Variable-temperature one-step method detection system based on weak-effect PAM, establishment method of system and application of system in detection of chlamydia psittaci

Abstract

The invention provides a weak-effect PAM-based variable-temperature one-step method detection system, an establishment method thereof and application of the weak-effect PAM-based variable-temperature one-step method detection system in chlamydia psittaci detection, and belongs to the technical field of detection. The sequences of the chlamydia psittaci RPA primer pair are as shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the sequence of the crRNA molecule is as shown in SEQ ID NO. 3. Based on in-depth study on PAM recognition characteristics of AapCas12b protein, the PAM restriction of the AapCas12b protein has remarkable temperature dependence, namely, the restriction is relatively weak under a high-temperature condition and is remarkably enhanced under a low-temperature condition. The important discovery provides a theoretical basis for developing a novel detection method.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a variable-temperature one-step detection system based on weak PAM, its establishment method, and its application in the detection of Chlamydia psittaci. Background Technology

[0002] Chlamydia psittaci ( Chlamydia psittaci Macrolides are an important zoonotic pathogen that can cause influenza-like symptoms and respiratory illnesses. Although macrolide antibiotics are effective in treating them, misdiagnosis and treatment delays often occur in clinical practice due to the high similarity of their clinical manifestations to influenza and the limitations of current detection technologies.

[0003] Currently, quantitative real-time PCR is the main method for detecting this pathogen, but it requires sophisticated experimental equipment and technical skills, making it difficult to widely apply in primary healthcare institutions, emergency rooms, or home testing scenarios. While high-throughput next-generation sequencing can achieve accurate identification, its long testing cycle, high cost, and reliance on specialized bioinformatics analysis also make it unsuitable for routine testing needs. Therefore, developing a simple, low-cost, rapid, sensitive detection technology suitable for grassroots deployment is of significant practical importance for the early diagnosis and precise control of Chlamydia psittaci.

[0004] In recent years, the CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR associated) system, derived from the adaptive immune mechanism of prokaryotes, has brought revolutionary breakthroughs to the field of molecular diagnostics due to its high developability, simplicity, and efficiency. This system consists of CRISPR RNA and a Cas protein with nuclease activity guided by it. To achieve highly sensitive molecular detection, CRISPR technology typically needs to be combined with isothermal nucleic acid amplification technology. This combined strategy can achieve nucleic acid amplification and detection without thermal cycling, a characteristic that is of great value for developing simple nucleic acid detection methods and instruments.

[0005] Based on these characteristics, detection technology platforms such as DETECTR, HOLMES, and CDetection have emerged. However, apart from the nucleic acid extraction step, these platforms typically require a "two-step" approach (i.e., the amplification and detection processes are performed separately). The two-step approach requires opening the reaction tube and transferring the liquid, thus increasing the complexity of the detection steps, the difficulty of constructing the detection equipment, and the cost. Furthermore, the liquid transfer step is highly susceptible to aerosol contamination, leading to false positives and posing a significant obstacle to point-of-care testing (POCT). In contrast, the "one-step" approach (i.e., completing isothermal amplification and CRISPR reaction in a single tube with a single sample addition) not only reduces operational complexity and avoids aerosol contamination but also enables simultaneous amplification and detection, shortening the detection time.

[0006] The current challenge of one-step methods is that the isothermal amplification reaction and the CRISPR cleavage reaction compete for substrates, leading to delayed or even non-existent substrate amplicon production, which in turn causes detection delay, reduced sensitivity, and unstable results. To address this issue, mainstream technology platforms primarily employ the following regulatory strategies: First, physical isolation, separating reagents in the tube cap, tube wall, or microfluidic device; second, physical rate reduction, constructing a miscible multiphase system to delay contact between CRISPR and the amplification reaction; third, chemical isolation, detecting only the amplification product without cleaving the original template target, or using photo-regulation strategies to block or activate (off-on) the guide RNA; and fourth, chemical rate reduction, i.e., reducing the initial cleavage efficiency of the CRISPR system by using suboptimal PAM sequences or modifying Cas enzymes, thus avoiding premature cleavage of the isothermal amplicon. Through the rational application of these regulatory methods, one-step methods simplify the operation process while maintaining detection efficiency, highlighting their enormous application potential in POCT detection.

[0007] In previous studies of this invention, bacteria derived from acidophilic thermostable bacteria were utilized. Alicyclobacillus acidophilusA nucleic acid detection system was constructed using the Cas12b protein. Guided by single-guide RNA (sgRNA), this protein specifically cleaves the target double-stranded DNA in the presence of a protospacer adjacent motif (PAM) (5'-TTN-3'), with a cleavage reaction temperature reaching 60-65°C. Simultaneously, upon activation, this protein exhibits trans-cleavage activity, non-specifically cleaving the ssDNA probe, thereby converting the detection result into a fluorescent signal. Based on this, this invention establishes two-step and one-step LAMP-CRISPR / Cas12b detection methods for Chlamydia psittaci. The two-step method shows high consistency with qPCR detection results, but is cumbersome and prone to aerosol contamination; while the one-step method, although simple to operate, has significantly reduced sensitivity, limiting its application. These limitations prompted this invention to develop a more sensitive, specific, and rapid one-step CRISPR detection system. Summary of the Invention

[0008] The purpose of this invention is to propose a variable-temperature one-step detection system based on weak PAM, its establishment method, and its application in the detection of Chlamydia psittaci, which has a good anti-aging effect.

[0009] The technical solution of this invention is implemented as follows: This invention provides a combination of Chlamydia psittaci RPA primer pair and crRNA molecule, wherein the sequences of the Chlamydia psittaci RPA primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the crRNA molecule is shown in SEQ ID NO.3.

[0010] The present invention further protects the Chlamydia psittaci RPA-CRISPR / Cas12b reaction system, comprising: the above-mentioned Chlamydia psittaci RPA primer pair and crRNA molecule combination; and further comprising: ssDNA and AapCas12b protein, wherein the ssDNA is FAM-TTTTTTTTTTTT-BHQ1.

[0011] As a further improvement of the present invention, the final concentration ratio of AapCas12b protein to crRNA is 1:1-3.

[0012] As a further improvement of the present invention, the reaction system comprises: primers, enzyme preparations, magnesium acetate and rehydration buffer required for recombinase polymerase amplification (RPA) reaction, and AapCas12b protein, specific sgRNA, ssDNA-FQ fluorescent reporter molecule and corresponding reaction buffer required for CRISPR / Cas12b detection system; the RPA and CRISPR / Cas12b components are premixed before the reaction starts, and the target nucleic acid sequence is amplified first under isothermal conditions in the same reaction tube, and then the fluorescence signal is activated by the trans-cleavage activity of Cas12b.

[0013] This invention further protects the application of the above-mentioned Chlamydia psittaci RPA-CRISPR / Cas12b reaction system in the preparation of a one-step temperature-dependent detection system for Chlamydia psittaci based on weak PAM.

[0014] As a further improvement of the present invention, the temperature variation conditions are 30-40℃ for 15-25 min and 55-65℃ for 30-50 min.

[0015] The present invention further protects the temperature-controlled one-tube freeze-drying tube for detecting Chlamydia psittaci. The reaction system for detecting Chlamydia psittaci is fixed in the one-tube freeze-drying tube by freeze-drying. The reaction system for detecting Chlamydia psittaci contains the above-mentioned Chlamydia psittaci RPA-CRISPR / Cas12b reaction system.

[0016] The present invention further protects a Chlamydia psittaci detection kit, wherein the Chlamydia psittaci detection kit contains the above-mentioned Chlamydia psittaci RPA primer pair and crRNA molecule combination, or the above-mentioned Chlamydia psittaci RPA-CRISPR / Cas12b reaction system, or the above-mentioned temperature-controlled one-tube lyophilized tube.

[0017] The present invention has the following beneficial effects: This invention, based on in-depth research into the PAM recognition characteristics of the AapCas12b protein, reveals a significant temperature dependence in its PAM restriction: the restriction is weaker at high temperatures and significantly enhanced at low temperatures. This important finding provides a theoretical basis for developing novel detection methods. This invention successfully establishes WPTTS (one-step RPA-CRISPR / Cas12b assay using...) w eak P AM recognition with t wo- t emperature sThe hifting detection platform utilizes the AapCas12b protein to achieve high-sensitivity detection by recognizing a weak PAM sequence and adjusting temperature transition conditions. Specifically, in the initial low-temperature phase, the low activity of the weak PAM effectively inhibits the Cas12b reaction, avoiding interference with RPA amplification; subsequently, the transition to a high-temperature phase fully releases the activity of the weak PAM, ensuring detection sensitivity.

[0018] This invention establishes WPTTS (one-step RPA-CRISPR / Cas12b assay using...) w eak P AMrecognition with t wo- t emperature s The hifting detection platform's core design lies in precisely controlling the Cas12b cleavage activity through temperature programming to achieve optimal reaction conditions at different stages of the reaction. Specifically, in the low-temperature stage (RPA-dominant amplification phase), inefficient PAM is used to maximally suppress Cas12b cleavage activity, thus avoiding interference with RPA amplification; while in the high-temperature stage (detection-dominant phase), the full activity of Cas12b is released, ensuring efficient signal detection. This strategy effectively solves the technical problem of mutual interference between amplification and detection in traditional single-tube methods, providing a new approach for developing highly sensitive integrated molecular diagnostic systems. This platform is not only easy to operate and fast, but also exhibits excellent detection performance, making it of significant application value in the clinical diagnosis and field detection of Chlamydia psittaci. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 To achieve a template concentration of 10 3 Comparison of trans-cleavage activity between typical PAM (TTC) and atypical PAM (TGT, TCG, TGC, GTC, ATG) in a one-step copy / reaction LAMP-AapCas12b detection system. NTC was used as a non-target control. Error bars represent mean ± standard deviation, n=3; Figure 2This image compares the trans-cleavage activity of AapCas12b using 64 PAM sequences (NNN) at different temperatures. TTN represents the classic PAM sequence. The heatmap shows the average enhancement of fluorescence signal within 1 hour after target DNA addition. NTC is the non-target control group, n=3. Figure 3 Comparison of trans-cleavage activity of AapCas12b at different temperatures when using the classic PAM sequence (TTN). Error bars represent mean ± standard deviation, n = 3; Figure 4 This study compares the trans-cleavage activity of AapCas12b against 20 extended PAM variants (4 bases) at two temperatures (35°C and 60°C). These PAM variants are derived from five weaker PAM variants (3 bases) and the classic PAM. The heatmap shows the mean enhancement of fluorescence signal within 1 hour after target DNA addition. NTC is the non-target control group, n=3. Figure 5 (a) The core strategy of the WPTTS detection platform. At low temperatures, substrates containing classic PAM activate a certain level of AapCas12b cleavage activity, which competitively consumes the substrate required for RPA amplification, thus limiting amplification efficiency. Substrates containing weak PAM, however, cannot effectively activate AapCas12b cleavage, allowing the RPA amplification reaction to proceed rapidly without interference, accumulating a large amount of amplification products. When the temperature rises to 60°C, the trans-cleavage activity of AapCas12b is fully activated in both reaction systems—classic PAM can only generate a weak signal relying on limited initial amplification products, while weak PAM achieves superior detection performance thanks to the abundant amplification products accumulated in the early stages. (b, c) Comparison of one-step reaction sensitivity using classic PAM (b) and weak PAM (c). Target substrates were serially diluted tenfold to 10 μL per reaction. 6 -10 0 Copy concentration. NTC is the non-target control. Error bars represent mean ± standard deviation, n=3. (d) Through 10 2 Comparison of recognition efficiency between classic PAM and weak PAM at template concentrations of copy / reaction (data from 10 independent replicate experiments) to systematically evaluate the reliability of the one-step reaction. Figure 6 Quantitative ranking of AapCas12b trans-cleavage activity in different PAM sequences. The heatmap shows the average fluorescence signal increase within 1 hour after target DNA addition.

[0021] Figure 7To evaluate the mismatch tolerance of classic PAM and weak PAM to the matched or mismatched CPSIT_0429 activator, mut1-20 each contain a single mismatch relative to the target site. WT represents wild-type, and NTC represents the non-target control. Error bars represent mean ± standard deviation, n=3; Figure 8 (a) Schematic diagram of the WPTTS platform for detecting Chlamydia psittaci. (b) Heatmap showing the comparison results of one-step detection with weak PAM target, one-step detection with classic PAM target, two-step detection with classic PAM target, and qPCR. For the one-step and two-step detections, the color gradient represents the fold increase in fluorescence signal relative to the non-target control during the 40-minute reaction period; the color gradient in the qPCR results corresponds to the measured Ct value. White areas in the heatmap represent negative test results. The x-axis labels indicate the sample name and its corresponding Ct value. (c) Comparison of the consistency results between the three detection strategies—weak PAM (one-step), classic PAM (one-step), and classic PAM (two-step)—and the gold standard qPCR results. "+" indicates a positive test result, "+" indicates a positive result. "" indicates a negative result. The percentage of agreement between the true positive rate and the true negative rate is listed in parentheses, n = 20. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Material All RPA primers, templates, and ssDNA-FQ reporter genes were synthesized by Genewiz Biotechnology (Suzhou) Co., Ltd. Detailed information can be found in the supplementary materials. AapCas12b protein was purchased from Shanghai Tulugang Biotechnology Co., Ltd.; the TwistAmpBasic kit was purchased from TwistDX, UK; the Chlamydia psittaci nucleic acid detection kit was purchased from Nanjing Yonglang Biotechnology Co., Ltd.; and the rapid nucleic acid extraction kit (magnetic bead method) was purchased from Chongqing Zhongyuan Huiji Biotechnology Co., Ltd. Experiments were conducted using the Chlamydia psittaci genome (GenBank accession number: CP002549.1). CPSIT The _0429 site fragment was used as a target.

[0024] Sample preparation Sputum or bronchoalveolar lavage fluid samples were obtained from the Jiangsu Province Chlamydia psittaci surveillance project and used as pathogen templates. Pathogen nucleic acid templates were extracted using a fully automated nucleic acid extractor in conjunction with a rapid nucleic acid extraction kit (magnetic bead method).

[0025] CRISPR / Cas12b detection system Each CRISPR / Cas12b nucleic acid detection system contains: 0.5 μL AapCas12b (10 μM), 2 μL sgRNA (2.5 μM), 2 μL ssDNA-FQ reporter gene (10 μM), and 2 μL HOLMES Cas12b buffer (10×). After adding an appropriate amount of template DNA, bring the volume to 20 μL with nuclease-free water. After centrifugation, immediately place the system in an AppliedBiosystems® QuantStudio Q5 (ABI Q5) instrument and react at the set temperature for 1 hour, during which fluorescence signals are acquired once per minute.

[0026] Recombinase polymerase amplification (RPA) and CRISPR / Cas12b two-step detection RPA Reaction: Primer sequences were designed according to the TwistAmp™ Reaction Kit instructions, and the reaction was performed following the TwistAmp® Basic Kit (TwistDx) procedure. The reaction mixture was as follows: 29.5 μL of primer-free rehydration buffer, 13.7 μL of nuclease-free water, 2.4 μL of forward / reverse primers (10 μM), and 2 μL of template DNA were mixed in an EP tube, and then added to a reaction tube containing lyophilized RPA enzyme. Vortexed until the enzyme powder was completely dissolved, and briefly centrifuged. 14 μL of the mixed reaction solution was taken, and 1 μL of magnesium acetate (280 nM) was added to form a 15 μL single reaction system. The reaction was incubated at 37°C in a metal bath for 20 minutes.

[0027] CRISPR / Cas12b reaction: Prepare the following mixture: 1.3 μL AapCas12b (10 μM), 5 μL sgRNA (2.5 μM), 5 μL ssDNA-FQ reporter gene (10 μM), 5 μL HOLMES Cas12b buffer (10×), and 33.7 μL nuclease-free water. Combine 15 μL of this mixture with 15 μL of RPA reaction product, mix thoroughly to a total volume of 30 μL, and incubate at 60°C for 40 minutes using an ABIQ5 instrument.

[0028] RPA combined with CRISPR / Cas12b one-step detection The one-step method is based on the two-step method described above with slight modifications: First, add 15 μL of CRISPR / Cas12b reaction mixture to an EP tube, then add 15 μL of RPA reaction mixture (formulation same as the two-step method), and mix well to form a total system of 30 μL. After sealing the tube, briefly centrifuge at low speed, and react at 37°C for 20 minutes in an ABI Q5 instrument, followed by a reaction at 60°C for 40 minutes, with fluorescence signals acquired once per minute during the 60°C stage.

[0029] Quantitative polymerase chain reaction (qPCR) The Chlamydia psittaci nucleic acid detection kit was used according to the manufacturer's instructions. Each reaction system contained 16 μL of reaction solution I, 1 μL of reaction solution II, and 3 μL of sample nucleic acid, for a total volume of 20 μL. The reaction program was: 50℃ for 2 minutes; 95℃ for 10 minutes; 95℃ for 15 seconds, 60℃ for 40 seconds, for a total of 40 cycles. Fluorescence signals were acquired during the extension phase of each cycle using an ABI Q5 instrument. A Ct value ≤ 35 was considered positive, and > 35 was considered negative.

[0030] Example 1: The PAM confinement of AapCas12b exhibits a significant temperature dependence. Existing literature indicates that Cas12a's cleavage kinetics can be effectively modulated by employing unconventional PAM strategies, thereby significantly improving the sensitivity of one-pot detection. To explore the applicability of this strategy to other Cas12 homologous proteins and further optimize the previously developed LAMP and AapCas12b-based Chlamydia psittaci detection scheme, this invention systematically compared the recognition efficiency of AapCas12b for conventional PAM (TTC) and five unconventional PAM sequences under low-copy template conditions. Experimental results show that in the one-tube LAMP+cas12b detection system, the use of unconventional PAM did not exhibit superior detection performance compared to conventional PAM. Figure 1 This finding is consistent with the conclusions of other studies.

[0031] To investigate whether temperature can affect PAM recognition characteristics and thus improve detection performance, this invention further examined the variation of PAM selectivity within a temperature range of 35-60℃. This is based on previous designs targeting *Chlamydia psittaci*. CPSIT For the sgRNA targeting _0429, this invention constructed a target mutant library containing all 64 possible 3-base PAM sequences (NNN), and comprehensively evaluated its trans-cleavage activity at six temperature gradients (35, 40, 45, 50, 55, and 60 °C). The results showed that classic PAM (TTN) exhibited significant catalytic activity within this temperature range. Figure 3It is worth noting that as the temperature rises to 60℃, AapCas12b exhibits a significant relaxation of PAM recognition, manifested as a reduction in the difference in endpoint fluorescence signals between classical and non-classical PAMs; while at low temperatures, AapCas12b exhibits a more stringent PAM selectivity, with significant differences in the endpoint fluorescence signals between the two types of PAMs. Figure 2 These findings reveal that AacCas12b's PAM recognition exhibits a significant temperature-dependent characteristic, consistent with existing literature reports that increased temperature leads to decreased PAM restriction in Cas proteins, providing new experimental evidence for the temperature regulation mechanism of the CRISPR / Cas system.

[0032] Example 2: Temperature's Regulation of Inefficient PAM Activity Has Universality. To further explore the universality of temperature's regulation of PAM activity, this invention first quantitatively evaluated and ranked the cutting efficiency of all tested PAMs. Based on this quantitative result, this invention screened out the five PAM sequences (GCC, ACC, AAC, AGC, CAT) with the lowest cutting efficiency under low-temperature conditions and defined them as "inefficient PAMs" as the objects of subsequent in-depth research. Figure 6 The cleavage efficiency of inefficient PAM was extended from 3-position to 4-position, and validated using targets with different GC contents. Experimental results showed that the extension of inefficient PAM from 3-position to 4-position did not significantly change the cleavage efficiency at three targets with different GC contents, and this phenomenon was independent of the extended base type, suggesting that the core recognition site of weak PAM may be limited to 3-position. Furthermore, under low-temperature conditions, compared with classic PAM, these inefficient PAMs exhibited lower activity at targets with different GC contents. This indicates that the recognition pattern of inefficient PAM is not significantly correlated with the GC content of the target spacer region, suggesting that this pattern may have general applicability. When the temperature was increased to 60℃, the weak PAM regained its cleavage ability, achieving an efficiency similar to that of classic PAM. Figure 4 This temperature-induced unconstrained PAM phenomenon is highly consistent with the temperature-dependent characteristics of overall PAM selectivity previously observed in this invention.

[0033] Example 3: Development of a one-step detection system based on inefficient PAM with variable temperature control Based on the study of the temperature dependence of PAM activity (significant differences in activity at low temperatures and convergence at high temperatures), this invention innovatively proposes the WPTTS (one-step RPA-CRISPR / Cas12b assay using weak PAM recognition with two-temperature shifting) detection platform. The core design of this platform lies in: precisely controlling the cleavage activity of Cas12b through temperature programming to achieve optimal reaction conditions at different stages of the reaction. Figure 5 a) Specifically, during the low-temperature phase (RPA-dominant phase), inefficient PAM is used to maximally suppress Cas12b cleavage activity, thereby avoiding interference with RPA amplification; while during the high-temperature phase (detection-dominant phase), the full activity of Cas12b is released, ensuring efficient signal detection. This strategy effectively solves the technical problem of mutual interference between amplification and detection in traditional single-tube methods, providing a new approach for developing highly sensitive integrated molecular diagnostic systems.

[0034] To systematically evaluate the detection performance of this platform, this invention selected inefficient PAM (ACC) and conventional PAM (TTC) for comparative study. The experiment used conditions of 37℃ for 20 minutes and 60℃ for 40 minutes to perform a one-step detection method on the RPA-AapCas12b detection system. Figure 5 Parallel tests of (b, c) were conducted. The results showed ( Figure 5 (b, c) In one-step detection, the sensitivity of conventional PAM is 10. 3 While the detection performance of inefficient PAM is significantly improved, the sensitivity is increased by an order of magnitude to 10 copies / reaction. 2 The copies / reaction method is equivalent to the two-step method. To verify the stability of the detection system, this invention operates at a detection limit of 10. 2 Ten replicate experiments were conducted at a copy / reaction concentration, and statistical results showed that the detection system has excellent robustness. Figure 5 d).

[0035] Example 4: Inefficient PAM exhibited superior specificity compared to conventional PAM. To systematically evaluate the sequence recognition specificity of inefficient PAM variants, we designed a series of double-stranded DNA targets containing single-base mismatches in the spacer region 20 bp downstream of the PAM site. Experimental results showed that, compared to conventional PAM, inefficient PAM variants exhibited significantly reduced tolerance to target sequence mismatches, demonstrating more stringent sequence recognition specificity at most positions. Particularly at positions 10 and 11, even single-base resolution was achieved. This characteristic gives them a unique advantage in molecular diagnostic applications requiring high specificity.Figure 7 ).

[0036] Example 5: Validation of Clinical Samples To evaluate the clinical application performance of the WPTTS detection platform, we applied it to the detection of Chlamydia psittaci in clinical samples. Figure 8 a). This study included 20 qPCR-confirmed positive samples and 20 negative control samples for validation. Regarding the design of the detection system, we developed not only one-step and two-step detection schemes targeting the classic PAM sequence, but also designed a weak PAM target based on the CPSIT_0429 gene region. The detection efficacy of the weak PAM strategy was systematically evaluated by comparing three detection strategies (weak PAM one-step, classic PAM one-step, and classic PAM two-step). The detection threshold was set at 3 times the average fluorescence amplification of the non-target control. The results showed that the weak PAM one-step strategy was significantly superior to the classic PAM one-step strategy, especially in samples with low viral load (Ct≤30), with a higher detection rate. Its performance was comparable to the classic PAM two-step strategy. Figure 8 b). Compared with the gold standard qPCR results, the negative concordance rate of all three methods reached 100% (20 / 20). Notably, the positive concordance rate of the weak PAM one-step method reached 95% (19 / 20), which was better than the 75% (15 / 20) of the classic PAM two-step method, while maintaining consistency with the results of the classic PAM two-step method. Figure 8 (c) This strongly validates its excellent diagnostic performance and clinical translational value.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A combination of Chlamydia psittaci RPA primer pairs and crRNA molecules, characterized in that, The sequences of the Chlamydia psittaci RPA primer pair are shown in SEQ ID NO.1 and SEQ ID NO.2, and the sequence of the crRNA molecule is shown in SEQ ID NO.

3.

2. The psittacosis chlamydia RPA-CRISPR / Cas12b reaction system, characterized in that, include: The combination of Chlamydia psittaci RPA primer pair and crRNA molecule as described in claim 1; further comprising: ssDNA and AapCas12b protein, wherein the ssDNA is FAM-TTTTTTTTTTTT-BHQ1.

3. The psittacosis chlamydia RPA-CRISPR / Cas12b reaction system according to claim 2, characterized in that, The final concentration ratio of AapCas12b protein to crRNA is 1:1-3.

4. The psittacosis chlamydia RPA-CRISPR / Cas12b reaction system according to claim 3, characterized in that, The reaction system comprises: primers, enzyme preparations, magnesium acetate and rehydration buffer required for the recombinase polymerase amplification reaction, and AapCas12b protein, specific sgRNA, ssDNA-FQ fluorescent reporter molecule and corresponding reaction buffer required for the CRISPR / Cas12b detection system; the RPA and CRISPR / Cas12b components are premixed before the reaction starts and are used in the same reaction tube to realize a continuous process of first amplifying the target nucleic acid sequence under isothermal conditions, and then activating the fluorescence signal through the trans-cleavage activity of Cas12b.

5. The application of the Chlamydia psittaci RPA-CRISPR / Cas12b reaction system as described in any one of claims 2-4 in the preparation of a one-step temperature-dependent detection system for Chlamydia psittaci based on weak PAM.

6. The application according to claim 5, characterized in that, The temperature variation conditions are: 30-40℃ for 15-25 minutes, and 55-65℃ for 30-50 minutes.

7. A variable-temperature lyophilized tube for detecting Chlamydia psittaci, characterized in that, The reaction system for detecting Chlamydia psittaci is fixed in the single-tube freeze-drying tube by freeze-drying, and the reaction system for detecting Chlamydia psittaci contains the Chlamydia psittaci RPA-CRISPR / Cas12b reaction system according to any one of claims 2-4.

8. A psittacosis chlamydia detection kit, characterized in that, The Chlamydia psittaci detection kit contains the Chlamydia psittaci RPA primer pair and crRNA molecule combination as described in claim 1, or the Chlamydia psittaci RPA-CRISPR / Cas12b reaction system as described in any one of claims 2-4, or the variable temperature one-tube lyophilized tube as described in claim 7.

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