Method for quantitatively detecting chlamydia psittaci based on microdroplet type digital PCR (Polymerase Chain Reaction)

Abstract

The invention relates to the technical field of pathogenic microorganism detection, in particular to a method for quantitatively detecting chlamydia psittaci based on microdroplet type digital PCR (Polymerase Chain Reaction). The invention discloses a primer probe composition for detecting chlamydia psittaci. The primer probe composition consists of a forward primer, a reverse primer and a probe, the nucleotide sequence of the forward primer is as shown in SEQ ID NO: 1; the nucleotide sequence of the reverse primer is as shown in SEQ ID NO: 2; the nucleotide sequence of the probe is as shown in SEQ ID NO: 3. The detection method provided by the invention is high in sensitivity, strong in specificity, rapid in detection and simple and convenient to operate, the used primer pair and probe are optimally designed based on sequence site difference, the detection background value is low, and the result is easier to judge.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogenic microorganism detection, and particularly relates to a method for quantitatively detecting Chlamydia psittaci based on droplet digital PCR. Background Art

[0002] Chlamydia psittaci (C. psittaci) is a Gram-negative prokaryotic microorganism that parasitizes intracellularly and belongs to the genus Chlamydia in the family Chlamydiaceae. Its hosts include poultry, wild birds, humans, etc. It is the main chlamydia that causes avian chlamydiosis and human chlamydiosis, and is also a chlamydia that has the greatest impact on production economy. Since the 1930s, C. psittaci has been widely spread and has caused devastating blows to poultry farms. Currently, reports of human-to-human transmission and environmental contact transmission of C. psittaci have emerged, which poses risks to students, veterinarians, and staff during their work. Therefore, accurate and rapid detection of C. psittaci has become crucial.

[0003] Nowadays, the diagnosis and prevention and control of C. psittaci are not ideal. The reasons are as follows: First, the clinical manifestations of C. psittaci are difficult to detect and judge; second, there is a lack of accurate and rapid laboratory diagnostic methods; third, it is difficult to distinguish C. psittaci from other types of chlamydia in the same family. The commonly used diagnostic method in current laboratories is qPCR detection. However, the sensitivity of qPCR is relatively low, and there are often large deviations between the detection of early infection and low-abundance nucleic acids of C. psittaci and the actual situation. Droplet Digital PCR (ddPCR) technology is a technology that divides the PCR system into tens of thousands of droplets, performs PCR amplification on each droplet, and detects the number of droplets with fluorescent signals, which can achieve rapid and accurate absolute quantitative detection of nucleic acids.

[0004] In summary, the rapid, accurate detection and identification of C. psittaci are of great significance for etiological research, epidemiological investigation, and the production safety of related industries. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for quantitatively detecting Chlamydia psittaci based on droplet digital PCR. The detection method provided by the present invention has high sensitivity, strong specificity, rapid detection, simple operation, and the primer pairs and probes used are optimized and designed based on sequence site differences, with a low detection background value and being more conducive to the determination of results.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a primer-probe composition for detecting Chlamydia psittaci, which consists of a forward primer, a reverse primer, and a probe;

[0008] The nucleotide sequence of the forward primer is as shown in SEQ ID NO: 1;

[0009] The nucleotide sequence of the reverse primer is as shown in SEQ ID NO: 2;

[0010] The nucleotide sequence of the probe is as shown in SEQ ID NO: 3.

[0011] Preferably, the 5'-ends of the forward primer and the reverse primer are labeled with the phosphoramidite dye VIC; the 3'-end of the probe is labeled with the quenching group BHQ1.

[0012] The present invention also provides the application of the primer-probe composition in the preparation of a reagent for detecting Chlamydia psittaci.

[0013] Preferably, the detection reagent is a kit.

[0014] The present invention also provides a kit for detecting Chlamydia psittaci, which contains the primer-probe composition.

[0015] The present invention also provides a droplet digital PCR detection method for Chlamydia psittaci disease for non-diagnostic and therapeutic purposes, including the following steps:

[0016] (1) Extract the DNA of the object to be detected as a template;

[0017] (2) Use the primer-probe composition to perform a ddPCR reaction on the DNA obtained in step (1), and detect the fluorescence signal of each microreaction unit; if the droplet emits a fluorescence signal, it indicates that the droplet contains the target DNA, and the result is determined to be positive; if no fluorescence signal is emitted, it indicates that the droplet does not contain the target DNA, and the result is determined to be negative.

[0018] Preferably, the system of the ddPCR reaction is: a total of 22 μL per tube, containing 11 μL of 2×ddPCR Supermix for probes ddPCR reaction solution, 1 μL each of 10 μmol / mL forward primer and reverse primer, 0.6 μL of 10 μmol / mL probe, 7.4 μL of DEPC water, and 1 μL of DNA template.

[0019] Preferably, the amplification system of the ddPCR reaction is: the heating and cooling rate is 2°C / s, the annealing temperature is 60°C for 5 min, and 40 cycles.

[0020] Compared with the prior art, based on the droplet digital PCR technology, the present invention designs primers, probes and establishes a detection method for the CPSIT 0429 gene of Chlamydia psittaci, and realizes the detection of Chlamydia psittaci by monitoring the fluorescence signals generated by each micro-reaction unit, having the following beneficial effects:

[0021] (1) High sensitivity: The lowest detection copy value of this method is 0.32 copies / μL.

[0022] (2) Strong specificity: This method can specifically identify the C.psittaci nucleic acid sequence and does not produce cross-reactions with Chlamydia abortus, Brucella, Mycobacterium bovis, Listeria, Staphylococcus aureus, and Salmonella.

[0023] (3) Rapid detection: The entire amplification and detection process can be completed within 2 hours.

[0024] (4) Simple operation: After placing the corresponding consumables in the instrument, putting the prepared ddPCR reaction system into the digital PCR instrument can complete the entire amplification and result determination process, without the need to interpret the Ct value and identify by agarose gel electrophoresis. At the same time, the used primer pairs and probes are optimized based on sequence site differences, with a low detection background value and being more conducive to result determination. Description of the Drawings

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0026] Figure 1 ddPCR amplification diagrams with different primer concentrations; among them, 1A and 1B are the detection results at 10 μmol / mL; 1C and 1D are the detection results at 20 μmol / mL; 1E and 1F are the detection results at 30 μmol / mL; 1G and 1H are the detection results at 50 μmol / mL.

[0027] Figure 2 ddPCR amplification diagrams with different probe concentrations; among them, 1A and 1B are the detection results at 10 μmol / mL; 1C and 1D are the detection results at 30 μmol / mL; 1E and 1F are the detection results at 50 μmol / mL.

[0028] Figure 3 Sensitivity test results; among them, 1A is the detection result with 10 3 positive droplets / μL; 1B is the detection result with 10 2Detection results of copies / μL; 1C is the number of positive droplets 10 1 Detection results of copies / μL; 1D is the number of positive droplets 10 0 Detection results of copies / μL; 1E is the number of positive droplets 10 -1 Detection results of copies / μL; 1F is the negative control with DEPC water as the template.

[0029] Figure 4 Are the specific test results; among them, 2A: Chlamydia abortus; 2B: Brucella; 2C: Mycobacterium bovis; 2D: Listeria; 2E: Staphylococcus aureus; 2F: Salmonella; 2G: Chlamydia psittaci; 2H: DEPC water. Detailed implementation manners

[0030] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0031] Embodiment 1: Primer and probe design

[0032] Based on the droplet digital PCR technology, the present invention designs primers, probes and establishes a detection method for the CPSIT0429 gene of Chlamydia psittaci, and realizes the detection of Chlamydia psittaci by monitoring the fluorescence signals generated by each micro-reaction unit.

[0033] According to the Chlamydia psittaci gene sequence downloaded from NCBI, specific primers and probes were designed for the CPSIT0429 gene using SnapGene software.

[0034] Table 1 Primer and probe sequence information

[0035]

[0036] Among them, the 5' end of the primer pair for detecting Chlamydia psittaci is labeled with the phosphoramidite dye VIC, and the 3' end of the probe is labeled with the quenching group BHQ1.

[0037] Embodiment 2: Sensitivity of primers and probes

[0038] 1. Nucleic acid DNA preparation

[0039] The nucleic acid DNA of Chlamydia psittaci was provided by the China Animal Health and Epidemiology Center, and there were two different nucleic acid samples A and B, both of which were pigeon swabs from Jiangsu.

[0040] After measurement, the initial copy number of nucleic acid sample A was 1×10 4 copies / μL, and the Chlamydia psittaci nucleic acid was serially diluted to obtain 5 concentration working standards, namely working standard 1, containing 1×103 copies / μL positive nucleic acid; Working standard 2, containing 1×10 2 copies / μL positive nucleic acid; Working standard 3, containing 1×10 1 copies / μL positive nucleic acid; Working standard 4, containing 1×10 0 copies / μL positive nucleic acid; Working standard 5, containing 1×10 -1 copies / μL positive nucleic acid. Nucleic acid sample B is used as working sample 6, containing 1×10 3 copies / μL positive nucleic acid.

[0041] Establishment of reaction system: Each tube of the reaction solution is 22 μL, containing 11 μL of 2×ddPCR Supermix for probes ddPCR reaction solution; upstream and downstream primers with different concentrations: 10 μmol / mL, 20 μmol / mL, 30 μmol / mL, 50 μmol / mL, 1 μL; probes with different concentrations: 10 μmol / mL, 30 μmol / mL, 50 μmol / mL, 0.6 μL; DEPC water: 7.4 μL.

[0042] Amplification conditions: The heating and cooling rate is set to 2°C / s, the annealing temperature recommended by the instrument is 60°C for 5 min, 40 cycles, and the amplification reaction is carried out. Each program is detected 2 times. After the reaction, the DNA copy number is read. The reaction is carried out in a Sniper digital PCR all-in-one machine DQ 24, and the detection results at different primer and probe concentrations are as follows Figure 1 、 Figure 2 shown. According to Figure 1 and Figure 2 it can be seen that among different primer and probe concentration gradients, the difference in the number of detected fluorescent droplets is small, indicating that the primer and probe concentrations do not affect the detection results.

[0043] 3. Sensitivity test

[0044] Using the primers and probes screened in Example 1 to prepare the reaction system and detect the working standards. The specific steps are as follows:

[0045] 1) Configuration of ddPCR reaction system: Each tube of the reaction solution is 22 μL, containing 11 μL of 2×ddPCR Supermix for probes ddPCR reaction solution, 1 μL each of 10 μmol / mL C.psittaci forward and C.psittaci reverse, 0.6 μL of 10 μmol / mL C.psittaci probe, 7.4 μL of DEPC water, and 1 μL of the DNA template to be detected.

[0046] 2) ddPCR reaction amplification system: The heating and cooling rate is set to 2°C / s, the annealing temperature recommended by the instrument is 60°C for 5 min, and 40 cycles are performed for the amplification reaction.

[0047] 3) Result determination: If the droplet emits a fluorescence signal, it indicates that the droplet contains the target DNA, and the result is determined as positive; if no fluorescence signal is emitted, it means that the droplet does not contain the target DNA, and the result is determined as negative. After completing the PCR amplification, the fluorescence signal of each microreaction unit is detected, and the absolute copy number of the template is calculated according to the Poisson distribution.

[0048] Working standards 1 - 5 were used to evaluate the sensitivity of this method for detecting Chlamydia psittaci. The results are as Figure 3 shown. Positive droplets appeared in working standards 1 - 4, and the number of droplets decreased. No positive droplets appeared in working standard 5. The results indicate that the lowest detection limit of Chlamydia psittaci by this method is working standard 4, and at this time, the total number of detected positive droplets is 7, that is, 0.32 copies / μL.

[0049] Example 3: Specificity test

[0050] The reaction system was prepared using the primers and probes designed in Example 1, and nucleic acid samples of Chlamydia abortus, Brucella, Mycobacterium bovis, Listeria, Staphylococcus aureus, Salmonella, and Chlamydia psittaci were used as templates for detection. The specific steps are as follows:

[0051] Preparation of specific samples: Nucleic acid samples of Chlamydia abortus, Brucella, Mycobacterium bovis, Listeria, Staphylococcus aureus, Salmonella, and Chlamydia psittaci were provided by the Zoonosis Surveillance Room of the China Animal Health and Epidemiology Center.

[0052] Detection of specific samples:

[0053] 1) Each tube of the reaction solution is 22 μL, containing 11 μL of 2×ddPCR Supermix for probes ddPCR reaction solution, 1 μL each of 10 μmol / mL C.psittaci forward and C.psittaci reverse, 0.6 μL of 10 μmol / mL C.psittaci probe, 7.4 μL of DEPC water, and 1 μL of the DNA template to be detected.

[0054] 2) ddPCR reaction amplification system: The heating and cooling rate is set to 2°C / s, the annealing temperature recommended by the instrument is 60°C for 5 min, and 40 cycles are performed for the amplification reaction.

[0055] 3) Result determination: If the droplet emits a fluorescence signal, it indicates that the droplet contains the target DNA, and the result is determined as positive; if no fluorescence signal is emitted, it indicates that the droplet does not contain the target DNA, and the result is determined as negative.

[0056] The results are as Figure 4 , and in this experiment, Chlamydia psittaci was detected as positive, and all other results were negative. The results indicate that this method can specifically identify the C. psittaci nucleic acid sequence and does not cross-react with Chlamydia abortus, Brucella, Mycobacterium bovis, Listeria, Staphylococcus aureus, and Salmonella.

[0057] Example 4: Repeatability test

[0058] Using the primers and probes designed in Example 1 to prepare the reaction system, the working standards 1 and 6 prepared in Example 2 were repeatedly detected by ddPCR three times, and the within-group coefficient of variation was calculated; the working standard 1 was repeatedly detected by ddPCR three times, and the between-group coefficient of variation was calculated.

[0059] The coefficient of variation CV% = (standard deviation SD / mean Mean) × 100%, and the between-group and within-group repeatability coefficients of variation were calculated. The specific steps are as follows:

[0060] Configuration of the ddPCR reaction system:

[0061] 1) Each tube of the reaction solution is 22 μL, containing 11 μL of 2×ddPCR Supermix for probes ddPCR reaction solution, 1 μL each of 10 μmol / mL C. psittaci forward and C. psittaci reverse, 0.6 μL of 10 μmol / mL C. psittaci probe, 7.4 μL of DEPC water, and 1 μL of the DNA template to be detected.

[0062] 2) ddPCR reaction amplification system: The heating and cooling rate is set to 2°C / s, the annealing temperature recommended by the instrument is 60°C for 5 min, and 40 cycles are carried out for the amplification reaction.

[0063] 3) Result determination: If the droplet emits a fluorescence signal, it indicates that the droplet contains the target DNA, and the result is determined as positive; if no fluorescence signal is emitted, it indicates that the droplet does not contain the target DNA, and the result is determined as negative.

[0064] The working standards 1 and 6 were repeatedly detected, and the results are shown in Table 2. The within-group coefficients of variation obtained were all less than 5%, indicating that this method has good within-group repeatability; the between-group coefficient of variation was less than 5%, indicating that this method has good between-group repeatability.

[0065] Table 2 Results of within-group and between-group repeatability tests

[0066]

[0067] Example 5: Detection of Clinical Samples

[0068] Prepare a reaction system using the primers and probes designed in Example 1, and detect 8 cotton swab samples collected from animals suspected of being infected with Chlamydia psittaci, and compare the results with those detected by the fluorescence PCR method recommended by the World Organization for Animal Health (WOAH). The specific steps are as follows:

[0069] Preparation of clinical samples: A total of 8 cotton swab samples from clinically suspected infected animals. The samples were extracted for nucleic acid by boiling and stored at -20°C for later use.

[0070] Configuration of ddPCR reaction system:

[0071] 1) Each tube of the reaction solution is 22 μL, containing 11 μL of 2×ddPCR Supermix for probes ddPCR reaction solution, 1 μL each of 10 μmol / mL C. psittaci forward and C. psittaci reverse, 0.6 μL of 10 μmol / mL C. psittaci probe, 7.4 μL of DEPC water, and 1 μL of the DNA template to be detected.

[0072] 2) ddPCR reaction amplification system: The heating and cooling rate is set to 2°C / s, the annealing temperature recommended by the instrument is 60°C for 5 min, and 40 cycles are carried out for the amplification reaction.

[0073] 3) Result determination: If the droplet emits a fluorescence signal, it indicates that the droplet contains the target DNA, and the result is determined to be positive; if no fluorescence signal is emitted, it indicates that the droplet does not contain the target DNA, and the result is determined to be negative.

[0074] The results are shown in Table 3. Using this method to detect 8 clinical samples suspected of being infected with Chlamydia psittaci, the results are 2 positive and 6 negative. The detection rate is the same as that of the fluorescence PCR detection result, indicating that this method can effectively detect clinical samples.

[0075] Table 3 Detection results of clinical samples

[0076]

[0077] In summary, this study successfully established a highly sensitive and specific detection method for Chlamydia psittaci based on droplet digital PCR technology. This method does not cross-react with other pathogens when detecting Chlamydia psittaci and can be used to distinguish Chlamydia abortus from Chlamydia psittaci.

[0078] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A primer-probe composition for detecting Chlamydia psittaci, characterized in that: It consists of a forward primer, a reverse primer and a probe; The nucleotide sequence of the forward primer is shown in SEQ ID NO: 1; The nucleotide sequence of the reverse primer is shown in SEQ ID NO: 2; The nucleotide sequence of the probe is shown in SEQ ID NO:

3.

2. The primer-probe combination according to claim 1, characterized in that The 5' ends of the forward primer and the reverse primer are labeled with the phosphoramidite dye VIC; the 3' end of the probe is labeled with the quenching group BHQ1.

3. Use of the primer-probe combination according to claim 1 or 2 in the preparation of a Chlamydia psittaci detection reagent.

4. The use according to claim 3, characterized in that: The detection reagent is a kit.

5. A kit for detecting Chlamydia psittaci, characterized in that: The kit contains the primer-probe combination according to claim 1 or 2.

6. A droplet digital PCR detection method for psittacosis chlamydia for non-diagnostic and therapeutic purposes, characterized in that: The steps include: (1) extracting DNA of the object to be detected as a template; (2) The DNA obtained in step (1) is subjected to a ddPCR reaction using the primer-probe combination described in claim 1 or 2, and the fluorescent signal of each microreaction unit is detected; if the droplet emits a fluorescent signal, it indicates that the droplet contains the target DNA, and the result is judged as positive; if no fluorescent signal is emitted, it indicates that the droplet does not contain the target DNA, and the result is judged as negative.

7. The detection method according to claim 6, characterized in that: The ddPCR reaction system is as follows: each tube contains 22 μL in total, 11 μL of 2×ddPCR Supermix for probes ddPCR reaction solution, 1 μL of 10 μmol / mL forward primer and 1 μL of reverse primer, 0.6 μL of 10 μmol / mL probe, 7.4 μL of DEPC water, and 1 μL of DNA template.

8. The detection method according to claim 6, characterized in that: The amplification system of the ddPCR reaction is: heating and cooling rate 2°C / s, annealing temperature 60°C for 5 min, and 40 cycles.

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