitivities, allowing for the acquisition of an optimized combination of primers with the best specificity and sensitivity.

[0186] Specifically, for miR-16-5p, the stem-loop reverse transcription primer with an anchor sequence length of 8 nucleotides was selected. For miR-34c-5p, the stem-loop reverse transcription primer with an anchor sequence length of 6 nucleotides was selected. For miR-9-3p, the stem-loop reverse transcription primer with an anchor sequence length of 6 nucleotides was selected. For miR-9-5p, the stem-loop reverse transcription primer with an anchor sequence length of 11 nucleotides was selected. These stem-loop reverse transcription primers were combined to form a stem-loop reverse transcription primer combination, as shown in Table 9.

[0187] Table 9. Screened stem-loop reverse transcription primers

[0188]

[0189] This combination of stem-loop reverse transcription primers was set as an example to verify multi-specificity, sensitivity and stability.

[0190] Multi-specificity testing of the screened stem-loop reverse transcription primer combinations

[0191] Singleplex reverse transcription was performed using each of the selected stem-loop reverse transcription primers individually with a mixed target miRNA template (miR-16-5p, miR-34c-5p, miR-9-3p, miR-9-5p), and each singleplex reverse transcription system is shown in Table 10.

[0192] Table 10. Singleplex reverse transcription system for each stem-loop reverse transcription primer in the stem-loop reverse transcription primers

[0193]

[0194]

[0195] To verify the multi-specificity of the stem-loop reverse transcription primer combination, the reverse transcription products of each single-plex reverse transcription in the single-plex reverse transcription were subjected to multiplex qPCR to determine whether there was cross-reaction in the quantification of the four target miRNAs. The multiplex qPCR system is shown in Table 11.

[0196] Table 11. Multiplex qPCR system

[0197]

[0198] The forward primer combination and the reverse primer combination refer to a combination of four forward primers and four reverse primers shown in Table 2, and the probe combination refers to a combination of four probes shown in Table 3.

[0199] like Figure 3A -E, there is no cross-reaction in the quantification of each target miRNA in the target miRNA. The stem-loop reverse transcription primer combination, the forward / reverse primer combination and the probe combination all have good multi-specificity. Therefore, this stem-loop reverse transcription primer combination can be used for multiplex RT-qPCR quantification of the following four target miRNAs: miR-16-5p, miR-34c-5p, miR-9-3p and miR-9-5p. Based on the multi-specificity of the stem-loop reverse transcription primer combination for detecting four target miRNAs, any two or three of the stem-loop reverse transcription primers can form a sub-combination with multi-specificity for detecting two or three target miRNAs in the target miRNA, respectively.

[0200] Sensitivity test of the selected stem-loop reverse transcription primer combinations

[0201] In order to test the sensitivity of the screened stem-loop reverse transcription primer combination, the mixed target miRNA template (miR-16-5p, miR-34c-5p, miR-9-3p, miR-9-5p) was serially diluted. Each template with different concentrations of mixed target miRNA was subjected to multiple RT using each of the screened stem-loop reverse transcription primers, and then multiple qPCR was performed to determine the lowest concentration of the mixed target miRNA as the detection limit, i.e., sensitivity.

[0202] Specifically, the mixed target miRNA template with a concentration of 1 pg / μL was diluted 10 times to 0.1 pg / μL, 0.01 pg / μL, 1 fg / μL, and 0.1 fg / μL for single-plex RT, and then the obtained reverse transcription products were subjected to multiplex qPCR. Figure 4A -D. The detection limit of miR-9-5p was 1 fg / μL; the detection limit of miR-9-3p was 0.1 fg / μL; and the detection limit of miR-16-5p and miR-34c-5p was 1 fg / μL.

[0203] Comparison of singleplex and multiplex RT-qPCR of target miRNAs

[0204] With the informed consent of all participants, 5 mL of peripheral blood was taken from the venous blood collection tube using a coagulation separation gel, the peripheral blood was allowed to stand at room temperature for 30 minutes, centrifuged at 3000 x g for 10 minutes, the supernatant was taken out, and the supernatant was stored in a -80 ° C refrigerator. The Qiagen serum / plasma miRNA extraction kit was used to extract nucleic acids using 200 μL of serum by a column method and according to the instructions of the kit, and finally dissolved in 20 μL of ddH2O. The extracted miRNA samples were stored in a -80 ° C refrigerator for later use.

[0205] The best forward / reverse primer and probe combination was selected from the above-mentioned experimental results, and singleplex RT-qPCR and multiplex RT-qPCR of serum miRNA samples were performed separately according to the above-mentioned reaction system and thermal cycle. Figure 5A There were no significant differences in amplification curves and Ct values ​​between singleplex and multiplex RT-qPCR, specifically Fig. 5F shown.

[0206] Stability testing of multiplex RT-qPCR

[0207] The stability test was performed by expanding the sample size to 20 individuals, and the results are shown in Table 12. From the results, it can be concluded that the multiple stem-loop reverse transcription primers in this example can accurately detect miR-34c-5p, miR-9-3p, miR-16-5p and miR-9-5p in the collected human serum samples.

[0208] Table 12. Ct values ​​of target miRNAs using singleplex / multiplex RT-qPCR in 20 individuals

[0209]

[0210] Example 2 - Multiple stem-loop reverse transcription primers for quantification of hsa-miR-210-3p, hsa-miR-126-3p, hsa-miR-205-5p and hsa-miR-486-5p

[0211] In one embodiment, the method of the present invention screens for stem-loop reverse transcription primer combinations for detecting multiple target miRNAs, wherein the multiple target miRNAs include hsa-miR-210-3p, hsa-miR-126-3p, hsa-miR-205-5p and hsa-miR-486-5p. The sequences of these target miRNAs and other miRNAs mentioned herein are reported in the microRNA database www.mirbase.org. Specifically, the sequence of hsa-miR-210-3p is shown in SEQ ID No.29, the sequence of hsa-miR-126-3p is shown in SEQ ID No.30, the sequence of hsa-miR-205-5p is shown in SEQ ID No.31, and the sequence of hsa-miR-486-5p is shown in SEQ ID No.32.

[0212] Designed multiple stem-loop reverse transcription primers with different anchor sequence lengths (6 / 8 / 11nt) for each target miRNA in the above-mentioned target miRNA.The length of each stem-loop reverse transcription primer in the stem-loop reverse transcription primer is about 40-60 nucleotide.Every kind of stem-loop reverse transcription primer in the stem-loop reverse transcription primer has stem-loop sequence and anchor sequence successively from its 5 ' end to its 3 ' end.In one embodiment, the stem-loop structure of all stem-loop reverse transcription primers has identical sequence, and the anchor sequence of each kind of stem-loop reverse transcription primer in the stem-loop reverse transcription primer is complementary to the 3 ' sequence of each kind of target miRNA in the target miRNA, wherein the selected length is in the scope between 3-12nt.The sequence of the stem-loop reverse transcription primer in example 2 is shown in table 13.

[0213] Table 13. Sequences of stem-loop reverse transcription primers used to quantify various target miRNAs including hsa-miR-210-3p, hsa-miR-126-3p, hsa-miR-205-5p and hsa-miR-486-5p

[0214]

[0215] In this example, the stem-loop sequences of all stem-loop reverse transcription primers are the same and are shown as SEQ ID No.37.

[0216] The forward and reverse primers of the reverse transcription product (including cDNA) are designed for multiplex qPCR. The forward primer has a Tm enhancement tail and a specific complementary binding sequence from its 5' to 3' end. The Tm enhancement tail includes randomly arranged A, T, C or G to increase its Tm value. The specific binding sequence has an 8-12 nucleotide sequence and is identical to the DNA equivalent of the 5' sequence of the target miRNA. When any U in the target miRNA is replaced by T, the DNA equivalent of the target miRNA has the same sequence as the target miRNA. The length of the forward primer is about 15-22 nucleotides.

[0217] The sequence of reverse primer is identical with a part of the stem-loop sequence of stem-loop reverse transcription primer. In this embodiment, reverse primer is a universal primer. In one embodiment, universal reverse primer is located on the stem-loop sequence with about 16-25 nucleotide, and the length of reverse primer is in the scope of 15-22 nucleotide. In one embodiment, universal primer has the sequence identical with a part of " ring " in the stem-loop sequence. The sequence of primer is shown in Table 14.

[0218] Table 14. Forward and reverse primers for cDNA generated in reverse transcription of various target miRNAs including hsa-miR-210-3p, hsa-miR-126-3p, hsa-miR-205-5p and hsa-miR-486-5p

[0219]

[0220]

[0221] The sequence of each probe in the probe is partially identical to a portion of the DNA equivalent of each target miRNA in the target miRNA to be detected. Each specific probe contains 12-25 nucleotides, wherein the quenching group MGB is marked at the 3' end and the fluorescent reporter group is marked at the 5' end. Different fluorescent groups such as FAM, VIC, CY5 or ROX are used to mark probes targeting different target miRNAs. In this embodiment, MGB probes are used as quenching groups. The MGB group marked at the 3' end of the MGB probe has a quenching effect on the fluorescence of the reporter group and can increase the Tm value of the probe itself. This enables the probe to preferentially and selectively bind to the groove of the double-stranded DNA molecule, thereby forming a stable hybrid complex. This further improves the specificity of the detection method. The structure and sequence of each probe in the probe are shown in Table 15.

[0222] Table 15. Structure of probes used in multiplex qPCR for detecting multiple target miRNAs including hsa-miR-210-3p, hsa-miR-126-3p, hsa-miR-205-5p and hsa-miR-486-5p

[0223]

[0224] Specificity testing of primers and probes using target miRNA template and ddH2O

[0225] Singleplex reverse transcription was performed for each of the target miRNAs using its corresponding stem-loop reverse transcription primer. The reverse transcription system is shown in Table 16.

[0226] Table 16. System for singleplex reverse transcription of each of the target miRNAs

[0227]

[0228] The reverse transcription thermal cycle is shown in Table 17:

[0229] Table 17. Thermal Cycles for Reverse Transcription

[0230]

[0231] The reverse transcription products produced in each single reverse transcription in the single reverse transcription were individually subjected to multiplex qPCR. The forward / reverse primers and probes designed for each target miRNA in the target miRNA were used for multiplex qPCR. The multiplex qPCR amplification system is shown in Table 18:

[0232] Table 18. Multiplex qPCR amplification system

[0233]

[0234] The qPCR thermal cycle is shown in Table 19:

[0235] Table 19. qPCR thermal cycling

[0236]

[0237] The results of multiplex qPCR were Fig. 6A -H. Each of the multiple target miRNAs is subjected to single reverse transcription using a stem-loop reverse transcription primer, wherein the anchor sequence of the stem-loop reverse transcription primer has a specific length, and the anchor sequence length of each stem-loop reverse transcription primer may be different from the anchor sequence length of other stem-loop reverse transcription primers. The specificity and sensitivity test of each of the stem-loop reverse transcription primers is completed. According to Fig. 6A and6B , miR-210-3p can be detected using a reverse transcription primer with an anchor sequence of 6 / 8 nt. Figure 6C and 6D , miR-126-3p can be detected using reverse transcription primers with anchor sequences of 4 / 6 / 11 nt. Figure 5E and 5F , miR-205-5p can be detected using a reverse transcription primer with an anchor sequence of 4 / 6 nt. According to Figures 5G and 5F, miR-486-5p can be detected using a reverse transcription primer with an anchor sequence of 4 / 8 nt.

[0238] Construction of miRNA multiplex system

[0239] Each stem-loop reverse transcription primer in the stem-loop reverse transcription primer is selected for each target miRNA in the target miRNA. The length of the anchor sequence of each stem-loop reverse transcription primer in the stem-loop reverse transcription primer can be adjusted to form more than one possible combination of stem-loop reverse transcription primers. In one embodiment, the stem-loop sequences of all stem-loop reverse transcription primers are the same. In another embodiment, the stem-loop sequence of each stem-loop reverse transcription primer in the stem-loop reverse transcription primer is different from the stem-loop sequence of other stem-loop reverse transcription primers. Each combination of stem-loop reverse transcription primers is subjected to multi-specificity and sensitivity tests. When a combination passes the specificity test, it is considered to be a potential combination for multiple RT-qPCR for quantifying multiple target miRNAs. Thereafter, a sensitivity test can be performed to determine the detection limit of the target miRNA. In order to improve the screening efficiency, once the multi-specificity of a particular combination is confirmed, the selection of the stem-loop reverse transcription primer can be stopped. The combination is then used for multiple RT-qPCR detection of the target miRNA combination. In order to obtain an optimized stem-loop reverse transcription primer combination, the length of the anchor sequence of the stem-loop reverse transcription primer is adjusted, and more combinations with different anchor sequence lengths are formed. These combinations with different anchor sequences were tested for multiple specificities and sensitivities, allowing for the acquisition of an optimized combination of primers with the best specificity and sensitivity.

[0240] In the embodiment of Example 2, a reverse transcription primer with an anchor sequence of 8 nt is used to detect miR-210-3p, a reverse transcription primer with an anchor sequence of 6 nt is used to detect miR-126-3p, a reverse transcription primer with an anchor sequence of 6 nt is used to detect miR-205-5p, and a reverse transcription primer with an anchor sequence of 8 nt is used to detect miR-486-5p. These are combined to form a stem-loop reverse transcription primer combination, as shown in Table 20.

[0241] Table 20. Stem-loop reverse transcription primer combinations for detecting miR-210-3p, miR-126-3p, miR-205-5p, and miR-486-5p.

[0242]

[0243] This combination of stem-loop reverse transcription primers was set up as an example for multi-specificity, sensitivity and stability testing.

[0244] Multispecific detection of stem-loop reverse transcription primer combinations

[0245] Each stem-loop reverse transcription primer in the combination is used in a single RT of a mixed target miRNA template (miR-210-3p, miR-126-3p, miR-205-5p, and miR-486-5p). Each single RT system is shown in Table 21:

[0246] Table 21. Singleplex RT System

[0247]

[0248] The reverse transcription products obtained from the single-plex RT were subjected to multiplex qPCR to determine if there was any cross-reaction in the quantification of the four target miRNAs, thereby verifying the multi-specificity of the stem-loop reverse transcription primer combination. The qPCR system is shown in Table 22:

[0249] Table 22. qPCR system

[0250]

[0251] The forward primer and reverse primer combination is a combination of four forward primers and one universal reverse primer as shown in Table 14, and the probe combination is a combination of four probes as shown in Table 3. Fig. 7A As shown in Figure 1, there was no cross-reaction in the quantification of each target miRNA in the target miRNA, and the stem-loop reverse transcription primer, forward / reverse primer and probe all showed good multi-specificity. Therefore, this combination can be used for multiplex RT-qPCR detection of four target miRNAs, namely miR-210-3p, miR-126-3p, miR-205-5p and miR-486-5p.

[0252] Based on the multi-specificity of the stem-loop reverse transcription primer combination for detecting four target miRNAs, any two or three of the stem-loop reverse transcription primers can form a sub-combination having multi-specificity for detecting two or three of the target miRNAs, respectively.

[0253] Sensitivity test of the selected stem-loop reverse transcription primer combinations

[0254] To test the sensitivity of the screened stem-loop reverse transcription primer combination, the mixed target miRNA template (miR-210-3p, miR-126-3p, miR-205-5p, miR-486-5p) was serially diluted. Each template with different concentrations of the mixed target miRNA template was subjected to multiple RT using the screened stem-loop reverse transcription primer combination, followed by multiple qPCR to determine the lowest detection limit, i.e., sensitivity.

[0255] Specifically, the concentration was 2.0×10 3 fg / μL of the target miRNA template was diluted 10-fold to 2.0×10 1 fg / μL, 2.0×10 0 fg / μL, 2.0×10 -1 fg / μL, 2.0×10 -2 fg / μL and 2.0×10 -3 fg / μL, and then all the reverse transcription products obtained were subjected to multiplex RT-qPCR amplification.

[0256] The results are Fig. 8A -D. The detection limit of miR-210-3p, miR-126-3p, miR-205-5p and miR-486-5p was 2.0×10 -2 fg / μL.

[0257] Comparison of singleplex and multiplex RT-qPCR of target miRNAs

[0258] use The serum / plasma miRNA kit (Yeasen, Shanghai, China) was used to extract total RNA including miRNA from individuals.

[0259] Using the optimal forward / reverse primer combination and probe combination found in Example 2, and using the qPCR system presented above, multiplex RT-qPCR of target miRNAs in total RNA samples was completed, and the results were presented in Fig. 9 Shown in.

[0260] Specifically, the stem-loop reverse transcription primer combinations screened for miR-210-3p, miR-126-3p, miR-205-5p, and miR-486-5p have good multi-specificity and sensitivity to correctly quantify the target miRNAs by multiplex RT-qPCR.

[0261] Optimizing multiplex RT-qPCR by adjusting the length of anchor sequences

[0262] A mixed target miRNA template containing hsa-miR-210-3p, hsa-miR-126-3p, hsa-miR-205-5p and hsa-miR-486-5p was used. Each target miRNA to be quantified was subjected to multiplex RT individually using a combination of stem-loop reverse transcription primers with different anchor sequence lengths shown in Table 23; the multiplex reverse transcription products were then subjected to multiplex qPCR to screen for a combination with good sensitivity and specificity.

[0263] Table 23. Combinations of stem-loop reverse transcription primers with different anchor sequence lengths

[0264]

[0265] Fig. 10A -D shows the amplification plot of multiplex RT-qPCR of mixed target miRNA templates using stem-loop reverse transcription primer combinations according to Table 23, so as to optimize the anchor sequence length of each of the stem-loop reverse transcription primers. Fig. 10A Shown are the results of qPCR using the fluorescent reporter VIC. Fig. 10B Shown are the results of qPCR using the fluorescent reporter ROX. Fig. 10C Shown are the results of qPCR using the fluorescent reporter CY5. Fig. 10D Shown are the results of qPCR using the fluorescent reporter FAM.

[0266] according to Fig. 10A -D and Table 23, Group 6 exhibits the best multi-specificity and sensitivity by showing the lowest or relatively low Ct value for each target miRNA in the target miRNA. Therefore, a stem-loop reverse transcription primer with an anchor sequence length of 8nt was selected for miR-210-3p. A stem-loop reverse transcription primer with an anchor sequence length of 6nt was selected for miR-126-3p. A stem-loop reverse transcription primer with an anchor sequence length of 6nt was selected for miR-205-5p. A stem-loop reverse transcription primer with an anchor sequence length of 8nt was selected for miR-486-5p.

[0267] Any two or three of hsa-miR-210-3p, hsa-miR-126-3p, hsa-miR-205-5p and hsa-miR-486-5p are mixed to form a mixed RNA template, and multiple reverse transcription is performed separately with the screened stem-loop reverse transcription primer combination; the multiple reverse transcription products are then subjected to multiple qPCR to verify the specificity and sensitivity of the screened stem-loop reverse transcription primer combination.

[0268] To summarize Example 1, the sequences of stem-loop reverse transcription primers, forward / reverse primers, and probes used for multiplex RT-qPCR quantification of miR-16-5p, miR-34c-5p, miR-9-3p, and miR-9-5p are listed below in Table 24.

[0269] Table 24. Primers and probes used for multiplex RT-qPCR

[0270]

[0271] To summarize Example 2, the sequences of stem-loop reverse transcription primers, forward / reverse primers, and probes used for multiplex RT-qPCR quantification of hsa-miR-210-3p, hsa-miR-126-3p, hsa-miR-205-5p, and hsa-miR-486-5p are listed below in Table 25.

[0272] Table 25. Primers and probes used for multiplex RT-qPCR

[0273]

[0274] Tables 26 and 27 list stem-loop reverse transcription primers with different anchor lengths.

[0275] Table 26. Stem-loop primer variants with different anchor lengths for miR-16-5p, miR-34c-5p, miR-9-3p, and miR-9-5p

[0276]

[0277]

[0278] Table 27. Stem-loop primer variants with different anchor lengths for miR-210-3p, miR-126-3p, miR-205-5p, and miR-486-5p

[0279]

[0280] In another embodiment of the present invention, Fig.11 As shown, the multiple stem-loop reverse transcription primer combination is used to determine the physiological or mental condition of a living subject by quantifying multiple target miRNAs in the living subject. This allows the multiple stem-loop reverse transcription primer combination to be used for disease diagnosis in a clinical setting such as a hospital.

[0281] First, a biological sample 911 is collected from a living subject in a collection process 901. A miRNA sample 913 having a plurality of target miRNAs is extracted 903 from the biological sample 911. Multiplex RT-qPCR 905 is then performed on the miRNA sample 913. Specifically, a reverse transcription process 905 is performed on the miRNA sample 913 using a multiplex stem-loop reverse transcription primer combination 110, and a collection of multiplex reverse transcription products is collected 915. Next, multiplex qPCR 907 is performed on the collection of reverse transcription products 915 to obtain a quantitative result for each of the plurality of target miRNAs, and a collection 917 of quantitative results for the plurality of target miRNAs is formed.

[0282] The physiological or mental condition of the living subject is determined 919 by comparing the collection of quantitative results for the plurality of target miRNAs to a set of standard values ​​(eg, quantitative results for the plurality of target miRNAs in one or more healthy human individuals).

[0283] In one embodiment, multiple target miRNAs are selected based on the physiological or mental condition to be determined. In one embodiment, multiple target miRNAs have two target miRNAs. In one embodiment, multiple target miRNAs have three target miRNAs. In one embodiment, multiple target miRNAs have four target miRNAs. In one embodiment, multiple target miRNAs have five target miRNAs. In one embodiment, multiple target miRNAs have six target miRNAs. In one embodiment, multiple target miRNAs have seven target miRNAs. In one embodiment, multiple target miRNAs have eight target miRNAs. In one embodiment, multiple target miRNAs have nine target miRNAs. In one embodiment, multiple target miRNAs have ten target miRNAs.

[0284] 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 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 stem-loop reverse transcription primers among the plurality of stem-loop reverse transcription primers are different from each other. 2 . The multiplex reverse transcription primer combination according to claim 1 , wherein the length of each stem-loop reverse transcription primer in the plurality of stem-loop reverse transcription primers is about 40-60 nt. 3 . The multiplex reverse transcription primer combination according to claim 1 , 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.

4. The multiple reverse transcription primer combination according to claim 1, wherein the plurality of 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.

5. The multiple reverse transcription primer combination of claim 4, 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.

6. The multiple reverse transcription primer combination according to claim 5, wherein when multiple reverse transcription is performed using a mixed target miRNA template comprising the first target miRNA and the second target miRNA and the multiple reverse transcription primer combination, The first stem-loop reverse transcription primer efficiently and only reverse transcribes the first target miRNA; and The second stem-loop reverse transcription primer efficiently and only reverse transcribes the second target miRNA. 7 . The multiple reverse transcription primer combination according to claim 1 , wherein the first stem-loop sequence of the first stem-loop reverse transcription primer and the second stem-loop sequence of the second stem-loop reverse transcription primer are the same.

8. The multiple reverse transcription primer combination according to claim 4, 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 anchor sequence is complementary to a unique 3' sequence of a third target miRNA among the plurality of target miRNAs.

9. The multiple reverse transcription primer combination according to claim 8, 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 anchor sequence is complementary to a unique 3' sequence of a fourth target miRNA among the plurality of target miRNAs.

10. The multiple reverse transcription primer combination of claim 9, 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. 11 . The multiple reverse transcription primer combination according to claim 10 , wherein at least two of the first stem-loop structure, the second stem-loop structure, the third stem-loop structure and the fourth stem-loop structure are the same. 12 . The multiple reverse transcription primer combination according to claim 11 , wherein all of the first stem-loop structure, the second stem-loop structure, the third stem-loop structure and the fourth stem-loop structure are the same.

13. A kit for simultaneously quantifying the expression levels of multiple target miRNAs, the kit comprising: A multiple reverse transcription primer combination, wherein the multiple reverse transcription primer combination has at least 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; a first forward primer and a second forward primer, wherein the first forward primer comprises a first Tm enhancing tail and a first forward primer sequence, and the second forward primer comprises a second Tm enhancing tail and a second forward primer sequence; at least one reverse primer; wherein the at least one reverse primer is complementary to the first stem-loop sequence and the second stem-loop sequence; 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; The first fluorescent reporter group is different from the second fluorescent reporter group. 14 . The kit according to claim 13 , wherein the length of each of the first stem-loop reverse transcription primer and the second stem-loop reverse transcription primer is about 40-60 nt.

15. The kit of claim 14, wherein each of the first anchor sequence and the second anchor sequence is about 3-12 nt in length.

16. The kit of claim 15, 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. 17 . The kit according to claim 16 , wherein the first stem-loop sequence of the first stem-loop reverse transcription primer and the second stem-loop sequence of the second stem-loop reverse transcription primer are the same.

18. The kit according to claim 16, wherein the length of the first anchor sequence of the first stem-loop reverse transcription primer is different from the length of the second anchor sequence of the second stem-loop reverse transcription primer.

19. The kit according to claim 16, wherein the multiple reverse transcription primer combination further comprises a third stem-loop reverse transcription primer and a fourth stem-loop reverse transcription primer; wherein the third stem-loop reverse transcription primer has a third stem-loop sequence and a third anchor sequence; and the fourth stem-loop reverse transcription primer has a fourth stem-loop sequence and a fourth anchor sequence; a third forward primer and a fourth forward primer, wherein the third forward primer comprises a third Tm enhancing tail and a third forward primer sequence, and the fourth forward primer comprises a fourth Tm enhancing tail and a fourth forward primer sequence; as well as A third probe and a fourth probe, wherein the third probe comprises a third probe sequence, a third fluorescent reporter group and a third quencher group; and the fourth probe comprises a fourth probe sequence, a fourth fluorescent reporter group and a fourth quencher group; Each of the first fluorescent reporter group, the second fluorescent reporter group, the third fluorescent reporter group and the fourth fluorescent reporter group is different from the other fluorescent reporter groups.

20. The kit according to claim 19, wherein each of the first fluorescent reporter group, the second fluorescent reporter group, the third fluorescent reporter group and the fourth fluorescent reporter group comprises one of VIC, CY5, ROX, and FAM, and each of the first quencher group, the second quencher group, the third quencher group and the fourth quencher group comprises one of BHQ1, BHQ2, BHQ3 and MGB.

Images