Primer probe combination and kit for digital PCR (polymerase chain reaction) detection of candida auricula and application

Abstract

The invention relates to the field of pathogen detection, in particular to a primer probe combination for digital PCR detection of candida auricula, a kit and application. The primer probe combination provided by the invention is a primer probe combination designed by taking a candida auricula genetic conserved site ITS as a target gene for amplification, can be used for digital PCR detection of candida auricula, can directly perform quantitative detection on candida auricula, can detect the content of candida auricula in a sample to be detected under an extremely low copy number, and has a good application prospect. The Candida auricula loading capacity in an environment sample can be accurately quantified, the method can be suitable for clinical early diagnosis, hospital infection monitoring and basic-level rapid detection, and accurate technical support is provided for prevention and control of multi-drug-resistant Candida auricula.

Description

Technical Field

[0001] This invention relates to the field of pathogen detection, and in particular to a primer-probe combination, kit, and application for digital PCR (dPCR) detection of Candida auris. Background Technology

[0002] Candida auris ( Candida auris Candida auris is a multidrug-resistant fungal pathogen. However, most medical institutions have not yet established active screening mechanisms for this pathogen, and the actual number of infections may be severely underestimated. Candida auris has the ability to survive for extended periods on patient skin and surfaces in the healthcare environment, and can be transmitted indirectly through direct contact or contaminated surfaces, easily causing nosocomial outbreaks. Currently, commonly used identification methods for invasive Candida include microscopy, histopathological examination, and culture. Microscopy and histopathological examination have low positive rates and may result in false negatives; culture methods are time-consuming, require high levels of technical expertise and operator skill, and because Candida auris shares high homology with Candida hemorrhagicum, identification using culture alone may lead to errors; serological testing can only determine the presence of a fungal infection, but cannot differentiate between fungal species. Therefore, it is essential to develop an accurate and rapid method for detecting Candida auris. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a primer-probe combination, kit, and application for digital PCR detection of Candida auris. The primer-probe combination provided by this invention enables accurate and rapid detection of Candida auris.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a primer-probe combination for detecting Candida auris, comprising: primers and probes for detecting Candida auris and primers and probes for detecting an internal reference gene. The nucleotide sequence of the upstream primer for detecting Candida auris is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5; the nucleotide sequence of the probe for detecting Candida auris is shown in SEQ ID NO.6. The nucleotide sequence of the upstream primer for detecting the internal reference gene is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8; the nucleotide sequence of the probe for detecting the internal reference gene is shown in SEQ ID NO.9. The probes are all labeled with fluorescent groups and quenching groups, and the fluorescent groups labeled on the probes for detecting Candida auris and the internal reference gene are different.

[0005] Preferably, the fluorescent groups labeled on different probes are selected from any one of ATTO, FAM, VIC, ROX, CY5 or CY5.5.

[0006] Preferably, the quenching groups labeled on different probes are selected from any one of BHQ1, BHQ2, BHQ3 or MGB.

[0007] Preferably, the probe for Candida auris is labeled with the fluorescent group FAM at its 5' end and the quencher group BHQ1 at its 3' end; the probe for the internal reference gene is labeled with the fluorescent group ROX at its 5' end and the quencher group BHQ2 at its 3' end.

[0008] This invention provides a kit for detecting Candida auris, comprising the primer-probe combination described in the above technical solution.

[0009] Preferably, the kit further includes digital PCR reaction buffer and nuclease-free water; the digital PCR reaction buffer contains Taq DNA polymerase, UDG enzyme, dNTPs, and Mg. 2+ One or more of the following in water.

[0010] This invention provides the application of the primer-probe combination or the kit described in the above-described technical solutions in the detection of Candida auris for non-diagnostic and non-therapeutic purposes.

[0011] Preferably, the detection method includes: Using the DNA of the sample to be tested as a template, digital PCR amplification was performed using the primer and probe combination described in the above technical solution. The fluorescence signal of the PCR amplification product was collected, and the results were quantified and analyzed. If the fluorescent signal on the probe of Candida auris is positive and the fluorescent signal on the probe of the internal reference gene is positive, then the sample to be tested contains Candida auris. If there are no positive fluorescent signals on the probe of the internal reference gene, the test is considered abnormal and needs to be repeated.

[0012] Preferably, the working concentrations of different probes are 150-300 nM; the working concentrations of the upstream primers of different primers are 300-600 nM; and the working concentrations of the downstream primers of different primers are 300-600 nM.

[0013] Preferably, the reaction program for digital PCR amplification includes: 95℃ for 5 min; 95℃ for 20 s, 54℃ for 30 s, for 45 cycles.

[0014] Beneficial effects: The primer-probe combination provided by this invention is designed with the conserved genetic site ITS of Candida auris as the target gene for amplification. It can be used for digital PCR detection of Candida auris, enabling rapid, efficient, sensitive, and specific quantitative detection of Candida auris. Using the primer-probe combination provided by this invention in conjunction with a dPCR platform, the reaction system is divided into tens of thousands of independent reaction units, greatly improving the sensitivity of the reaction and enabling rapid detection (the total time from reaction system preparation to result analysis is approximately 2 hours). Simultaneously, it can absolutely quantify the copy number of Candida auris in the sample. It achieves rapid detection of Candida auris in samples such as sputum, bronchoalveolar lavage fluid, urine, and blood. The detection process requires no culture, has a short detection cycle, greatly reduces manpower, and improves detection efficiency. In addition, the primers and probes provided by this invention can accurately detect the content of Candida auris in the sample at extremely low copy numbers, which is more sensitive and accurate than other quantitative detection methods. It has high detection sensitivity, does not require the setting of a standard curve, and can directly interpret the nucleic acid copy number of Candida auris in the sample based on the detection results, which greatly simplifies the operation steps. No culture is required, which reduces the workload of personnel and shortens the detection cycle, making it suitable for rapid detection of clinical samples. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly introduced below.

[0016] Figure 1 Example diagram for interpreting digital PCR results; Figure 2 These are the results of primer and probe screening. Figure 3 The results of the primer and probe working concentration screening; Figure 4 This is a specific test result for Candida auris; Figure 5 The results of the Candida auris sensitivity test; Figure 6 These are the results of strain testing for clinical samples. Detailed Implementation

[0017] This invention provides a primer-probe combination for detecting Candida auris, comprising: primers and probes for detecting Candida auris and primers and probes for detecting an internal reference gene. The nucleotide sequence of the upstream primer for detecting Candida auris is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5; the nucleotide sequence of the probe for detecting Candida auris is shown in SEQ ID NO.6. The nucleotide sequence of the upstream primer for detecting the internal reference gene is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8; the nucleotide sequence of the probe for detecting the internal reference gene is shown in SEQ ID NO.9. The probes are all labeled with fluorescent groups and quenching groups, and the fluorescent groups labeled on the probes for detecting Candida auris and the internal reference gene are different.

[0018] In one implementation, the fluorescent groups labeled on different probes are selected from any one of ATTO, FAM, VIC, ROX, CY5, or CY5.5.

[0019] In one implementation, the quenching groups labeled on different probes are selected from any one of BHQ1, BHQ2, BHQ3 or MGB.

[0020] In one embodiment, the probe of the Candida auris is labeled with the fluorescent group FAM at its 5' end and the quencher group BHQ1 at its 3' end; the probe of the internal reference gene is labeled with the fluorescent group ROX at its 5' end and the quencher group BHQ2 at its 3' end.

[0021] The primer-probe combination provided by this invention is a primer-probe combination designed with the conserved genetic site ITS of Candida auris as the target gene for amplification. It can be used for digital PCR detection of Candida auris, and can directly quantify Candida auris with high specificity (it can distinguish other Candida species and other closely related bacteria). It can detect the content of Candida auris in the test sample at extremely low copy numbers (1 copies / μL). It can accurately quantify Candida auris in environmental samples without the need for a standard curve. It is suitable for early clinical diagnosis, hospital infection monitoring and rapid testing at the grassroots level, and provides precise technical support for the prevention and control of multidrug-resistant Candida auris.

[0022] Based on the above advantages, the present invention provides a kit for detecting Candida auris, comprising the primer and probe combination described in the above technical solution.

[0023] In one embodiment, the kit further includes digital PCR reaction buffer and nuclease-free water; the digital PCR reaction buffer contains Taq DNA polymerase, UDG enzyme, dNTPs, and Mg. 2+ One or more of the following in water.

[0024] Based on the above advantages, this invention provides the application of the primer-probe combination or the kit described in the above technical solution in the detection of Candida auris for non-diagnostic and non-therapeutic purposes. The detection can be for non-diagnostic and non-therapeutic purposes, merely to obtain an intermediate value indicating whether the sample contains Candida auris, and to quantify the concentration of Candida auris in the sample. The sample can be food or soil from the environment, etc.

[0025] As one implementation method, the detection method includes: Using the DNA of the sample to be tested as a template, digital PCR amplification was performed using the primer and probe combination described in the above technical solution. The fluorescence signal of the PCR amplification product was collected, and the results were quantified and analyzed. If the fluorescent signal on the probe of Candida auris is positive and the fluorescent signal on the probe of the internal reference gene is positive, then the sample to be tested contains Candida auris. If there are no positive fluorescent signals on the probe of the internal reference gene, the test is considered abnormal and needs to be repeated.

[0026] In one implementation method, the working concentrations of different probes are 150-300 nM; the working concentrations of the upstream primers of different primers are 300-600 nM; and the working concentrations of the downstream primers of different primers are 300-600 nM.

[0027] As one implementation, the digital PCR amplification reaction program includes: 95℃ for 5 min; 95℃ for 20 s, 54℃ for 30 s, for 45 cycles.

[0028] In one implementation, the sample to be tested may include one or more of sputum, bronchoalveolar lavage fluid, urine, and blood.

[0029] As one implementation method, such as Figure 1 As shown, the fluorescence signal of the PCR amplification product is presented as a two-dimensional scatter plot. The upper shaded area represents the gray value (brightness) of positive points, and the lower shaded area represents the gray value (brightness) of negative points. A high gray value for positive points and a low gray value for negative points, i.e., a large gap between positive and negative points, indicates a good effect. When the gray values ​​of negative points are consistent, a larger gray value for positive points indicates a larger gap between positive and negative points, indicating even better results.

[0030] As one implementation method, instruments for collecting fluorescence signals of PCR amplification products and performing quantitative and analytical results include digital PCR readers. The instrument system calculates the true concentration of the sample according to the statistical formula Poisson distribution and outputs the results in copies / μL.

[0031] To further illustrate the present invention, the primer and probe combination, kit, and application for digital PCR detection of Candida auris provided by the present invention are described in detail below with reference to embodiments and accompanying drawings, but these should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1 The genetically conserved site ITS was selected as the target gene for Candida auris amplification. Primers were manually designed for conserved sequence regions within the species, and probes were manually designed for variable regions between species. Two pairs of primers and probes were designed. The internal reference gene was the conserved LBD sequence of the model plant Arabidopsis thaliana. Primer and probe sequence information and their corresponding numbers are shown in Table 1.

[0033] Table 1 Primer and probe sequence information

[0034] Among them, probe EN-P1 is labeled with the fluorescent group FAM at its 5' end and the quencher group MGB at its 3' end; probe EN-P2 is labeled with the fluorescent group FAM at its 5' end and the quencher group BHQ1 at its 3' end; probe P internal reference is labeled with the fluorescent group ROX at its 5' end and the quencher group BHQ2 at its 3' end.

[0035] The primers and probes of Candida auris strain in Table 1 were used to form combination 1 (EN-F1, EN-R1 and EN-P1) and combination 2 (EN-F2, EN-R2 and EN-P2).

[0036] The detection method is as follows: 1) Provide the nucleic acid sample to be tested; 2) The PCR reaction system was prepared using primer and probe combinations 1 and 2 respectively, as follows: 1.5 μL of dPCR premix (10×), 1.5 μL of primer-probe mix (10×), 7.5 μL of sample DNA, and 15 μL of nuclease-free water; the working concentrations of primers and probes were 500 nM and 250 nM respectively.

[0037] 3) Load the reaction system described in step 2) using an electric pipette to distribute 13 μL of the reaction system evenly onto a 20,000-well chip.

[0038] 4) Digital PCR workflow.

[0039] Amplification and reading were performed using the AccuAuto Pro all-in-one digital PCR platform manufactured by Accubio (Shanghai) Co., Ltd.

[0040] Amplification: Place the prepared chip on the AccuAuto Pro all-in-one machine and perform PCR amplification according to the reaction conditions in Table 2.

[0041] Table 2 dPCR amplification program

[0042] Note: PCR reaction solution with added samples or chips that have been loaded should be used for experiments immediately. If, due to special reasons, the PCR reaction solution or chips that have been loaded cannot be used for experiments in time, they should be stored at 4°C for no more than 12 hours.

[0043] Chip reading and analysis: The expanded chip is automatically read and analyzed.

[0044] 5) Result Judgment Criteria: The reading results are presented as a two-dimensional scatter plot, such as... Figure 1 As shown, the upper part represents the grayscale value (brightness) of positive points, and the lower shaded area represents the grayscale value (brightness) of negative points. A high grayscale value for positive points and a low grayscale value for negative points, i.e., a large gap between positive and negative points, indicates a good effect. When the grayscale values ​​of negative points are the same, a larger grayscale value for positive points results in a larger gap between positive and negative points, indicating an even better effect.

[0045] Fixed value: The instrument system will calculate the true concentration of the sample according to the statistical formula Poisson distribution and output the result in copies / μL.

[0046] Quality control: An internal reference gene primer probe is introduced into the reaction system for each test. A positive result will always be found in the ROX channel in each output. If no positive result is found, the test is invalid and must be repeated.

[0047] 6) Interpretation of test results: If both the fluorescence channel and ROX channel of the Candida auris probe show a positive spot, the sample is determined to contain Candida auris; if the fluorescence channel of the Candida auris probe shows no positive spot but the ROX channel shows a positive spot, the sample is determined to be free of Candida auris; if both the fluorescence channel and ROX channel of the Candida auris probe show no positive spot, the test is considered abnormal and needs to be repeated.

[0048] The primer-probe combination test results are shown below Figure 2 Select its corresponding combination 2.

[0049] Example 2 In this embodiment, based on the *Candida auris* primer-probe combination 2 and the primer-probe for the internal reference gene determined in Example 1, the working solution concentrations of the above primers and probes were screened. Four gradients were set for the probes (150 nM, 200 nM, 250 nM, 300 nM), and four gradients were set for the primers (300 nM, 400 nM, 500 nM, 600 nM) for digital PCR testing. Results are shown below. Figure 3 The numbers from left to right represent probe concentrations of 150 nM, 200 nM, 250 nM, and 300 nM, and primer concentrations of 300 nM, 400 nM, 500 nM, and 600 nM, respectively, when the target is used as the sample to be tested.

[0050] from Figure 3 The results showed that, based on the instrument interpretation criteria, the gap value between positive and negative points was largest when the probe working concentration was 250 nM and the primer concentration was 500 nM. Ultimately, the working concentrations of the Candida auris and internal reference gene probes were selected as 250 nM and 500 nM, respectively.

[0051] Example 3: Specificity test of Candida auris kit In this embodiment, specificity testing was performed based on the primer-probe combination and optimal working concentration of the probe determined in Examples 1 and 2. The test strains are as follows: Pichia pastoris (fermented yeast) Pichiafermentans CICC32388), Hansenula polymorpha ( Debaryomyces hanserill CICC1266), Omkoda yeast ( Kodamaea ohmeri CICC32993), Candida dublin ( Candids dublinensis NCPF3949), N1 [Staphylococcus aureus ( Staphylococcus aureus ATCC25923), Serratia marcescens ( Serratia marcescens ATCC 8100), Corynebacterium bandingense ( Corynebacterium striatum ATCC BAA-1293), Enterococcus faecalis ( Enterococcus faecalis ATCC 29212)], N2 [Klebsiella pneumoniae ( Klebsiella pneumoniae ATCC 700603), Staphylococcus epidermidis ( Staphylococcus epidermidis ATCC 12228 ） Acinetobacter baumannii ( Acinetobacter baumannii ATCC 19606), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ATCC 27853), Gert's Hidden Sphere ( Cryptococcus gattii [ATCCMYA-45260] and five Candida species [Candida glabrata ( , ATCCMYA-45260) ... Candida glabrata, MY2950), Candida krusei ( Candida krusei, ATCC 6258), Candida tropicalis ( Candida tropicalis, ATCC 1369), Candida albicans ( Candidaalbicans, ATCC 14053), Candida parapsilosis ( Candida parapsilosis [ATCC22019], N1, N2, and five Candida species are mixed strains. Except for Candida dublin, all of the above strains were purchased from […]. Microbiologics, Lnc All other strains were purchased from the China Industrial Microbial Culture Collection Center.

[0052] Test results are available Figure 4And Table 3.

[0053] Table 3 Results of Candida auris specificity test

[0054] The results showed that the primer-probe combination provided by this invention did not amplify in other fungi and bacteria, demonstrating good specificity.

[0055] Example 4: Sensitivity test of the Candida auris kit In this embodiment, sensitivity testing was performed based on the primer-probe combination and optimal working concentration of the probe determined in Examples 1 and 2. The test content is as follows: The *Candida auris* nucleic acid at a concentration of 40 copies / μL was diluted 100-fold to ensure the final concentration was below the limit of quantitation (1 copy / μL) of the Zhenzhun Bio digital PCR platform. The reaction solution without sample addition was prepared according to the method in Example 1. 7.5 μL of the reaction system was aliquoted into EP tubes, and then 7.5 μL of the sample nucleic acid to be tested was added to each tube. Ten parallel tests were performed for each group. The detection results are shown below. Figure 5 The detection rate for all samples was 100%.

[0056] The results showed that the detection limits of the primer and probe sets of the detection kit for Candida auris of the present invention were all no higher than 1.0 copies / μL, indicating that the kit has high sensitivity and can detect samples with very low template content normally.

[0057] Example 5: Detection of clinical sample strains using the Candida auris kit. In this embodiment, clinical sample strains were tested based on the primer-probe combination and optimal working concentration of the probes determined in Examples 1 and 2. The test content is as follows: (1) Eleven strains were isolated from the clinical samples to be tested. Each sample was tested in parallel, resulting in a total of 11 test results. (2) Extract nucleic acid from the sample to be tested using a fully automated nucleic acid extraction instrument; (3) Prepare the reaction solution without adding the sample according to the method of Example 1, take 7.5 μL of the reaction system and dispense it into EP tubes, and then add 7.5 μL of the nucleic acid of the sample to be tested. (4) Perform dPCR detection according to the digital PCR workflow in Example 1.

[0058] See results Figure 6 The samples to be tested are numbered 1 to 11 from left to right.

[0059] The results showed that the primer-probe combination for Candida auris provided by this invention could successfully detect the Candida auris pathogen in the test samples. This is consistent with the results of known first-generation sequencing.

[0060] Sample 1 tested showed the presence of Candida auris at a concentration of 116.19 copies / μL; Sample 2 tested showed the presence of Candida auris at a concentration of 77.99 copies / μL; Sample 3 tested showed the presence of Candida auris at a concentration of 87.68 copies / μL; Sample 4 tested showed the presence of Candida auris at a concentration of 188.91 copies / μL; Sample 5 tested showed the presence of Candida auris at a concentration of 150.07 copies / μL; Sample 6 tested showed the presence of Candida auris at a concentration of 211.37 copies / μL; Sample 7 tested showed the presence of Candida auris at a concentration of 161.41 copies / μL; Sample 8 tested showed the presence of Candida auris at a concentration of 123.02 copies / μL; Sample 9 tested showed the presence of Candida auris at a concentration of 187.14 copies / μL; Sample 10 tested showed the presence of Candida auris at a concentration of 470.34 copies / μL; Sample 11 tested showed the presence of Candida auris at a concentration of 247.28 copies / μL.

[0061] In summary, the primer-probe combination provided by this invention enables rapid, efficient, sensitive, and specific quantitative detection of *Candida auris*. Based on the dPCR platform, the reaction system is divided into tens of thousands of independent reaction units, greatly improving the sensitivity of the reaction and enabling rapid detection (the total time from setting up the reaction system to analyzing the results is approximately 2 hours). It also allows for absolute quantification of the *Candida auris* copy number in the sample. This invention enables rapid detection of *Candida auris* in samples such as sputum, bronchoalveolar lavage fluid, urine, and blood. The detection process requires no culture, has a short cycle, significantly reduces manpower, and improves detection efficiency. The primer-probe combination provided by this invention can accurately detect the content of *Candida auris* in the sample at extremely low copy numbers, making it more sensitive and accurate than other quantitative detection methods. Its high sensitivity eliminates the need for a standard curve; the nucleic acid copy number of *Candida auris* in the sample can be directly interpreted based on the detection results, greatly simplifying the operation. The absence of culture reduces workload and shortens the detection cycle, making it suitable for rapid detection of clinical samples.

[0062] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting Candida auris ( Candida auris The primer-probe combination is characterized by, include: Primers and probes for detecting Candida auris and primers and probes for detecting the internal reference gene: The nucleotide sequence of the upstream primer for detecting Candida auris is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5; the nucleotide sequence of the probe for detecting Candida auris is shown in SEQ ID NO.

6. The nucleotide sequence of the upstream primer for detecting the internal reference gene is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8; the nucleotide sequence of the probe for detecting the internal reference gene is shown in SEQ ID NO.

9. The probes are all labeled with fluorescent groups and quenching groups, and the fluorescent groups labeled on the probes for detecting Candida auris and the internal reference gene are different.

2. The primer-probe combination according to claim 1, characterized in that, The fluorescent groups labeled on different probes are selected from any one of ATTO, FAM, VIC, ROX, CY5, or CY5.

5.

3. The primer-probe combination according to claim 1, characterized in that, The quenching groups labeled on different probes are selected from any one of BHQ1, BHQ2, BHQ3 or MGB.

4. The primer-probe combination according to any one of claims 1-3, characterized in that, The probe for Candida auris is labeled with the fluorescent group FAM at its 5' end and the quencher group BHQ1 at its 3' end; the probe for the internal reference gene is labeled with the fluorescent group ROX at its 5' end and the quencher group BHQ2 at its 3' end.

5. A kit for detecting Candida auris, characterized in that, Includes the primer-probe combination as described in any one of claims 1-4.

6. The reagent kit according to claim 5, characterized in that, The kit also includes digital PCR reaction buffer and nuclease-free water; the digital PCR reaction buffer contains Taq DNA polymerase, UDG enzyme, dNTPs, and Mg. 2+ One or more of the following in water.

7. The use of the primer-probe combination according to any one of claims 1-4 or the kit according to claim 5 or 6 in the detection of Candida auris for non-diagnostic and non-therapeutic purposes.

8. The application according to claim 7, characterized in that, The detection method includes: Using the DNA of the sample to be tested as a template, digital PCR amplification is performed using the primer and probe combination described in any one of claims 1-4, and the fluorescence signal of the PCR amplification product is collected and the results are quantified and analyzed. If the fluorescent signal on the probe of Candida auris is positive and the fluorescent signal on the probe of the internal reference gene is positive, then the sample to be tested contains Candida auris. If there are no positive fluorescent signals on the probe of the internal reference gene, the test is considered abnormal and needs to be repeated.

9. The application according to claim 8, characterized in that, The working concentrations of different probes were 150-300 nM; the working concentrations of the upstream primers of different primers were 300-600 nM; and the working concentrations of the downstream primers of different primers were 300-600 nM.

10. The application according to claim 8, characterized in that, The digital PCR amplification reaction program includes: 95℃ for 5 min; 95℃ for 20 s, 54℃ for 30 s, for 45 cycles.

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