A fluorescence detection method for the prostate cancer marker PCA3 based on gold nanorods and CRISPR Cas12a amplification

Abstract

This invention belongs to the field of biodetection technology and discloses a fluorescence detection method for the prostate cancer biomarker PCA3 based on gold nanorods and CRISPR Cas12a amplification. This invention provides a detection kit for the ultrasensitive detection of the long non-coding RNA PCA3, a tumor marker for prostate cancer. This kit utilizes the paracleavage property of CRISPR Cas12a activated by the target sequence to cleave the fluorescent reporter probe, releasing the fluorophore. This removes the signal quenching effect of the gold nanorods on the fluorophore, restoring the fluorescence signal and achieving ultrasensitive detection of the target sequence. The limit of detection (LOD) of this kit is as low as 1.65 pM, achieving highly selective quantitative detection of PCA3.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a fluorescence detection method for the prostate cancer marker PCA3 based on gold nanorods and CRISPR Cas12a amplification. Background Technology

[0002] Prostate cancer is the second most common malignant tumor among men worldwide. Early diagnosis is crucial for improving patient survival, optimizing treatment options, and improving prognosis. For a long time, clinical practice has relied heavily on the detection of prostate-specific antigen (PSA) in serum for screening and preliminary diagnosis of prostate cancer. PSA is a protease secreted by prostate epithelial cells, and an elevated concentration in the blood usually indicates that there may be a lesion in the prostate. However, PSA as a biomarker has significant limitations, which seriously affect the accuracy and specificity of diagnosis. (1) High false positive rate: Elevated PSA levels are not a specific indicator of prostate cancer. Many benign pathological conditions, such as benign prostatic hyperplasia (BPH) and prostatitis, can lead to a significant increase in PSA levels. This results in a large number of unnecessary and invasive prostate biopsies, causing patients physical and mental pain, economic burden, and potential risks of complications such as bleeding and infection. (2) Sensitivity and specificity are difficult to achieve simultaneously: Lowering the positive threshold of PSA can improve detection sensitivity, but it will introduce more false positives; raising the threshold to enhance specificity will miss some patients with early or low-grade prostate cancer. This inherent contradiction makes PSA-based screening strategies controversial in terms of balancing risks and benefits. (3) Inability to effectively distinguish between aggressive and indolent cancers: PSA levels cannot reliably distinguish between clinically significant, aggressive prostate cancers that require active intervention and slow-growing, indolent cancers that may not require immediate treatment, which can easily lead to "overdiagnosis" and "overtreatment".

[0003] To overcome the shortcomings of PSA, the search for more specific new molecular markers has become a research hotspot. Among them, prostate cancer antigen 3 (PCA3) has shown great potential. PCA3 is a long non-coding RNA whose gene is highly specifically overexpressed in prostate cancer tissue, but its expression level is extremely low or undetectable in normal prostate tissue or benign prostatic hyperplasia tissue. Compared with PSA, PCA3 has extremely high tissue specificity, and its expression level is basically unaffected by benign lesions such as prostatitis and hyperplasia, thus it is expected to reduce false positive results from the root. Clinical studies have shown that PCA3 detection is significantly more specific than PSA in differentiating between prostate cancer and benign prostate diseases. Although PCA3 has theoretical advantages, its widespread clinical application is still limited by the bottleneck of existing detection technology: (1) The procedure is cumbersome and the degree of automation is low: Traditional PCA3 detection mainly relies on reverse transcription quantitative polymerase chain reaction (RT-qPCR). This method requires multiple steps, including RNA extraction, reverse transcription into cDNA, and multiple rounds of thermal cycling PCR amplification. The operation process is complex and time-consuming (usually several hours), requiring high technical skills from the experimenters and making it difficult to achieve rapid, point-of-care, or immediate detection in primary healthcare institutions. (2) Sensitivity and cost challenges: The sensitivity of RT-qPCR is highly dependent on efficient nucleic acid extraction, high-quality reverse transcriptase, and precise temperature control equipment. For PCA3 RNA in trace samples or degraded samples, there may be missed detection. At the same time, the expensive enzyme preparations, specialized instruments, and consumables keep the detection cost high, limiting its accessibility as a screening method. (3) Potential cross-contamination: Amplification-based technologies have the risk of product aerosol contamination, which may lead to false positives and require a high level of laboratory environment.

[0004] In recent years, the CRISPR-Cas system, particularly CRISPR-Cas12a (such as LbCas12a and AsCas12a), has been revolutionaryly applied in the field of molecular diagnostics due to its unique "trans-cleavage" activity. When the Cas12a / crRNA complex specifically binds to target double-stranded DNA, its non-specific single-stranded DNA cleavage activity is activated, enabling indiscriminate cleavage of single-stranded DNA reporter molecules in the reaction system. By labeling the reporter molecules with fluorescent and quenching groups, ultrasensitive and rapid detection of target nucleic acids can be achieved through the release of fluorescent signals induced by cleavage. CRISPR-Cas diagnostic technology, with its advantages of high specificity, high sensitivity, speed (typically completed within one hour), and room-temperature operation, provides a completely new pathway for developing next-generation molecular diagnostic tools. Summary of the Invention

[0005] The first aspect of the present invention is to provide a detection kit.

[0006] The second objective of this invention is to provide the application of the detection kit of the first aspect of this invention in the preparation of products for detecting PCA3.

[0007] A third aspect of the present invention is to provide a method for detecting PCA3 for non-disease diagnostic purposes.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a detection kit comprising gold nanorods, a fluorescent reporter probe, a Cas protein, and crRNA complementary to a target RNA gene.

[0009] In some embodiments of the present invention, the nucleotide sequence of the crRNA complementary to the target RNA gene is shown in SEQ ID NO:2.

[0010] In some embodiments of the present invention, the nucleotide sequence of the fluorescent reporter probe is shown in SEQ ID NO:1.

[0011] In some embodiments of the present invention, the 5' end of the nucleotide sequence of the fluorescent reporter probe is labeled with a fluorescent group.

[0012] In some embodiments of the present invention, the fluorescent group is at least one of FAM, HEX, HTX, VIC, TAMRA, ROX and CY5; more specifically, it is FAM.

[0013] In some embodiments of the present invention, the Cas protein includes the Cas12 protein.

[0014] In some embodiments of the present invention, the Cas protein is selected from at least one of Cas12i, Cas12j, Cas12a, Cas12b, Cas12d, Cas12e, Cas12f, Cas12g, and Cas12h.

[0015] In some embodiments of the present invention, the kit further includes a reaction buffer, such as HEPES buffer.

[0016] In some embodiments of the present invention, the diameter of the gold nanorods is 10-20 nm, such as any value or a range formed by any combination of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nm.

[0017] In some embodiments of the present invention, the aspect ratio of the gold nanorods is 8:3.

[0018] In some embodiments of the present invention, the gold nanorods can be prepared using conventional techniques in the art or commercially available.

[0019] In some embodiments of the present invention, the kit further includes a positive control and a negative control.

[0020] A second aspect of the present invention provides the application of the detection kit of the first aspect of the present invention in the preparation of products for detecting PCA3.

[0021] A third aspect of the present invention provides a method for detecting PCA3 for non-disease diagnostic purposes, comprising the steps of using a kit from the first aspect of the present invention.

[0022] In some embodiments of the present invention, the method specifically includes the following steps: Obtain the nucleic acid from the sample to be tested; A solution containing gold nanorods and a solution containing a fluorescent reporter probe were mixed and incubated to obtain a fluorescent probe system. A solution containing Cas protein and a solution containing crRNA complementary to the target RNA gene were mixed and reacted to obtain the CRISPR Cas12a system. The fluorescent probe system, CRISPR Cas12a system, and nucleic acid of the sample to be tested are mixed, and the CRISPR reaction is performed to detect and read the fluorescence signal. The presence of PCA3 in the sample is determined based on the fluorescence signal.

[0023] In some embodiments of the present invention, the incubation conditions are: incubation at room temperature for 40-60 min; further, incubation at room temperature for 45-60 min; and even further, incubation at room temperature for 50-55 min.

[0024] In some embodiments of the present invention, the fluorescent reporter probe is adsorbed onto the surface of the gold nanorods through incubation.

[0025] In some embodiments of the present invention, the solvent of the solution containing gold nanorods is HEPES buffer.

[0026] In some embodiments of the present invention, the solvent of the solution containing the fluorescent reporter probe is HEPES buffer.

[0027] In some embodiments of the present invention, the reaction conditions are 35-40°C for 40-80 min; further, 35-38°C for 50-70 min; and even further, 36-38°C for 55-65 min.

[0028] In some embodiments of the present invention, the final concentration of the Cas12a protein in the reaction system is 70-90 nM, further 75-90 nM; even further 75-85 nM; and more preferably 80 nM.

[0029] In some embodiments of the present invention, the final concentration of the crRNA in the reaction system is 200-300 nM, further 230-260 nM; even further 235-250 nM; and more preferably 240 nM.

[0030] In some embodiments of the present invention, the solvent for both the solution containing the Cas protein and the solution containing crRNA complementary to the target RNA gene is HEPES buffer.

[0031] In some embodiments of the present invention, the conditions for CRISPR reaction detection are: 35-40°C for 20-60 min; further, 36-40°C for 30-50 min; and even further, 36-38°C for 35-45 min.

[0032] In some embodiments of the present invention, a fluorescence spectrophotometer is used to read the fluorescence signal.

[0033] In some embodiments of the present invention, the excitation wavelength of the fluorescence spectrophotometer is 490 nm and the emission wavelength is 525 nm.

[0034] This invention utilizes the trans-cutting properties of CRISPR Cas12a to achieve rapid detection of the target analyte PCA3, avoiding false positive signals found in traditional methods and improving diagnostic accuracy. The introduction of a fluorescence detection system simplifies the operation and enhances the sensitivity of the detection results. Compared to traditional blood tests, detecting prostate cancer through urine samples simplifies the traditional testing process.

[0035] The beneficial effects of this invention are: This invention provides a highly sensitive detection kit for the long non-coding RNA PCA3, a tumor marker in prostate cancer. This kit utilizes the target sequence to activate the paracleavage property of CRISPR Cas12a, cleaving the fluorescent reporter probe, releasing the fluorophore, and relieving the signal quenching effect of the gold nanorod on the fluorophore, thus restoring the fluorescence signal and achieving highly sensitive detection of the target sequence. The core lies in the trans-cleavage property of the CRISPR Cas12a system: a portion of the PCA3 sequence undergoes base complementary pairing with the guide crRNA, altering the conformation of Cas12a and activating its non-specific trans-cleavage activity, cleaving the fluorescent reporter probe ssDNA-FAM. The fluorophore moves away from the gold rod, resulting in enhanced fluorescence signal. This detection kit achieves a limit of detection (LOD) as low as 1.65 pM, enabling highly selective quantitative detection of PCA3.

[0036] This invention provides a fluorescence detection method for the prostate cancer biomarker PCA3 based on gold nanorods and CRISPR Cas12a amplification. Traditional detection methods for PSA in serum are cumbersome and prone to false positives. The method provided in this invention utilizes the trans-cleavage properties of CRISPR Cas12a. Through the interaction of PCA3 with crRNA, the conformation of Cas12a is altered, activating its trans-cleavage properties, cleaving ssDNA-FAM, releasing a fluorescent group, and activating the fluorescence signal. This enables rapid and ultrasensitive detection of PCA3 in urine, avoiding false positives in traditional methods and simplifying the detection process. It meets the current demand for ultrasensitive molecular diagnostics of trace biomarkers in complex biological samples. Attached Figure Description

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram illustrating the detection principle of the detection method in Embodiment 1 of the present invention.

[0038] Figure 2 This is an electron microscope image of the AuNRs prepared in Example 1 of the present invention.

[0039] Figure 3 Fluorescence spectrum of the assembled fluorescent probe.

[0040] Figure 4 This is a CRISPR Cas12a trans-shearing electrophoresis image.

[0041] Figure 5 The results of the feasibility assessment of a CRISPR Cas12a-mediated detection protocol for the prostate cancer marker PCA3 based on fluorescence signal amplification.

[0042] Figure 6This is a standard curve diagram of the response of the detection method of Example 1 of the present invention to different concentrations of target substances.

[0043] Figure 7 This is a specificity identification diagram of the detection method in Embodiment 1 of the present invention. Detailed Implementation

[0044] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0045] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0046] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0047] Example 1 A fluorescence detection method for the prostate cancer marker PCA3 based on gold nanorods and CRISPR Cas12a amplification (flow diagram shown) Figure 1 (As shown), including the following steps: (1) Assembly of PCA3 detection probe 1) Synthesis of AuNRs: 5 mL of 0.5 mM HAuCl4 and 5 mL of 0.2 M CTAB (hexadecyltrimethylammonium bromide) solution were mixed and vigorously stirred on a magnetic stirrer. During stirring, 1 mL of 6 mM NaBH4 solution was rapidly added. The solution changed from yellow to brownish-yellow after 2 min, and stirring was stopped. The solution was then left at room temperature for 30 min to obtain the seed solution. 7.0 g of CTAB and 1.234 g of C... 17 H 33COONa was dissolved in 250 mL of 50 °C pure water and cooled to 30 °C. Then, 24 mL of 4 mM AgNO3 solution was added. The mixture was allowed to stand at 30 °C for 15 min, followed by the addition of 250 mL of 1 mM HAuCl4 solution. After magnetic stirring for 90 min, the solution became colorless, and then 2.1 mL of 12 M HCl was added. After slow stirring with a magnetic stirrer for 15 min, 1.25 mL of 64 mM ascorbic acid (AA) was added, followed by vigorous stirring for 30 s. Finally, 0.8 mL of seed solution was added to the above solution. The resulting mixture was stirred for 30 s and allowed to stand at 30 °C for 12 h for AuNRs growth. The final product, AuNRs, was obtained by centrifugation at 7000 rpm for 30 min and removal of the supernatant.

[0048] Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are powerful characterization techniques that help observe the morphology and structure of the prepared AuNRs. SEM was used to analyze the morphology of the AuNRs, and the results are as follows: Figure 2 As shown, AuNRs were successfully prepared. The prepared AuNRs are uniform in size and have good morphology, with a length of about 40 nm, a bottom diameter of 15 nm, and an aspect ratio of 8:3.

[0049] 2) Probe preparation: Take 20 μL of 1.0 nM AuNRs solution (solvent: HEPES buffer, 20 mM) and 20 μL of 500 nM fluorescent reporter molecule ssDNA-FAM (5'-FAM-CATGAATGGATGTAGAGTTG-3', SEQ ID NO:1) solution (solvent: HEPES buffer, 20 mM) and incubate at room temperature for 50 min to obtain the fluorescent probe.

[0050] 20 μL of 200 nM Cas12a protein and 20 μL of 600 nM crRNA (5'-UAAUUUCUACUAAGUGUAGAUCCCAGGGAUCUCUGUGCUUCC-3', SEQ ID NO:2) were reacted in 10 μL of 20 mM HEPES buffer for 60 min at 37 °C to obtain the CRISPR Cas12a system. The Cas12a protein was purchased from Beijing Kexin Hengye Biotechnology Co., Ltd., catalog number KX-E-002 (2000 pmol).

[0051] The PCA3-specific sequence fragment is 5'-GGAAGCACAGAGATCCCTGGG-3' (SEQ ID NO:3).

[0052] (2) CRISPR Cas12a reverse shear triggering 1) RNA extraction: RNA samples were extracted from urine using standard RNA extraction methods. For example, following the instructions for the ZR Urine RNA Isolation Kit™, 30 μL of RNA was extracted from 3 mL of urine. The ZR Urine RNA Isolation Kit™ was purchased from Guangzhou Qiyun Biotechnology Co., Ltd., catalog number R1038.

[0053] 2) Activate CRISPR Cas12a trans-cleavage: Mix the fluorescent reporter molecule and CRISPRCas12a system from step (1) with 10 μL of extracted RNA sample and incubate for 40 min at 37℃.

[0054] (3) Signal detection: The fluorescence intensity of the solution after the reaction in step (2) was detected using a fluorescence spectrophotometer. The excitation wavelength was 490 nm and the emission wavelength was 525 nm. The detection was repeated three times at room temperature.

[0055] Example 2 This embodiment uses a fluorescence spectrophotometer as a powerful technique to determine the intensity of the fluorescent probe signal and characterize the preparation of the fluorescent probe in Example 1. Specifically, 70 μL of 500 nM ssDNA-FAM was used for fluorescence detection at an excitation wavelength of 490 nm. 70 μL of AuNRs solution was also used for fluorescence detection at an excitation wavelength of 490 nm. 40 μL of AuNRs solution was incubated with 40 μL of 500 nM ssDNA-FAM solution for 50 min, and 70 μL of the reaction solution was used for fluorescence detection at an excitation wavelength of 490 nm.

[0056] The results are as follows Figure 3 As shown, the fluorescent reporter molecule ssDNA-FAM has a strong emission wavelength at 525 nm. AuNRs show no fluorescent signal. When ssDNA-FAM is adsorbed onto the surface of AuNRs, the FAM fluorescence signal is quenched, and the emission intensity at 525 nm is significantly reduced.

[0057] Example 3 This embodiment rigorously verifies the CRISPR Cas12a trans-shearing properties in step (2) of Example 1 using a polyacrylamide gel system. Specifically: 10 μL of Maker was added to the first lane. ① 10 μL of 10 mM ssDNA-FAM and 10 μL of 10 mM PCA3 (SEQ ID NO:3) were mixed with 2 μL of 3× loading buffer containing nucleic acid dye, and 10 μL of each was added to the second and third lanes respectively. ② 6 μL of ssDNA-FAM was mixed with 3 μL of 200 nM Cas12a protein, 3 μL of 600 nM crRNA and 2 μL of 3× loading buffer containing nucleic acid dye, and 10 μL of each was added to the fourth lane. ③ Mix 6 μL of ssDNA-FAM with 3 μL of 200 nM Cas12a protein, 3 μL of 600 nM crRNA, 3 μL of 10 mMPCA3, and 2 μL of 3× loading buffer containing nucleic acid dye. Add 10 μL of the mixture to lane 5. Then perform polyacrylamide gel electrophoresis.

[0058] The results are as follows Figure 4 As shown, lanes two and three each have only one distinct band, lane four has three distinct bands, and lane five has only two distinct bands. This indicates that with the addition of PCA3, the CRISPR Cas12a trans-cleavage property is activated, and ssDNA-FAM is cleaved.

[0059] Example 4 This embodiment rigorously verified the feasibility of PCA3 detection using CRISPR Cas12a trans-shearing-mediated fluorescence signal amplification as described in Example 1 using a fluorescence spectrophotometer. Specifically: ① 20 μL of 200 nM Cas12a protein and 20 μL of 600 nM crRNA were reacted in 10 μL of 20 mM HEPES buffer for 60 min at a reaction temperature of 37 ℃. ② 20 μL of AuNRs solution and 20 μL of 500 nM ssDNA-FAM solution were incubated for 50 min. After the reaction, the two solutions were mixed, and 70 μL was used for fluorescence detection at an excitation wavelength of 490 nm. ③ 20 μL of 200 nM Cas12a protein and 20 μL of 600 nM crRNA were reacted in 10 μL of 20 mM HEPES buffer for 60 min at a reaction temperature of 37 ℃. ④ Incubate 20 μL of AuNRs solution with 20 μL of 500 nM ssDNA-FAM solution for 50 min. Mix the two solutions after the reaction with 10 μL of 10 nM PCA3, and take 70 μL for fluorescence detection at an excitation wavelength of 490 nm. ⑤ Incubate 20 μL of AuNRs solution with 20 μL of 500 nM ssDNA-FAM solution for 50 min. Mix the two solutions after the reaction with 10 μL of 40 nM PCA3, and take 70 μL for fluorescence detection at an excitation wavelength of 490 nm.

[0060] The results are as follows Figure 5 As shown, in the absence of the PCA3 fragment, the trans-cleavage property of the CRISPR Cas12a system is not activated, resulting in a low fluorescence signal intensity. With the addition of PCA3, the trans-cleavage property of the CRISPR Cas12a system is activated, cleaving ssDNA-FAM to release fluorophores, thus enhancing the fluorescence signal. The fluorescence signal of the detection system increases with increasing PCA3 concentration.

[0061] Example 5 In this embodiment, the detection method of Example 1 was used to detect PCA3 series diluents (5 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM) to evaluate the analytical performance of the detection method.

[0062] The results showed that the fluorescence signal intensity of the detection system increased with increasing PCA3 concentration. The average fluorescence intensity as a percentage of the logarithm of PCA3 concentration (lgCPCA3) was within the range of 5 × 10⁻⁶. -12 M to 5×10 -7 Linear correlation is observed within the range of M. Figure 6 The regression equation is y = 115.15x + 856.84925 (R²). 2 = 0.9917), where x is the logarithm of the PCA3 concentration.

[0063] Three blank samples (i.e., samples without PCA3) were tested, and the mean X0 and standard deviation P0 were calculated. S0 was then calculated, which is the sum of the mean of the blank samples and three times the standard deviation (the formula is S0 = X0 + 3 × P0). Substituting S0 into the above regression equation, the detection limit of the detection method in Example 1 was found to be 1.65 pM.

[0064] Example 6 This embodiment uses the detection method of Example 1 to detect different biomarkers (including M1, M3, c-myc, let-7a, TK1, and PCA3, etc.) to evaluate the specificity of the detection method. During the detection process, the incubated CPCA3 RNA was replaced with RNA from other different biomarkers, while other experimental conditions remained unchanged.

[0065] The results are as follows Figure 7 As shown, the average fluorescence signal intensity of each non-target sequence was comparable to that of the blank signal, while the fluorescence signal intensity observed in the mixture containing PCA3 and all four interfering substances (in which the concentration of PCA3 was 500 nM and the concentration of each of the four interfering substances was 5 µM) was very close to that of PCA3 alone, suggesting that the detection method of Example 1 has excellent selectivity for the target analyte PCA3.

[0066] Example 7: Detection of Clinical Samples This embodiment uses the detection method of Example 1 to test clinical samples (including urine samples from patients with renal cell carcinoma, bladder cancer, benign prostatic hyperplasia, and prostate cancer) to evaluate the application value of the detection method.

[0067] The test results are shown in Table 1. PCA3 was detected only in the urine of patients with bladder cancer, while it was not detected in other samples. This indicates that the detection method in Example 1 has high application potential in the clinical detection of prostate cancer.

[0068] Table 1. Detection results of clinical samples

[0069] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A detection kit comprising gold nanorods, a fluorescent reporter probe, a Cas protein, and crRNA complementary to a target RNA gene.

2. The detection kit according to claim 1, characterized in that, The nucleotide sequence of the crRNA complementary to the target RNA gene is shown in SEQ ID NO:

2.

3. The detection kit according to claim 1, characterized in that, The nucleotide sequence of the fluorescent reporter probe is shown in SEQ ID NO:

1.

4. The detection kit according to any one of claims 1-3, characterized in that, The Cas protein includes the Cas12 protein; Preferably, the Cas protein is selected from at least one of Cas12i, Cas12j, Cas12a, Cas12b, Cas12d, Cas12e, Cas12f, Cas12g, and Cas12h.

5. The detection kit according to claim 4, characterized in that, The kit also includes a reaction buffer.

6. The use of the test kit according to any one of claims 1-5 in the preparation of a product for detecting PCA3.

7. A method for detecting PCA3 for non-disease diagnostic purposes, comprising the steps of using the kit according to any one of claims 1-5.

8. The method according to claim 7, characterized in that, The method specifically includes the following steps: Obtain the nucleic acid from the sample to be tested; A solution containing gold nanorods and a solution containing a fluorescent reporter probe were mixed and incubated to obtain a fluorescent probe system. A solution containing Cas protein and a solution containing crRNA complementary to the target RNA gene were mixed and reacted to obtain the CRISPR Cas12a system. The fluorescent probe system, CRISPR Cas12a system, and nucleic acid of the sample to be tested are mixed, and the CRISPR reaction is performed to detect and read the fluorescence signal. The presence of PCA3 in the sample is determined based on the fluorescence signal.

9. The method according to claim 8, characterized in that, The incubation conditions are room temperature incubation for 40-60 min; and / or, the reaction conditions are 35-40℃ reaction for 40-80 min.

10. The method according to claim 8, characterized in that, The CRISPR reaction detection conditions are 35-40℃ for 20-60 min.

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