A tcccas13a-rt-lamp-based cxcl9 mrna detection kit and its application in cervical cancer screening

Abstract

The application discloses a TccCas13a-RT-LAMP-based CXCL9 mRNA detection kit and application thereof in cervical cancer screening. The kit comprises a reverse transcriptase, a T7 promoter-dependent specific LAMP primer group, a TccCas13a protein, a crRNA and an ssRNA-FQ reporter molecule, and can complete continuous reactions from target RNA to detection signals under single-tube closed tube conditions. The kit comprises a liquid dosage form and a freeze-dried bead dosage form, wherein the freeze-dried bead dosage form adopts a tube cover and tube bottom split cavity pre-packaging structure, and the reaction procedure is 37 DEG C pre-reaction for 15 min and 60 DEG C reaction for 45 min; the reaction condition of the liquid dosage form is 58 DEG C constant temperature reaction for 60 min. The detection limit of the kit is not higher than 1000 copies / µL, the total detection time is not longer than 60 min, the kit has the characteristics of simple operation, strong specificity and high sensitivity, and is suitable for rapid screening of cervical cancer.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology and relates to a nucleic acid detection technology based on the combination of CRISPR-Cas13a and loop-mediated isothermal amplification (LAMP). Specifically, it relates to a single-tube closed-tube nucleic acid detection system based on thermostable TccCas13a protein and RT-LAMP. This system integrates reverse transcription, isothermal amplification, and CRISPR-mediated signal detection in the same reaction tube, and is used to rapidly, sensitively, and specifically detect the expression level of CXCL9 mRNA in biological samples to assist in the early screening of cervical cancer. Background Technology

[0002] Cervical cancer is the fourth most common malignant tumor among women worldwide, and persistent human papillomavirus (HPV) infection is the main causative factor. Currently, commonly used cervical cancer screening methods mainly include HPV DNA testing, liquid-based thin-layer cytology testing (TCT), and HPV testing combined with TCT screening.

[0003] However, existing screening technologies have significant shortcomings in terms of detection methods and biomarker selection:

[0004] 1. Deficiencies in detection technology

[0005] (1) Existing molecular detection methods have complex operation procedures, usually requiring multiple steps such as nucleic acid extraction, reverse transcription, amplification and detection. They have problems such as high risk of sample contamination, long detection cycle and strict requirements on experimental environment and operators, making it difficult to achieve rapid on-site detection (POCT).

[0006] (2) Conventional PCR amplification technology relies on precision temperature control equipment (such as thermal cyclers), which places high demands on equipment configuration and power supply in primary healthcare institutions and remote areas, thus limiting the widespread application of screening technology;

[0007] (3) Although existing isothermal amplification techniques (such as LAMP) can be performed under isothermal conditions, they lack an effective signal amplification mechanism when used alone, have limited detection sensitivity, and are prone to false positive results caused by non-specific amplification, which cannot meet the accuracy requirements of early cervical cancer screening.

[0008] (4) Existing CRISPR detection technologies often require separate steps from conventional amplification methods, which are cumbersome. Furthermore, some Cas proteins have poor thermal stability and are difficult to be compatible with high-temperature isothermal amplification systems, thus limiting the realization of single-tube integrated detection.

[0009] 2. Deficiencies in the detection of biomarkers

[0010] (1) HPV DNA, as the most commonly used screening marker, can only reflect the viral infection status and cannot directly assess the carcinogenesis process of host cells. It is not specific enough to distinguish transient infection from precancerous lesions and cervical cancer, leading to overdiagnosis and treatment.

[0011] (2) Existing cell morphology-based markers (such as TCT) rely on subjective interpretation, lack objective molecular quantitative indicators, and have low sensitivity for detecting cervical adenocarcinoma and early small lesions.

[0012] (3) As a molecular marker reflecting changes in the host immune response and tumor microenvironment, the expression regulation mechanism of CXCL9 mRNA in the occurrence and development of cervical cancer and its application value as a screening marker have not been fully explored. At present, there is a lack of simple, rapid and accurate detection methods for CXCL9 mRNA.

[0013] Therefore, there is an urgent need to develop a single-tube integrated detection technology that combines novel isothermal amplification technology with a CRISPR signal amplification system, and to establish a new cervical cancer screening method using host-derived molecules such as CXCL9 mRNA as markers, so as to achieve cervical cancer screening that is simple to operate, low in cost, highly specific, and suitable for rapid on-site detection.

[0014] 3. Problems or deficiencies of existing technologies most similar to the invention

[0015] The differences between the prior art and the technical solution of this invention, as determined by a search, are as follows:

[0016] 3.1 Nucleic acid detection method based on the combination of RT-LAMP and CRISPR technologies

[0017] Existing technologies disclose various nucleic acid detection methods that combine loop-mediated isothermal amplification (LAMP) with the CRISPR-Cas system. For example, Chinese invention patent applications CN202511589480.4 and CN202411651826.4 disclose detection methods based on RT-LAMP combined with Cas12a or Cas12b, respectively. However, the above-mentioned prior art differs from the present invention in the following significant ways:

[0018] (1) Different types of core enzymes: The Cas12a or Cas12b proteins used in the above-mentioned prior art are DNA-targeting Cas proteins, while the present invention uses TccCas13a protein, which belongs to the RNA-targeting Cas13 family. The two are fundamentally different in terms of target recognition type, cleavage mechanism and protein structure.

[0019] (2) Insufficient temperature compatibility: In the above-mentioned existing technologies, the optimal reaction temperature of Cas12a / Cas12b does not match the RT-LAMP amplification temperature (60℃–65℃). Therefore, a step-by-step operation mode is adopted, that is, RT-LAMP amplification is performed first, and then the amplification product is transferred to a detection system containing Cas protein and reporter probe for signal detection. This step-by-step operation mode has the following drawbacks: the operation is cumbersome, which increases the risk of cross-contamination of samples; the detection cycle is long, which is not conducive to the implementation of point-of-care testing (POCT).

[0020] (3) First use of TccCas13a protein: TccCas13a protein is derived from the thermophilic bacterium Thermoclostridium caenicola. It maintains stable RNA cleavage activity within the temperature range of 37℃–70℃, and is compatible with the reaction temperature of RT-LAMP (55℃–65℃), providing a key technical basis for realizing single-tube integrated detection. There are no existing reports on the use of TccCas13a protein in conjunction with RT-LAMP technology.

[0021] 3.2 Research on tumor detection based on CXCL9

[0022] Existing research indicates that CXCL9, as a chemokine, is abnormally expressed in various tumor tissues and may participate in the regulation of the tumor immune microenvironment. However, current research on CXCL9 in cervical cancer mainly focuses on the following aspects:

[0023] (1) Most studies only use immunohistochemistry or Western blot to verify the expression difference of CXCL9 at the protein level. These methods require tissue samples, are invasive tests, and are not suitable for large-scale population screening.

[0024] (2) A few studies involved the detection of CXCL9 mRNA, but all of them used the conventional real-time quantitative PCR (qRT-PCR) method, which requires precise temperature control equipment and professional laboratory conditions. Moreover, the operation steps are numerous and the detection cycle is long, which makes it difficult to meet the needs of primary medical institutions and rapid on-site detection.

[0025] (3) There are no reports in the existing technology on using CXCL9 mRNA as a biomarker for cervical cancer screening and establishing a single-tube integrated rapid detection method by combining isothermal amplification and CRISPR technology.

[0026] In summary, existing technologies do not yet include a method for combining the thermostable TccCas13a protein with RT-LAMP technology to complete reverse transcription, isothermal amplification, and CRISPR detection in the same reaction tube, and for the non-invasive qualitative detection of CXCL9 mRNA in cervical samples to achieve cervical cancer screening. Therefore, there is an urgent need in this field to develop a new cervical cancer screening technology that is easy to operate, rapid, low-cost, and suitable for rapid on-site detection. Summary of the Invention

[0027] The purpose of this invention is to provide a CXCL9 mRNA detection kit based on TccCas13a-RT-LAMP and its application in cervical cancer screening.

[0028] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0029] Application of CXCL9 mRNA as a detection biomarker in the preparation of products for cervical cancer screening.

[0030] A CXCL9 mRNA detection kit, which is based on TccCas13a-RT-LAMP technology and integrates reverse transcription components, loop-mediated isothermal amplification components and TccCas13a CRISPR fluorescence detection components, and can complete the amplification and signal detection of CXCL9 mRNA under single-tube closed conditions;

[0031] The kit includes reaction buffer, Bst 3.0 DNA polymerase, WarmStart RTx reverse transcriptase, thermostable Hi-T7 RNA polymerase, LAMP primer set, MgSO4, TccCas13a protein, crRNA, and ssRNA-FQ reporter molecule.

[0032] The sequence of the LAMP primer set is as follows:

[0033] F3: 5'-GCTGTTCCTGCATCAGCA-3',

[0034] B3: 5'-CCTTGGGTGGTGCTGATGCA-3',

[0035] FIP: 5'-TAATACGACTCACTATAGGGAGTTTGTTTCAGGAGATTCTGCTGAATCTGGGTTT-3',

[0036] BIP: 5'-AATTTCTCCGCAGGAAAGGCTTTCAAGGATTGTAGGTGGAT-3',

[0037] LoopF:5'-TGGGTTCCTTGCACTCCAA-3',

[0038] LoopB: 5'-AATCAGTCCTTCACATCTG-3';

[0039] The sequence of the crRNA is 5'-UAGGUUAGUAUCCUCUAAGUACAGUUUGAACUCCAUUCUU-3';

[0040] The sequence of the ssRNA-FQ reporter molecule is 5'-FAM-rUrUrUrUrU-BHQ1-3';

[0041] The kit is in liquid or lyophilized bead form;

[0042] The reaction conditions for the liquid dosage form are a constant temperature reaction at 58°C for 60 minutes.

[0043] The lyophilized beads formulation adopts a pre-packaging structure with separate cavities at the cap and bottom. The reaction conditions are: first, a pre-reaction at 37°C for 15 minutes, then a reaction at 60°C for 45 minutes.

[0044] The lyophilized beads are pre-packaged in a single reaction tube. The pre-packaged components in the tube cap include reaction buffer, Bst 3.0 DNA polymerase, WarmStart RTx reverse transcriptase, thermostable Hi-T7 RNA polymerase, and LAMP primer set. The pre-packaged components in the tube bottom include MgSO4, TccCas13a protein, crRNA, and ssRNA-FQ reporter molecule.

[0045] In the reaction system of the kit, the final concentration of Bst 3.0 DNA polymerase is 0.25~0.5 U / µL, the final concentration of WarmStart RTx reverse transcriptase is 0.25~0.5 U / µL, the final concentration of thermostable Hi-T7 RNA polymerase is 2~5 U / µL, the final concentration of FIP and BIP is 2.0µM, the final concentration of F3 and B3 is 0.2µM, the final concentration of LoopF ​​and LoopB is 0.8µM, the final concentration of the additionally added MgSO4 is 5mM, the final concentration of TccCas13a protein is 0.3µM, the final concentration of crRNA is 0.9µM, and the final concentration of ssRNA-FQ reporter molecule is 0.5µM.

[0046] The above-mentioned CXCL9 mRNA detection kit is used in the preparation of products for cervical cancer screening.

[0047] The significant advantages of this invention are:

[0048] (1) This invention integrates the heat-resistant TccCas13a protein with RT-LAMP technology in the same reaction tube. The entire process does not require opening the cap. Reverse transcription, isothermal amplification and CRISPR signal detection are completed under single-tube closed conditions, which effectively avoids false positive problems caused by aerosol contamination. The operation is simple and suitable for rapid on-site detection.

[0049] (2) The total detection time of this invention does not exceed 60 minutes and the detection limit is not higher than 1000 copies / µL, which meets the dual requirements of speed and sensitivity for rapid clinical screening.

[0050] (3) The kit of the present invention includes two forms: liquid dosage form and lyophilized beads dosage form. The lyophilized beads dosage form adopts a pre-dispensing structure with separate cavities at the cap and bottom of the tube, eliminating the need for manual preparation of the reaction system. The components can be mixed by centrifugation or gentle tapping, further simplifying the operation and facilitating storage and transportation.

[0051] (4) This invention is the first to use host-derived CXCL9 mRNA as a molecular marker for cervical cancer screening. Compared with traditional HPV DNA detection, it can better reflect the host immune response status and changes in the tumor microenvironment, and has higher clinical relevance.

[0052] (5) Clinical sample validation results show that the overall concordance rate between the kit of the present invention and the qRT-PCR method is 95.0%, the detection sensitivity for cervical cancer samples is 95.0%, and the specificity is 95.0%, which can effectively meet the clinical needs of cervical cancer screening. Attached Figure Description

[0053] Figure 1 Venn diagram of differentially expressed genes upregulated and downregulated in the GSE7410, GSE63514, and GSE67522 cervical cancer datasets.

[0054] Figure 2 Survival analysis and ROC diagnostic curve of CXCL9 mRNA in cervical cancer.

[0055] Figure 3 This invention is based on a schematic diagram illustrating the reaction principle of detecting CXCL9 mRNA using TccCas13a-RT-LAMP technology.

[0056] Figure 4 : A schematic diagram of the detection operation process of the TccCas13a-RT-LAMP kit in lyophilized bead formulation of this invention.

[0057] Figure 5 The amplification curves of the liquid formulation TccCas13a-RT-LAMP kit of this invention for positive plasmids and template-free controls.

[0058] Figure 6The amplification curves of the TccCas13a-RT-LAMP kit in lyophilized beads of this invention for positive plasmids and template-free controls are shown. Detailed Implementation

[0059] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0060] Example 1: Screening and validation of CXCL9 as a novel biomarker for cervical cancer

[0061] CXCL9 (CXC motif chemokine ligand 9, also known as MIG) is an important chemokine. In this embodiment, bioinformatics methods were used to screen and verify its feasibility as a novel biomarker for cervical cancer. The specific process is as follows.

[0062] 1.1 Screening for the CXCL9 gene from the GEO public database

[0063] 1.1.1 Description of Specific Technical Approach

[0064] Data source: Three independent cervical cancer gene expression profile datasets, GSE7410, GSE63514, and GSE67522, were selected from the GEO public database as the basic data for screening differentially expressed genes, ensuring the representativeness and independence of the data.

[0065] Screening criteria: Differential expression analysis was performed on approximately 60,000 genes in the dataset. The screening threshold was strictly set as expression change fold |FC|>1.5 and statistical significance P<0.05 to exclude meaningless gene expression differences.

[0066] Cross-validation: such as Figure 1 As shown, Venn diagram analysis was used to cross-compare the differentially expressed genes upregulated in three independent datasets. From hundreds of differentially expressed genes, three conserved genes, CXCL9, GBP5, and MMP9, which were consistently upregulated in all three datasets, were selected to ensure the stability of the association between the selected genes and cervical cancer.

[0067] CXCL9 was the preferred choice among the three genes that were upregulated. CXCL9 has a clear immune regulatory function and can recruit immune effector cells such as CD8+ T cells and NK cells through the CXCR3 receptor. It is also closely related to the immune microenvironment of cervical cancer. After comprehensive consideration, it was selected as a novel biomarker for cervical cancer, laying the foundation for the development of subsequent detection kits.

[0068] 1.1.2 CXCL9 is a novel biomarker for potential cervical cancer screening.

[0069] To verify the effectiveness of CXCL9 as a biomarker for cervical cancer screening, bioinformatics analysis was performed on CXCL9 mRNA expression data from cervical cancer samples in a public database. Survival curve and ROC curve analyses were used to clarify its diagnostic and prognostic value. The results are as follows:

[0070] Correlation analysis of CXCL9 expression level with prognosis of cervical cancer patients ( Figure 2 A): Using Kaplan-Meier survival curve analysis, the results showed that the overall survival rate of patients in the CXCL9 high expression group was significantly higher than that in the low expression group (HR=0.66, 95% CI: 0.48-0.92, P=0.013). This result indicates that CXCL9 expression level is significantly positively correlated with the prognosis of cervical cancer patients, and high CXCL9 expression suggests better survival outcomes, making it a potential molecular indicator for prognostic stratification of cervical cancer.

[0071] Evaluation of the diagnostic and differential diagnostic ability of high CXCL9 expression levels in cervical cancer ( Figure 2 B): Receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) for CXCL9 expression levels in distinguishing between cervical cancer and non-cancerous states was 0.787 (95% CI: 0.753–0.820). This AUC value is greater than 0.75 and significantly higher than the random level (AUC=0.5), indicating that CXCL9 has a moderately high diagnostic ability for cervical cancer, and its expression level can effectively distinguish cervical cancer patients from non-cancerous individuals.

[0072] In summary, CXCL9 mRNA, as a host-derived molecular marker, possesses both diagnostic discrimination ability (AUC=0.787) and prognostic predictive value (HR<1, P<0.05). Its expression level is closely related to the occurrence and development of cervical cancer, making it a highly promising candidate marker for cervical cancer screening. Currently, detection methods for CXCL9 mRNA still suffer from problems such as cumbersome operation, slow detection speed, and insufficient accuracy. Therefore, developing a simple, rapid, and accurate single-tube integrated CXCL9 mRNA detection system has significant clinical application value for achieving early cervical cancer screening.

[0073] Example 2: CXCL9 mRNA detection kit based on TccCas13a-RT-LAMP (liquid form)

[0074] Based on the validation results of Example 1, in order to achieve rapid and accurate detection of CXCL9 mRNA, a CXCL9 mRNA detection kit (liquid form) based on TccCas13a-RT-LAMP technology was developed.

[0075] 2.1 Reagent Kit Working Principle

[0076] This kit integrates three steps: reverse transcription, isothermal amplification, and CRISPR detection. Its working principle consists of two stages: isothermal amplification and CRISPR detection.

[0077] (1) Isothermal amplification stage: First, WarmStart RTx reverse transcriptase uses RNA in the sample as a template to synthesize cDNA under the guidance of specific primers; then, Bst 3.0 DNA polymerase performs loop-mediated isothermal amplification (LAMP) under isothermal conditions to produce a large amount of double-stranded DNA product containing the T7 promoter sequence; finally, thermostable Hi-T7 RNA polymerase recognizes the T7 promoter sequence on the double-stranded DNA and transcribes it into a large amount of target RNA, providing sufficient RNA substrate for the subsequent CRISPR detection stage and ensuring the sensitivity of the detection.

[0078] (2) CRISPR detection stage: TccCas13a protein is pre-mixed with crRNA to form a ribonucleoprotein (RNP) complex. When the target RNA generated during the isothermal amplification stage is present, crRNA specifically recognizes and binds to the target RNA through base complementarity, thereby activating the trans-cleavage of TccCas13a. The activated TccCas13a non-specifically cleaves the free ssRNA-FQ reporter molecule in the system, causing the 5' fluorescent group (FAM) of the reporter molecule to separate from the 3' quencher group (BHQ1), releasing a detectable fluorescent signal. The intensity of the fluorescent signal is positively correlated with the initial CXCL9 mRNA content in the sample. Qualitative or semi-quantitative detection of the target RNA can be achieved through real-time fluorescence monitoring.

[0079] 2.2 Reagent Kit Composition

[0080] The specific components of this kit are as follows:

[0081] (1) Reaction buffer system: 10× custom buffer solution, pH 8.8, with the following components and concentrations: Tris-HCl 200mM, (NH4)2SO4 100mM, BSA 20mg / mL, RNase inhibitor 10U / µL, KCl 500mM, MgSO4 20mM, Tween®-20 1%, NTP Mix (ATP, UTP, GTP, CTP 0.8mM each), dNTP Mix (dATP, dTTP, dGTP, dCTP 0.8mM each). Note: The buffer solution contains MgSO4 as the background magnesium ions for the reaction. The 5mM MgSO4 mentioned in all technical parameters of this invention refers only to the exogenous MgSO4 added to the system and does not include the magnesium ions contained in the buffer solution.

[0082] (2) Reverse transcription and amplification enzyme system: including Bst 3.0 DNA polymerase, WarmStart RTx reverse transcriptase and thermostable Hi-T7 RNA polymerase. Since T7 transcription, LAMP amplification and CRISPR detection need to be performed simultaneously, the activities of each enzyme need to be balanced. The final concentration of each enzyme was determined by experimental optimization: the final concentration of Bst 3.0 DNA polymerase is 0.25~0.5U / µL, preferably 0.3U / µL; the final concentration of WarmStart RTx reverse transcriptase is 0.25~0.5U / µL, preferably 0.3U / µL; the final concentration of thermostable Hi-T7 RNA polymerase is 2~5U / µL, preferably 4U / µL; the ratio of the activity units of WarmStart RTx to Hi-T7 RNA polymerase is 1:10~1:20, preferably 1:13.3.

[0083] (3) LAMP primer set: Specific LAMP primers were designed targeting the conserved region of CXCL9 mRNA (NM_002416.3). The 5' end of the FIP primer carries the T7 promoter sequence (5'-TAATACGACTCACTATAGGG-3') to ensure that the LAMP amplification product can be recognized by Hi-T7 RNA polymerase and transcribed into RNA. The specific primer sequences are shown in the table below:

[0084]

[0085] (4) LAMP primer MIX preparation: i.e., 10× primer mixture. When using the reaction system, the final concentration of FIP and BIP is 2µM, the final concentration of F3 and B3 is 0.2µM, and the final concentration of LoopF ​​and LoopB is 0.8µM.

[0086] (5) crRNA primer: Its spacer sequence targets the CXCL9-specific region in the T7 transcript, and the specific sequence is 5'-UAGGUUAGUAUCCUCUAAGUACAGUUUGAACUCCAUUCUU-3'. To ensure efficient formation of the RNP complex, the molar ratio of crRNA to TccCas13a is set at 3:1 (0.9µM:0.3µM).

[0087] (6) Signal reporter molecule: An ssRNA-FQ reporter molecule with a 5' end labeled with a FAM fluorescent group and a 3' end labeled with a BHQ1 quenching group is used for the detection of fluorescence signal. The specific sequence is 5'-FAM-rUrUrUrUrU-BHQ1-3'; where rU represents uracil ribonucleoside residues.

[0088] (7) Positive control: In vitro transcribed RNA or recombinant plasmid containing the CXCL9 mRNA sequence, used to verify the effectiveness of the reaction system.

[0089] (8) Negative control: RNase-free water, used to exclude interference from non-specific reactions.

[0090] 2.3 Reaction Conditions

[0091] The detection method, reaction temperature, and time of this kit have been optimized to ensure detection efficiency and accuracy, as detailed below:

[0092] Detection method: Real-time fluorescence monitoring is used, which can be achieved using a quantitative PCR instrument or a portable fluorescence detector, making it convenient for use in different scenarios;

[0093] Reaction conditions: React at a constant temperature of 58℃ for 60 minutes to complete the detection.

[0094] 2.4 Composition of the reaction system and experimental procedures

[0095] The total volume of the reagent kit reaction system is 25µL, as detailed below:

[0096]

[0097] Note: In the 25µL total reaction system, 2.5µL of 10× custom buffer solution was added to bring the final concentration of MgSO4 to 2mM; an additional 1µL of 125mM MgSO4 stock solution was added, bringing the final concentration of this exogenous component to 5mM. Throughout this invention, "final concentration of 5mM MgSO4" refers to the exogenously added portion; magnesium ions present in the buffer solution are background ions and are not included in this statistical value.

[0098] Experimental steps:

[0099] (1) Preparation of reaction system: Add each component in sequence according to the table above on ice, ensuring that each component reaches the specified final concentration. After addition, perform instantaneous centrifugation (2,000×g, 5s).

[0100] (2) Set reaction conditions: Place the reaction tube in a real-time fluorescence PCR instrument or a portable fluorescence detector, set the reaction temperature to 58℃, the reaction time to 60 min, collect the fluorescence signal every 1 min, and monitor the reaction process in real time.

[0101] (3) Result interpretation: The positive criterion is when the fluorescence signal value exceeds the threshold (Threshold=Mean_NTC+3×SD_NTC, where Mean_NTC is the average fluorescence signal value of the template-free control and SD_NTC is the standard deviation of the fluorescence signal of the template-free control) and an “S”-shaped amplification curve appears; if the value does not exceed the threshold or there is no “S”-shaped amplification curve, it is judged as negative.

[0102] Example 3: CXCL9 mRNA detection kit based on TccCas13a-RT-LAMP (lyophilized bead formulation)

[0103] To address the technical shortcomings of liquid reagent kits, such as demanding storage conditions, high low-temperature transportation costs, and poor stability due to the susceptibility of multi-enzyme activity to environmental temperature fluctuations, this embodiment optimizes the formulation process based on liquid reagent kits and develops a CXCL9 mRNA detection kit with pre-packaged lyophilized beads in dual-bead form.

[0104] 3.1 Overall design and protective agent system of freeze-dried beads

[0105] This kit uses a pre-packaged structure of lyophilized beads for the RT-LAMP system in the tube cap and lyophilized beads for the Cas13a detection system in the tube bottom. A dedicated 5× lyophilization protectant stock solution was added during the preparation of all lyophilized beads.

[0106] 3.1.1 Formulation of Lyophilization Protectant Mother Liquor

[0107] The formula of the 5× freeze-drying protectant stock solution (w / v) used in this invention is as follows: trehalose 0.5 g / mL, mannitol 0.25 g / mL, dextran-40 0.15 g / mL, PEG-20000 0.375 g / mL, and bovine serum albumin 0.005 g / mL.

[0108] 3.1.2 Components and final concentration of lyophilized beads

[0109] All reaction components from Example 2, excluding sample RNA and enzyme-free water, were divided into a cap pre-dispensing system and a bottom pre-dispensing system. These were then mixed with a lyophilization protectant to prepare lyophilized beads. The final concentrations of each component in the final reaction system were identical to those of the liquid system in Example 2. The specific components are as follows:

[0110] (1) Pre-filled lyophilized beads with tube caps (RT-LAMP amplification system)

[0111] Components and final concentrations: 1× custom buffer (pH 8.8), Bst 3.0 DNA polymerase 0.3 U / µL, WarmStart RTx reverse transcriptase 0.3 U / µL, thermostable Hi-T7 RNA polymerase 4 U / µL, LAMP primer MIX (FIP / BIP 2µM, F3 / B3 0.2µM, LoopF / B 0.8µM).

[0112] (2) Pre-packing of freeze-dried beads at the bottom of the tube (CRISPR detection system)

[0113] Components and final concentrations: MgSO4 5mM, TccCas13a protein 0.3µM, crRNA 0.9µM, ssRNA-FQ reporter molecule 0.5µM.

[0114] 3.2 Preparation process of freeze-dried beads

[0115] This embodiment employs a preparation process involving low-temperature premixing on ice, rapid freezing in liquid nitrogen, and segmented gradient freeze-drying. The specific steps are as follows:

[0116] 3.2.1 Preparation of premixed solution

[0117] Premixed solutions were prepared separately for the RT-LAMP system at the tube cap and the CRISPR detection system at the tube bottom. All operations were performed on ice. Both premixed solutions were mixed at a volume ratio of 4:1 (reaction component: 5 × lyophilization protectant stock solution), i.e., 2 µL of 5 × lyophilization protectant stock solution was added to every 8 µL of reaction component system, and the mixture was thoroughly and gently blown to mix.

[0118] 3.2.2 Droplet quick-freezing molding

[0119] The two premixed liquids were dispensed using a precision dropper, with each liquid being dispensed at a standard rate of 10µL / particle. Liquid nitrogen was placed in an insulated container beforehand, and the premixed liquid droplets were added drop by drop into the liquid nitrogen. The principle of ultra-low temperature instantaneous freezing was used to rapidly solidify the droplets into freeze-dried microspheres of uniform size and dense structure.

[0120] 3.2.3 Microsphere Collection Pretreatment

[0121] After the liquid nitrogen has completely evaporated, the shaped freeze-dried microspheres are collected using a sterile sieve and quickly transferred to a pre-cooled freeze dryer tray.

[0122] 3.3 Segmented gradient freeze-drying program

[0123] The freeze-drying process employs a four-stage gradient process: pre-freezing → annealing → sublimation drying → desorption drying. Specific parameters are shown in the table below.

[0124]

[0125] 3.4 Finished Product Packaging Process

[0126] After the lyophilization process is completed, quickly complete the aseptic packaging operation: take a standard PCR tube, pre-fill each tube with one Cas13a system lyophilization bead at the bottom and one RT-LAMP system lyophilization bead at the top, and immediately seal the tube after filling; put the packaged reaction tube into an aluminum foil packaging bag with built-in desiccant, vacuum seal it, and store it in a cool, dry place away from light.

[0127] 3.5 Reagent Kit Detection Procedure

[0128] This lyophilized bead kit exhibits low RNA cleavage activity of TccCas13a at 37°C, but strong binding affinity of crRNA to target RNA. The system employs a stepwise dosing design: specific recognition and binding are first achieved at low temperature in a system without RT-LAMP amplification reagents; after pre-binding, RT-LAMP reaction components are added, and the temperature is raised to 60°C to simultaneously initiate nucleic acid amplification and Cas13a cleavage-mediated fluorescence signal amplification. The specific experimental steps are as follows:

[0129] (1) Sample addition: Take out the vacuum-sealed pre-filled reaction tube, add 2µL of the RNA sample to be tested to the bottom of the tube, and add an equal amount of RNase-free water to the negative control tube, so that the sample liquid is fully wetted and in contact with the Cas13a system lyophilized beads at the bottom of the tube.

[0130] (2) Low temperature pre-reaction: Place the reaction tube in a constant temperature metal bath and incubate at 37°C for 15 min to allow the TccCas13a / crRNA ribonucleoprotein complex to fully recognize and bind to the CXCL9 target RNA sequence in the sample, thus completing the specific target recognition pre-binding.

[0131] (3) Construction of the main reaction system: After the pre-reaction is completed, the reaction tube is centrifuged briefly (2000×g, 10s) or inverted and gently tapped to make the RT-LAMP system lyophilized beads at the tube cap fall completely into the liquid at the bottom of the tube and mix thoroughly; add RNase-free water to make up to a total system volume of 25µL to form a complete detection reaction system.

[0132] (4) Isothermal fluorescence detection: Place the reaction tube in a real-time fluorescence PCR instrument or a portable fluorescence detector, set the reaction temperature to 60℃ and the reaction time to 45min, and automatically collect the fluorescence signal every 1min to monitor the reaction amplification process in real time.

[0133] (5) Result interpretation: A positive result is determined when the fluorescence signal value exceeds the threshold (Threshold=Mean_NTC+3×SD_NTC) and a typical “S”-shaped amplification curve appears; a negative result is determined when the fluorescence signal does not reach the threshold and there is no “S”-shaped amplification curve.

[0134] Example 4: Construction of positive control plasmid and validation of kit performance

[0135] To facilitate standardized evaluation of the amplification and detection performance of the kit and eliminate interference from the instability of the RNA template itself and fluctuations in reverse transcription efficiency, this embodiment first uses the recombinant plasmid pUC57-CXCL9 containing the CXCL9 target sequence as a standard template to verify the performance of the kit's LAMP amplification and CRISPR detection modules.

[0136] 4.1 Construction of positive control plasmid

[0137] The positive control plasmid was constructed using conventional molecular cloning methods, with the specific steps as follows:

[0138] (1) Target gene synthesis: Based on the 1-1260nt sequence of CXCL9 mRNA (NM_002416.3), a double-stranded DNA fragment was synthesized by Sangon Biotech (Shanghai) Co., Ltd. To facilitate subsequent vector ligation, EcoRI and HindIII restriction sites were introduced at both ends of the fragment.

[0139] (2) Vector construction: The synthesized double-stranded DNA fragment was digested with EcoRI / HindIII and then ligated with the pUC57 vector, which had also been digested with the same enzymes. The ligation product was transformed into DH5α competent cells and plated on antibiotic-containing medium for culture. Positive clones were picked, and plasmids were extracted and sequenced to verify that the inserted fragment sequence was correct. The recombinant plasmid with correct sequencing was named pUC57-CXCL9 and used as a positive control template for subsequent experiments.

[0140] (3) Plasmid quantification: The concentration of pUC57-CXCL9 plasmid was determined using a NanoDrop nucleic acid concentration analyzer. The concentration was converted to copy number based on the molecular weight of the plasmid and then aliquoted and stored at -80℃.

[0141] 4.2 Reagent kit performance validation

[0142] This experiment verified the efficacy of the liquid dosage form in Example 2 and the lyophilized bead dosage form in Example 3, respectively. The specific experimental design and results are as follows:

[0143] Experimental design: Reaction systems were prepared according to those in Example 2 (liquid dosage form) and Example 3 (lyophilized beads dosage form), respectively, with 2 µL of 10... 6 pUC57-CXCL9 plasmid standard (copies / µL) was used as a template, with four replicate wells for each concentration. RNase-free water was also included as a template-free control (NTC).

[0144] Reaction conditions: Both liquid and lyophilized bead formulations were reacted according to their respective specified conditions. The liquid formulation was reacted at a constant temperature of 58℃ for 60 min, while the lyophilized bead formulation was reacted at 37℃ for 15 min followed by a reaction at 60℃ for 45 min. The total reaction time for both was 60 min, and fluorescence signals were collected every 1 min.

[0145] Result Interpretation: A positive result was determined by a fluorescence signal value exceeding the threshold (Threshold = Mean_NTC + 3 × SD_NTC) and the appearance of an "S"-shaped amplification curve. The number of positive detections and the detection rate at each time point were statistically analyzed, and the results are shown in the table below:

[0146]

[0147] Option 1: Mean_NTC=950RFU, SD_NTC=77 RFU, Threshold=949+3×81=1192RFU

[0148] Option 2: Mean_NTC = 1287 RFU, SD_NTC = 93 RFU, Threshold = 1287 + 3 × 91 = 1560 RFU

[0149] As shown in the table, both the liquid and lyophilized bead kits of this invention achieved a 100% positive detection rate for CXCL9 at 50 minutes of reaction. Specifically, the lyophilized bead kit demonstrated a 50% positive detection rate in the early detection phase (45 minutes), significantly higher than the 25% of the liquid kit. This indicates that the lyophilized bead kit has a faster signal accumulation rate under the same conditions, thus enabling earlier detection of positive samples even with a higher threshold. Furthermore, both kits showed zero NTC positive detections at all time points, with a detection rate of 0% and a specificity of 100%, indicating no significant non-specific reactions and stable, reliable detection results. The detection efficacy established based on the plasmid template in this embodiment lays the methodological foundation for the real RNA detection in subsequent Examples 6 (cellular RNA) and 7 (clinical samples).

[0150] Example 5: Determination of Limit of Detection (LOD)

[0151] This embodiment uses a lyophilized bead-based kit with recombinant plasmid DNA as a template to determine the theoretical minimum detection level of the kit's LAMP amplification and CRISPR signal detection modules, characterizing the basic sensitivity of the amplification system itself. The actual clinical detection sensitivity of the kit for CXCL9 mRNA (including the reverse transcription step) is detailed in the verification results of cell RNA samples in Example 6 and clinical cervical exfoliated cell samples in Example 7. The specific experimental process and results are as follows.

[0152] (1) Preparation of standard: pUC57-CXCL9 plasmid was serially diluted 10-fold with RNase-free water to prepare a concentration of 10. 7 10 6 10 5 10 4 10 3 10 2 10 1 10 0 A series of standard products in copies / µL.

[0153] (2) Reaction system: The reaction system was prepared according to the lyophilized bead kit of Example 3. 2µL of each concentration of standard was added as a template. Eight replicate wells were set for each concentration. At the same time, a template-free control (NTC) was set.

[0154] (3) Reaction conditions: The reaction was carried out according to the reaction conditions specified for the lyophilized beads, that is, the reaction was carried out at 37℃ for 15 min and then at 60℃ for 45 min, with a total reaction time of 60 min. Fluorescence signals were collected every 1 min.

[0155] (4) Result determination: The positive determination criterion was that the fluorescence signal value exceeded the threshold (Threshold=Mean_NTC+3×SD_NTC) and an "S"-shaped amplification curve appeared. The number of positive detections and the detection rate of each concentration of standard were counted. The results are shown in the table below:

[0156]

[0157] As can be seen from the table, the detection limit (LOD) of the kit of the present invention for CXCL9 is... 95 The concentration corresponding to a 95% detection rate is ≤1000 copies / µL, indicating that the kit has high detection sensitivity and can meet the needs of low-concentration detection in clinical samples.

[0158] Example 6: Detection of cervical cancer cell samples

[0159] To verify the kit's ability to detect actual cell samples, this embodiment uses cervical cancer cells (HeLa) as the detection target and employs a lyophilized bead-type kit for detection to evaluate the kit's applicability in cell samples. The specific experimental process and results are as follows.

[0160] Experimental steps:

[0161] (1) RNA extraction from HeLa cells: Total RNA was extracted from cultured cervical cancer cells HeLa using a commercial total RNA extraction kit (TRIzol method). The RNA concentration was determined by NanoDrop and then adjusted to 50 ng / µL.

[0162] (2) Preparation of reaction system: Prepare the reaction system according to the lyophilized bead kit of Example 3, and add 2µL of HeLa cell total RNA (equivalent to 100ng total RNA) after adjusting the concentration as a template.

[0163] (3) Control settings: Both positive and negative controls were set up. The positive control was 2µL 10 6 The pUC57-CXCL9 plasmid was prepared in copies / µL, with 2µL of RNase-free water as the negative control, to verify the effectiveness and specificity of the reaction system.

[0164] (4) Reaction conditions: The reaction was carried out according to the reaction conditions specified for the lyophilized beads dosage form. The reaction was carried out at 37℃ for 15 min and then at 60℃ for 45 min. The total reaction time was 60 min. Fluorescence signal was collected every 1 min.

[0165] (5) Result Interpretation: According to the threshold standard (Threshold=Mean_NTC+3×SD_NTC) determined in Example 2, samples with fluorescence signals exceeding the threshold and exhibiting an "S"-shaped amplification curve within the valid time were judged as positive; samples that did not exceed the threshold or did not exhibit an "S"-shaped amplification curve were judged as negative. The experimental results are shown in the table below:

[0166]

[0167] As can be seen from the table, the lyophilized bead-based kit of this invention, using 100 ng of total RNA from HeLa cells as a template, can effectively detect target RNA with a positive rate of 100%. The results of both the positive and negative controls met expectations, with no false positives or false negatives, further verifying the reliability and stability of the kit and indicating that this kit is suitable for the detection of CXCL9 mRNA in cervical cancer cell samples.

[0168] Example 7: Clinical sample validation and comparative evaluation with the qRT-PCR gold standard method

[0169] To verify the clinical accuracy, specificity, and practical application value of the CXCL9 mRNA detection kit in lyophilized bead form of this invention, this embodiment conducts blinded detection verification based on clinical cervical exfoliated cell samples. Simultaneously, it uses the clinically common qRT-PCR method as the gold standard for comparative analysis to clarify the clinical diagnostic efficacy of the kit and provide data support for its clinical application in cervical cancer auxiliary screening.

[0170] 7.1 Establishment of Clinical Diagnostic Thresholds for the Reagent Kit

[0171] To adapt to clinical sample testing and effectively distinguish between healthy individuals and cervical cancer patients, this invention establishes a clinical diagnostic threshold based on the background expression level of CXCL9 mRNA in cervical exfoliated cell samples from normal healthy individuals. The specific method for establishing this threshold is as follows:

[0172] (1) Normal sample inclusion: 50 cervical exfoliated cell samples were collected from healthy women aged 25-65. All samples were clinically examined, tested for HPV and verified by cytology to confirm that there were no cervical lesions, gynecological malignancies and related inflammatory interference, and to ensure that the samples were negative control samples with normal background.

[0173] (2) Sample pretreatment: Total RNA was extracted from all samples using a commercial total RNA extraction kit. After quantification by UV spectrophotometer, the RNA concentration of all samples was adjusted to 50 ng / μL.

[0174] (3) Detection method: The lyophilized bead-type reagent kit of the present invention was used for detection. The standard reaction conditions were: pre-incubation at 37℃ for 15 min, instantaneous centrifugation, and constant temperature reaction at 60℃ for 45 min. Fluorescence signal was collected every 1 min throughout the process, and the peak value of fluorescence signal of each sample was recorded.

[0175] (4) Threshold statistics and setting: Statistical analysis of the fluorescence peak values ​​of 50 normal samples showed that the average peak value of the sample fluorescence signal was Mean=2685RFU, the standard deviation was SD=245RFU, and the overall fluctuation range was 2280~3150RFU. Based on the principle of controlling the false positive rate to about 2.5%, the clinical diagnostic threshold of this kit was set as: Mean+2SD=3175RFU.

[0176] (5) Clinical outcome interpretation criteria:

[0177] Positive: The peak fluorescence signal of the sample is >3175 RFU, and a typical "S"-shaped amplification curve is observed, indicating abnormally high expression of CXCL9 mRNA. Further colposcopy and histopathological examination are recommended for diagnosis.

[0178] Negative: The peak fluorescence signal of the sample is ≤3175RFU, and there is no typical "S"-shaped amplification curve, indicating that CXCL9 mRNA expression is normal. Routine follow-up is recommended, and combined screening with HPV DNA testing can be carried out.

[0179] 7.2 Clinical Sample Validation Experiment Design

[0180] A case-control study was conducted, collecting 100 clinical cervical exfoliated cell samples. All samples were clinically submitted and had complete clinical records and pathological diagnostic evidence. The subjects were divided into two groups: a cervical cancer case group (n=50, all samples were pathologically confirmed as cervical cancer) and a normal control group (n=50, all healthy subjects with normal clinical examination, cytology, and HPV testing, and no cervical lesions or related medical history).

[0181] All clinical samples underwent standardized processing: total RNA was extracted using the same batch of commercially available RNA extraction kits, and the concentration was uniformly calibrated after quantification. Each sample was divided into two parallel samples, one for testing using the lyophilized bead kit of this invention and the other for testing using the clinical reference method (qRT-PCR). The two tests were performed independently and interpreted blindly.

[0182] 7.3 Detection Results and Comparative Analysis

[0183] The results were strictly interpreted according to the established clinical thresholds of this invention and the standard interpretation procedures of commercial qRT-PCR kits (covering 21 HPV subtypes). The positive detection rates of the two groups of samples were statistically analyzed, and the diagnostic efficacy of the two detection methods was compared. The specific results are shown in the table below:

[0184]

[0185] Experimental results show that the lyophilized bead-form kit of this invention has a diagnostic sensitivity of 98.0% for clinical cervical cancer samples, which is basically the same as the detection performance of the clinical reference method (the commercially available qRT-PCR kit). It can accurately detect the vast majority of cervical cancer positive samples confirmed by histopathology. Only one false positive was detected in the normal control group, and the diagnostic specificity was 98.0%.

[0186] Statistical analysis showed that the overall detection concordance rate between the kit of this invention and the qRT-PCR method reached 98.0% (98 / 100), demonstrating excellent consistency. Compared to the traditional qRT-PCR method, which is cumbersome to operate, time-consuming, and relies on sophisticated instruments, the kit of this invention is simple to operate, rapid to detect, and requires no complex sample processing. It also balances high sensitivity and high specificity, meeting the rapid screening needs of large-scale cervical samples in clinical settings. It has extremely high application value and promising prospects in the early auxiliary diagnosis and clinical triage and follow-up of cervical cancer.

[0187] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. Application of CXCL9 mRNA as a detection biomarker in the preparation of products for cervical cancer screening.

2. A CXCL9 mRNA detection kit, characterized in that: The kit is based on TccCas13a-RT-LAMP technology and integrates reverse transcription components, loop-mediated isothermal amplification components, and TccCas13a CRISPR fluorescence detection components, enabling the amplification and signal detection of CXCL9 mRNA under single-tube closed conditions.

3. The reagent kit according to claim 2, characterized in that: The kit includes reaction buffer, Bst 3.0 DNA polymerase, WarmStart RTx reverse transcriptase, thermostable Hi-T7 RNA polymerase, LAMP primer set, MgSO4, TccCas13a protein, crRNA, and ssRNA-FQ reporter molecule.

4. The reagent kit according to claim 3, characterized in that: The sequence of the LAMP primer set is as follows: F3: 5'-GCTGTTCCTGCATCAGCA-3', B3: 5'-CCTTGGGTGGTGCTGATGCA-3', FIP: 5'-TAATACGACTCACTATAGGGAGTTTGTTTCAGGAGATTCTGCTGAATCTGGGTTT-3', BIP: 5'-AATTTCTCGCAGGAAAGGCTTTCAAGGATTGTAGGTGGAT-3', LoopF:5'-TGGGTTCCTTGCACTCCAA-3', LoopB: 5'-AATCAGTCCTTCACATCTG-3'.

5. The reagent kit according to claim 3, characterized in that: The sequence of the crRNA is 5'-UAGGUUAGUAUCCUCUAAGUACAGUUUGAACUCCAUUCUU-3'.

6. The reagent kit according to claim 3, characterized in that: The sequence of the ssRNA-FQ reporter molecule is 5'-FAM-rUrUrUrUrU-BHQ1-3'.

7. The reagent kit according to claim 3, characterized in that: The kit is in liquid or lyophilized bead form; The reaction conditions for the liquid dosage form are a constant temperature reaction at 58°C for 60 minutes. The lyophilized beads formulation adopts a pre-packaging structure with separate cavities at the cap and bottom. The reaction conditions are: first, a pre-reaction at 37°C for 15 minutes, then a reaction at 60°C for 45 minutes.

8. The reagent kit according to claim 7, characterized in that: The lyophilized beads are pre-packaged in a single reaction tube. The pre-packaged components in the tube cap include reaction buffer, Bst 3.0 DNA polymerase, WarmStart RTx reverse transcriptase, thermostable Hi-T7 RNA polymerase, and LAMP primer set. The pre-packaged components in the tube bottom include MgSO4, TccCas13a protein, crRNA, and ssRNA-FQ reporter molecule.

9. The reagent kit according to claim 7, characterized in that: In the reaction system of the kit, the final concentration of Bst 3.0 DNA polymerase is 0.25~0.5 U / µL, the final concentration of WarmStart RTx reverse transcriptase is 0.25~0.5 U / µL, the final concentration of thermostable Hi-T7 RNA polymerase is 2~5 U / µL, the final concentration of FIP and BIP is 2.0µM, the final concentration of F3 and B3 is 0.2µM, the final concentration of LoopF ​​and LoopB is 0.8µM, the final concentration of the additionally added MgSO4 is 5mM, the final concentration of TccCas13a protein is 0.3µM, the final concentration of crRNA is 0.9µM, and the final concentration of ssRNA-FQ reporter molecule is 0.5µM.

10. The use of the CXCL9 mRNA detection kit according to any one of claims 2 to 9 in the preparation of products for cervical cancer screening.

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