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

Abstract

The invention relates to the technical field of molecular biological detection. The invention provides a primer pair and a probe for detecting chlamydia abortus, and further provides a kit which comprises the primer pair and the probe and is used for quantitatively detecting the chlamydia abortus through micro-droplet digital PCR, and a non-diagnostic method for quantitatively detecting the chlamydia abortus based on the micro-droplet digital PCR. And negative and positive results are judged according to the fluorescence signal condition of the microdroplet in the amplification product. According to the technical scheme, rapid and effective identification of the chlamydia abortus disease can be achieved, cross reaction with other common pathogens can be avoided while the chlamydia abortus is detected, and the method has good specificity, good repeatability and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biological detection, and in particular to a method for quantitatively detecting Chlamydia abortus based on droplet digital PCR. Background Art

[0002] Chlamydia abortus (C. abortus) is a common pathogen of endemic abortion in small ruminants. It can not only cause disease in common livestock such as cattle, sheep, and pigs, but also cause infection in a variety of wild animals and special economic animals, and even infect marine mammals. However, there are no typical clinical symptoms in the early stages of infection, so it is often overlooked, resulting in abortion, stillbirth, and weak offspring in the late stages of pregnancy in affected animals. Infected animals can become a source of infection and infect humans, causing similar symptoms, leading to miscarriage, premature birth, or stillbirth in pregnant women. Chlamydia abortus is usually transmitted through contact with secretions or contaminants of infected animals, but due to the non-specificity of its symptoms, it is difficult to make an accurate diagnosis in the early stages of infection, which poses a challenge to disease prevention and control.

[0003] As a type of obligate intracellular parasite, Chlamydia abortus can survive in host cells, latently infect and escape immunity, and can also cause symptoms such as abortion and infertility in animals. For Chlamydia abortus disease, current epidemiological surveys mostly use indirect hemagglutination tests, lacking effective pathogen detection methods, which often results in large deviations in the results obtained by these studies. Droplet Digital PCR (ddPCR), as a third-generation PCR technology, can achieve absolute quantification of target DNA through the oil-in-water emulsion droplet method, and has the advantages of rapidity, sensitivity and specificity.

[0004] In summary, the rapid and effective identification of abortion chlamydiosis not only helps to analyze the causes of animal abortion, but also has important significance for understanding the epidemic situation of abortion chlamydiosis and thus formulating and implementing targeted prevention and control strategies. Summary of the invention

[0005] The purpose of the present invention is to provide a method for quantitatively detecting Chlamydia abortus based on droplet digital PCR. The technical scheme of the present invention can achieve rapid and effective identification of Chlamydia abortus disease, and can detect Chlamydia abortus without cross-reacting with other common pathogens. It has good specificity, good repeatability and high sensitivity.

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

[0007] The present invention provides a primer pair and a probe for detecting Chlamydia abortus. The primer pair comprises a forward primer and a reverse primer. The sequence of the forward primer is shown in SEQ ID NO:1, and the sequence of the reverse primer is shown in SEQ ID NO:2; the sequence of the probe is shown in SEQ ID NO:3.

[0008] Preferably, the 5' end of the probe is connected to a fluorescent group, and the 3' end is connected to a quencher group; the fluorescent group includes CY5, and the quencher group includes BHQ2.

[0009] The invention provides a kit for quantitatively detecting Chlamydia abortus by droplet digital PCR, comprising the primer pair and the probe.

[0010] Preferably, ddPCR reaction solution and double distilled water are also included.

[0011] The present invention also provides a method for quantitatively detecting Chlamydia abortus based on droplet digital PCR for non-diagnostic purposes, comprising the following steps:

[0012] (1) using the genomic DNA of the sample to be tested as a template, performing droplet digital PCR amplification using the primer pair and the probe to obtain an amplification product;

[0013] (2) judging negative or positive results based on the fluorescence signal of the droplets in the amplified product;

[0014] When the droplets emit a fluorescent signal, the result is judged as positive; when the droplets do not emit a fluorescent signal, the result is judged as negative.

[0015] Preferably, the total reaction system of the droplet digital PCR amplification in step (1) is 22 μL, including 11 μL of 2×ddPCR reaction solution, 1 μL of 20 μM / L forward primer, 1 μL of 20 μM / L reverse primer, 0.6 μL of 10 μM / L probe, 1 μL of DNA template of the sample to be tested and 7.4 μL of double distilled water.

[0016] Preferably, the reaction procedure of the droplet digital PCR amplification in step (1) is: the heating and cooling rate is set to 2°C / s, the droplet stabilization stage is 60°C for 5min; pre-denaturation is 95°C for 15min; denaturation is 95°C for 20s, annealing is 60°C for 30s, and 40 cycles.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects:

[0018] 1. The technical solution of the present invention can realize the rapid and effective identification of Chlamydia abortus with high sensitivity. It has been verified that the minimum detection limit of Chlamydia abortus nucleic acid can reach 1.76 copies / μL.

[0019] 2. The technical solution of the present invention can detect Chlamydia abortus without cross-reaction with other common pathogens, and has good specificity. It has been verified that there is no cross-reaction with nucleic acid samples such as Coxiella burnetii, Mycobacterium bovis, Brucella, Listeria, Staphylococcus aureus, and Chlamydia psittaci.

[0020] 4. The technical solutions of the present invention have good repeatability, and the coefficient of variation is verified to be less than 10%.

[0021] 3. The technical solution of the present invention is simple to operate. At the same time, the primer pairs and probes used are optimized based on the differences in the ompA gene sequence sites of Chlamydia abortus, and the detection background value is low, making it easier to determine the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the ddPCR amplification diagram of different primer concentrations;

[0023] Figure 2 It is the ddPCR amplification diagram of different probe concentrations;

[0024] Figure 3 is the sensitivity test result;

[0025] Figure 4 The specificity test results ( Figure 4 1A represents DNA of Chlamydia abortus, 1B represents DNA of Coxiella burnetii, 1C represents DNA of Mycobacterium bovis, 1D represents DNA of Brucella, 1E represents DNA of Listeria monocytogenes, 1F represents DNA of Staphylococcus aureus, 1G represents DNA of Chlamydia psittaci, and 1H represents double distilled water). DETAILED DESCRIPTION

[0026] The present invention provides a primer pair and a probe for detecting Chlamydia abortus, wherein the primer pair and the probe are designed based on the ompA gene sequence of Chlamydia abortus. The primer pair of the present invention comprises a forward primer and a reverse primer, wherein the sequence of the forward primer is shown in SEQ ID NO:1, and the specific sequence is 5'-GAATTTCAGCAGACCGGTTATTC-3'; the sequence of the reverse primer is shown in SEQ ID NO:2, and the specific sequence is 5'-GTTGCCAAGCAAGCTCATTAT-3'. The sequence of the probe of the present invention is shown in SEQ ID NO:3, and the specific sequence is 5'-AGGAACAGCCGATAAACTCTGGCC-3'.

[0027] In the present invention, the 5' end of the probe is connected to a fluorescent group, and the 3' end is connected to a quenching group; the fluorescent group includes CY5, and the quenching group includes BHQ2.

[0028] The invention provides a kit for quantitatively detecting Chlamydia abortus by droplet digital PCR, comprising the primer pair and the probe.

[0029] In the present invention, the kit also includes ddPCR reaction solution and double distilled water.

[0030] The present invention also provides a method for quantitatively detecting Chlamydia abortus based on droplet digital PCR for non-diagnostic purposes, comprising the following steps:

[0031] (1) using the genomic DNA of the sample to be tested as a template, performing droplet digital PCR amplification using the primer pair and the probe to obtain an amplification product;

[0032] (2) judging negative or positive results based on the fluorescence signal of the droplets in the amplified product;

[0033] When the droplets emit a fluorescent signal, the result is judged as positive; when the droplets do not emit a fluorescent signal, the result is judged as negative.

[0034] In the present invention, the total reaction system of the droplet digital PCR amplification in step (1) is 22 μL, including 11 μL of 2×ddPCR reaction solution, 1 μL of 20 μM / L forward primer, 1 μL of 20 μM / L reverse primer, 0.6 μL of 10 μM / L probe, 1 μL of DNA template of the sample to be tested, and 7.4 μL of double distilled water.

[0035] In the present invention, the reaction procedure of the droplet digital PCR amplification in step (1) is: the heating and cooling rate is set to 2°C / s, the droplet stabilization stage is 60°C for 5min; pre-denaturation is 95°C for 15min; denaturation is 95°C for 20s, annealing is 60°C for 30s, and 40 cycles.

[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] Example 1 Primer and probe design

[0038] The ompA gene sequence of Chlamydia abortus (GEN ID: NZ_CP158101.1) was downloaded from NCBI, and the primers and probes were designed using PrimerQuest primer design software. The nucleotide sequences of the primers and probes are shown in Table 1.

[0039] Table 1 Primer and probe sequence information

[0040] Primer and probe name sequence serial number Forward primer 5'-GAATTTCAGCAGACCGGTTATTC-3' SEQ ID NO.1 Reverse primer 5'-GTTGCCAAGCAAGCTCATTAT-3' SEQ ID NO.2 Probe 5'-AGGAACAGCCGATAAACTCTGGCC-3' SEQ ID NO.3

[0041] The 5' end of the probe for detecting Chlamydia abortus is labeled with cyanine dye CY5, and the 3' end of the probe is labeled with quencher group BHQ2.

[0042] Example 2 Sensitivity

[0043] (I) Preparation of Chlamydia abortion nucleic acid DNA

[0044] The nucleic acid DNA of Chlamydia abortus was provided by the China Animal Health and Epidemiology Center.

[0045] The copy number of the nucleic acid was determined to be 1×10 4 The abortion Chlamydia nucleic acid was diluted in a gradient manner to obtain five concentrations of working standards, namely working standard 1, containing 1×10 3 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 1.03×10 0 copies / μL positive nucleic acid; working standard 5, containing 1×10 -1 copies / μL positive nucleic acid.

[0046] (II) Establishment of reaction system

[0047] Each tube of reaction solution was 22 μL, containing 11 μL of 2×ddPCR Supermix for probes (ddPCR reaction solution); 1 μL of forward and reverse primers of different concentrations: 10 μmol / L, 20 μmol / L, 50 μmol / L, 100 μmol / L; 0.6 μL of probes of different concentrations: 10 μmol / L, 30 μmol / L, 50 μmol / L, 70 μmol / L; and 7.4 μL of double distilled water.

[0048] Amplification conditions: the heating and cooling rate was set at 2°C / s, the droplet stabilization stage was 60°C for 5 min; pre-denaturation was 95°C for 15 min; denaturation was 95°C for 20 s, annealing was 60°C for 30 s, and 40 cycles were performed.

[0049] Each procedure was repeated twice. The DNA copy number was read after the reaction was completed. The reaction was carried out in the Sniper digital PCR machine DQ 24, and the test results at different primer and probe concentrations were counted.

[0050] according to Figure 1 and Figure 2It can be seen from the test results that when the concentrations of the forward primer and reverse primer are 20 μmol / L and the probe concentration is 10 μmol / L, the fluorescence signal boundary between the negative and positive droplets is obvious, the fluorescence of the positive droplets is concentrated, and the positive signal reaches the strongest. At this time, the primer and probe concentrations reach the optimal reaction concentration.

[0051] (III) Sensitivity test

[0052] A reaction system was prepared based on the primers and probes screened in Example 1, and the working standard was tested. The specific steps are as follows:

[0053] 1) ddPCR reaction system configuration: Each tube contains 22 μL of reaction solution, including 11 μL of 2× ddPCR reaction solution, 1 μL of 20 μM / L forward primer and reverse primer, 0.6 μL of 10 μmol / L probe, 7.4 μL of double distilled water, and 1 μL of DNA template to be detected.

[0054] 2) ddPCR reaction amplification system: the heating and cooling rate was set at 2°C / s, the droplet stabilization stage was 60°C for 5 min, pre-denaturation was 95°C for 15 min, denaturation was 95°C for 20 s, annealing was 60°C for 30 s, and 40 cycles were performed. Each procedure was repeated twice.

[0055] 3) Result determination: If the droplet emits a fluorescent signal, it means that the droplet contains the target DNA and the result is determined to be positive; if the droplet does not emit a fluorescent signal, it means that the droplet does not contain the target DNA and the result is determined to be negative.

[0056] After PCR amplification, the fluorescence signal of each micro-reaction unit was detected, and the absolute copy number of the template was calculated according to the Poisson distribution. Working standards 1-5 were used to evaluate the sensitivity of this method in detecting Chlamydia abortus.

[0057] The results are as follows Figure 3 As shown, working standards 1-4 showed positive droplets, and the number of droplets decreased. Working standard 5 did not show positive droplets. This shows that the minimum detection limit of Chlamydia abortus using the scheme of the present invention is working standard 4, and the sensitivity is 1.76 copies / μL.

[0058] Example 3 Specificity test

[0059] Based on the primers and probes designed in Example 1, a reaction system was prepared to detect nucleic acid samples of Chlamydia abortus, Coxiella burnetii, Mycobacterium bovis, Brucella, Listeria monocytogenes, Staphylococcus aureus, and Chlamydia psittaci, and double distilled water was used as a negative control. The specific steps are as follows:

[0060] Preparation of specific samples: The nucleic acid samples of Chlamydia abortus, Coxiella burnetii, Mycobacterium bovis, Brucella, Listeria monocytogenes, Staphylococcus aureus, and Chlamydia psittaci were provided by the Zoonotic Disease Monitoring Office of the Chinese Animal Health and Epidemiology Center.

[0061] Detection of specific samples: 1) ddPCR reaction system configuration: 22 μL of reaction solution per tube, containing 11 μL of 2×ddPCR Supermix forprobes ddPCR reaction solution, 1 μL of 20 μM / L forward primer and reverse primer, 0.6 μL of 10 μmol / L probe, 7.4 μL of double distilled water, and 1 μL of DNA template to be detected. 2) ddPCR reaction amplification system: The heating and cooling rate is set to 2℃ / s, the droplet stabilization stage is 60℃ for 5min; pre-denaturation is 95℃ for 15min; denaturation is 95℃ for 20s, annealing is 60℃ for 30s, and 40 cycles. 3) Result determination: If the droplet emits a fluorescent signal, it means that the droplet contains the target DNA, and the result is determined to be positive; if no fluorescent signal is emitted, it means that the droplet does not contain the target DNA, and the result is determined to be negative.

[0062] The results are as follows Figure 4 In this experiment, positive droplets appeared in the nucleic acid of Chlamydia abortus samples, while no positive droplets appeared in the nucleic acid of Coxiella burnetii, Mycobacterium bovis, Brucella, Listeria monocytogenes, Staphylococcus aureus, and Chlamydia psittaci samples, and the experiment was established. The results show that the scheme of the present invention can detect Chlamydia abortus without cross-reaction with other common pathogens, and has good specificity.

[0063] Example 4 Repeatability Test

[0064] Based on the primers and probes designed in Example 1, a reaction system was prepared. The working standards 1 and 2 prepared in Example 2 were tested three times by ddPCR, and the coefficient of variation between groups was calculated. In the same ddPCR, 3 was tested three times to calculate the coefficient of variation within the group. The coefficient of variation between and within the group was calculated according to the coefficient of variation CV% = (standard deviation SD / mean value Mean) × 100%.

[0065] The specific steps are as follows:

[0066] ddPCR reaction system configuration: 1) Each tube of reaction solution is 22μL, containing 11μL of 2×ddPCR Supermixfor probesddPCR reaction solution, 1μL of 20μM / L forward primer and reverse primer, 0.6μL of 10μmol / L probe, 7.4μL of double distilled water, and 1μL of DNA template to be detected. 2) ddPCR reaction amplification system: the heating and cooling rate is set to 2℃ / s, the droplet stabilization stage is 60℃ for 5min; pre-denaturation is 95℃ for 15min; denaturation is 95℃ for 20s, annealing is 60℃ for 30s, and 40 cycles. 3) Result determination: If the droplet emits a fluorescent signal, it means that the droplet contains the target DNA, and the result is determined to be positive; if no fluorescent signal is emitted, it means that the droplet does not contain the target DNA, and the result is determined to be negative. After completing PCR amplification, the fluorescent signal of each micro-reaction unit is detected, and the absolute copy number of the template is calculated according to the Poisson distribution.

[0067] Table 2 Results of inter-group and intra-group repeatability tests

[0068]

[0069] The working standards 1 and 2 were tested repeatedly. The results are shown in Table 2. The coefficients of variation of the obtained results were all less than 10%, indicating that the method has good repeatability.

[0070] Example 5 Detection of clinical samples

[0071] The reaction system was prepared using the primers and probes designed in Example 1, and 20 clinical tissue samples collected from animals suspected of being infected with Chlamydia abortus were tested, and the results were compared with those of the fluorescent PCR method recommended by the World Organization for Animal Health (WOAH). The specific steps are as follows:

[0072] Preparation of clinical samples: A total of 20 samples of clinically suspected infected animal tissue samples (collected from slaughterhouses in different regions of Xinjiang, stored and provided by the China Animal Health and Epidemiology Center) were collected. The tissue samples were first ground, then boiled to extract nucleic acid, and stored at -20°C for later use.

[0073] Detection of clinical samples: 1) ddPCR reaction system configuration: 22 μL of reaction solution per tube, containing 11 μL of 2×ddPCR Supermix forprobes ddPCR reaction solution, 1 μL of 20 μM / L forward primer and reverse primer, 0.6 μL of 10 μmol / L probe, 7.4 μL of double distilled water, and 1 μL of DNA template to be detected. 2) ddPCR reaction amplification system: The heating and cooling rate is set to 2℃ / s, the droplet stabilization stage is 60℃ for 5min; pre-denaturation is 95℃ for 15min; denaturation is 95℃ for 20s, annealing is 60℃ for 30s, and 40 cycles. 3) Result determination: If the droplet emits a fluorescent signal, it means that the droplet contains the target DNA, and the result is determined to be positive; if no fluorescent signal is emitted, it means that the droplet does not contain the target DNA, and the result is determined to be negative.

[0074] Table 3 Clinical sample test results

[0075]

[0076] The results are shown in Table 3. The method was used to detect 20 clinical samples suspected of being infected with Chlamydia abortus. The results were 14 positive and 6 negative. The detection rate was higher than the fluorescence PCR detection result, indicating that the method can effectively detect clinical samples and has higher sensitivity than the fluorescence PCR method recommended by WOAH.

[0077] In summary, it can be seen that the primer pairs and probes of the present invention and the ddPCR method using the primer pairs and probes are highly specific, highly sensitive, and have good repeatability. They are simple and rapid, and can effectively identify individual animals with early infection, and are helpful for the accurate and rapid detection of Chlamydia abortus.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer pair and probe for detecting Chlamydia abortus, characterized in that: The primer pair includes a forward primer and a reverse primer, the sequence of the forward primer is shown in SEQ ID NO: 1, and the sequence of the reverse primer is shown in SEQ ID NO: 2; The sequence of the probe is shown in SEQ ID NO:

3.

2. The primer pair and probe according to claim 1, characterized in that: The 5' end of the probe is connected to a fluorescent group, and the 3' end is connected to a quenching group; The fluorescent group includes CY5, and the quenching group includes BHQ2.

3. A kit for quantitative detection of Chlamydia abortus by droplet digital PCR, characterized in that: Comprising the primer pair and probe according to claim 1 or 2.

4. The kit according to claim 3, characterized in that It also includes ddPCR reaction solution and double distilled water.

5. A method for quantitative detection of Chlamydia abortus based on droplet digital PCR for non-diagnostic purposes, characterized in that: The steps include: (1) using the genomic DNA of the sample to be tested as a template, and performing droplet digital PCR amplification using the primer pair and probe described in claim 1 or 2 to obtain an amplified product; (2) judging negative or positive results based on the fluorescence signal of the droplets in the amplified product; When the droplets emit a fluorescent signal, the result is judged as positive; when the droplets do not emit a fluorescent signal, the result is judged as negative.

6. The method according to claim 5, characterized in that The total reaction system of the droplet digital PCR amplification in step (1) is 22 μL, including 11 μL of 2×ddPCR reaction solution, 1 μL of 20 μM / L forward primer, 1 μL of 20 μM / L reverse primer, 0.6 μL of 10 μM / L probe, 1 μL of DNA template of the sample to be tested, and 7.4 μL of double distilled water.

7. The method according to claim 5, characterized in that The reaction procedure of the droplet digital PCR amplification in step (1) is: the heating and cooling rate is set to 2°C / s, the droplet stabilization stage is 60°C for 5min; pre-denaturation is 95°C for 15min; denaturation is 95°C for 20s, annealing is 60°C for 30s, and 40 cycles.

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