Primer probe combination and application thereof in simultaneous identification of chlamydia psittaci and chlamydia abortus

Abstract

The invention discloses a primer probe combination and application thereof in simultaneous identification of chlamydia psittaci and chlamydia abortus, and relates to the technical field of animal molecular biology test methods and test reagents. The method comprises the following steps: firstly, designing a universal primer probe for a common conserved region of chlamydia parrot (15 genotypes) and chlamydia abortus ompA genes, then designing an identification primer probe for conserved but different regions of ompA genes of two chlamydia, respectively marking FAM and VIC fluorophores by the two probes, then screening out an optimal primer probe combination and optimal PCR amplification reaction conditions, and finally identifying the ompA genes of the chlamydia parrot (15 genotypes) and the ompA genes of the chlamydia abortus. Finally, the sensitivity, specificity, repeatability and the like of the primer probe combination are verified through experiments. By using the detection method disclosed by the invention, the chlamydia psittaci and the chlamydia abortus can be identified at the same time, all genotypes of chlamydia psittaci can be detected, and the effect is obviously superior to that of the existing known detection method.

Description

Technical Field

[0001] This invention belongs to the field of animal molecular biology testing methods and reagents, and particularly relates to a primer-probe combination and its application in the simultaneous identification of Chlamydia psittaci and Chlamydia abortus. It also involves the use of specific detection methods and the preparation of detection kits. Background Technology

[0002] Chlamydia are obligate intracellular parasites, intermediate in size between bacteria and viruses, with a unique developmental cycle. Widely distributed worldwide, they are zoonotic pathogens that can cause various diseases in humans, mammals, and birds. The chlamydia species known to be associated with human and animal chlamydial diseases are mainly classified under the genus *Chlamydia* within the family Chlamydiaceae. Currently, 16 species have been identified, including *Chlamydia trachomatis*, *Chlamydia psittaci*, *Chlamydia pneumoniae*, and *Chlamydia abortus*. Both *Chlamydia abortus* and *Chlamydia psittaci* can cause abortion in cattle and sheep, making them important zoonotic pathogens. *Chlamydia abortus* primarily causes abortion in infected animals, especially in late pregnancy. *Chlamydia abortus* is prevalent in cattle and sheep in my country, particularly in sheep flocks, with an average positive rate of 3.21%–28.81%. Chlamydia psittaci is a zoonotic pathogen with a global distribution. Pigeons, parrots, and wild birds are its primary hosts, but it can also infect humans, cattle, sheep, and other mammals. It mainly causes respiratory diseases in birds and abortions or respiratory illnesses in mammals. With continuous evolution, the pathogen can be classified into 15 genotypes based on its ompA gene. Clinically, a preliminary diagnosis of chlamydia-related diseases can be made based on medical history, clinical symptoms, and pathological characteristics, but definitive diagnosis still requires laboratory testing. Therefore, nucleic acid testing for Chlamydia psittaci and Chlamydia abortus is of great significance for the treatment and prevention of these diseases.

[0003] Although there are reports of establishing quantitative real-time PCR detection methods for *Chlamydia psittaci* and *Chlamydia abortus*, with the genetic evolution of *Chlamydia psittaci*, the genotypes have gradually evolved to 15. Existing detection methods (such as application number: 201310540233.6) only target one or a few genotypes and can no longer meet the current needs for multi-genotype detection of *Chlamydia psittaci*. Furthermore, sequence alignment of the conserved gene *ompA* in *Chlamydia psittaci* and *Chlamydia abortus* revealed that the most conserved region of all *Chlamydia psittaci* genotypes is not 100% homologous, and there is some overlap with *Chlamydia abortus*. In other words, based on the sequence characteristics of the conserved region, *Chlamydia psittaci* can be divided into two main categories: A, B, D, E, F, E / B, and WC form one category; while C, Matt116, M56, 6N, 1V, R54, Daruma, and CPX0308 form another category, which shares 100% homology with *Chlamydia abortus*. Therefore, it is currently impossible to detect Chlamydia abortus and all genotypes of Chlamydia psittaci with high sensitivity and specificity.

[0004] Quantitative real-time PCR (qPCR) is a highly sensitive, specific, convenient, and rapid detection method suitable for infectious disease screening in human and animal disease control systems and related testing laboratories. It has been widely used for detecting human infectious diseases such as influenza and animal infectious diseases such as African swine fever. Therefore, using qPCR to detect *Chlamydia psittaci* and *Chlamydia abortus* is the best and most effective means for monitoring, screening, treating, and controlling these two infectious diseases. Although some reports have established qPCR detection methods for *Chlamydia psittaci* and *Chlamydia abortus* separately, in addition to the shortcomings mentioned above, these methods still involve single-reaction tube detection of a single disease, lack sufficient sensitivity, and are costly in terms of manpower, resources, and time, failing to meet the needs of rapid clinical screening and diagnosis. Therefore, the simultaneous identification of *Chlamydia psittaci* and *Chlamydia abortus* in a single reaction tube is of great significance for timely symptomatic treatment and control of disease progression. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention designs universal primers and probes targeting the conserved regions of the ompA gene shared by Chlamydia psittaci (15 genotypes) and Chlamydia abortus. Furthermore, it designs distinguishing primers and probes targeting the conserved but different regions of the ompA gene in both chlamydia species. The two probes are labeled with different fluorescent groups, FAM and VIC, respectively, ultimately achieving the goal of simultaneously identifying Chlamydia psittaci, Chlamydia abortus, and detecting all genotypes of Chlamydia psittaci.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] One objective of this invention is to provide a primer-probe combination, which includes a universal primer-probe combination and a distinguishing primer-probe combination. The sequence of the universal primer-probe combination is shown in SEQ ID NO.1~SEQ ID NO.3 or in SEQ ID NO.4~SEQ ID NO.6, and the sequence of the distinguishing primer-probe combination is shown in SEQ ID NO.7~SEQ ID NO.9.

[0008] Preferably, the primer-probe combination further includes fluorescent groups, including FAM fluorescent groups, VIC fluorescent groups, and BHQ1 fluorescent groups.

[0009] More preferably, the universal primer-probe combination comprises a FAM fluorescent group and a BHQ1 fluorescent group, and the identification primer-probe combination comprises a VIC fluorescent group and a BHQ1 fluorescent group.

[0010] More preferably, the universal primer-probe combination sequence is shown in SEQ ID NO.4~SEQ ID NO.6.

[0011] The second objective of this invention is to provide the application of any of the primer-probe combinations described above in the preparation of products capable of simultaneously identifying Chlamydia psittaci and Chlamydia abortus.

[0012] Preferably, the primer-probe combination is annealed at 57°C in the application.

[0013] Preferably, the final concentration of the primers in both the universal primer-probe combination and the identification primer-probe combination is 0.45 μM, and the final concentration of the probes is 0.2 μM.

[0014] Preferably, the mixing ratio of the universal primer-probe combination to the identification primer-probe combination is 1:1.

[0015] The third objective of this invention is to provide a product that can simultaneously identify Chlamydia psittaci and Chlamydia abortus, wherein the product contains any of the primer-probe combinations described above.

[0016] Preferably, the product form includes a reagent kit.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention first designs universal primers and probes targeting the conserved regions of the ompA gene shared by *Chlamydia psittaci* (15 genotypes) and *Chlamydia abortus*. Then, it designs distinguishing primers and probes targeting the conserved but different regions of the ompA gene from both chlamydia species. The two probes are labeled with FAM and VIC fluorescent groups, respectively. The optimal primer-probe combination and optimal PCR amplification conditions are then screened. Finally, the sensitivity, specificity, and repeatability of the primer-probe combination are experimentally verified. The kit prepared using the primer-probe combination of this invention can simultaneously identify *Chlamydia psittaci* and *Chlamydia abortus*, and can detect all genotypes of *Chlamydia psittaci*, with significantly better results than existing known detection methods. Attached Figure Description

[0019] Figure 1 This is the sequence alignment result in Embodiment 1 of the present invention.

[0020] Figure 2 This refers to the continuous base deletion region of the ompA gene of Chlamydia abortus in Example 1 of the present invention.

[0021] Figure 3 The image shows the primer / probe screening results in Example 1 of this invention, where A: universal primer / probe screening; B: identification primer / probe screening; 1: F2 / R2 / P2 combination; 2: F1 / R1 / P1 combination; red: Chlamydia psittaci nucleic acid; blue: Chlamydia abortus nucleic acid.

[0022] Figure 4 The sensitivity verification results are shown in Example 1 of this invention, where A: general detection sensitivity; B: discriminative detection sensitivity; 1-9: 10 8 copies / uL-10 0 copies / uL.

[0023] Figure 5 The specificity verification results in Example 1 of the present invention are as follows: 1: Chlamydia psittaci type A; 2: Chlamydia psittaci type B; 3: Chlamydia psittaci type C; 4: Chlamydia abortus; 5: Brucella; 6: Newcastle disease virus; 7: H9N2 subtype avian influenza virus; 8: Foot-and-mouth disease virus type O; 9: Pleistocene ruminant virus; 10: Escherichia coli; 11: Salmonella avianis. Detailed Implementation

[0024] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0025] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0026] Example 1

[0027] I. Sequence Alignment

[0028] The ompA gene sequences of *Chlamydia psittaci* and *Chlamydia abortus* (Table 1) were downloaded from the NCBI database and sequence alignment was performed using Megalign software. First, the homology of the upper, middle, and lower segments of the complete ompA gene sequences of the two chlamydia species was compared individually. Sequences of the same segments with high homology were selected for alignment, and the sequence with the highest homology between the ompA genes of the two chlamydia species was determined as the target sequence for universal primer probes. Subsequently, sequences with high homology to *Chlamydia abortus* but different from those of *Chlamydia psittaci* were selected as target sequences for specific primer probes.

[0029] Table 1. Reference sequences for Chlamydia psittaci and Chlamydia abortus.

[0030]

[0031] To simultaneously detect and identify *Chlamydia psittaci* and *Chlamydia abortus*, at least two characteristic targets need to be screened: a general target and a distinguishing target. This can be achieved through sequence comparison (…). Figure 1 The study found high homology in the upper region, and that loci 168, 171, and 174 (Chlamydia psittaci sequence number: CP003790.1) could serve as characteristic targets. Based on the differences in nucleotides at these three loci, all Chlamydia psittaci genotypes can be divided into two categories: one category includes A, B, D, E, F, E / B, and WC, and the other category includes C, Matt116, M56, 6N, 1V, R54, Daruma, and CPX0308. The latter category is identical to Chlamydia abortus. Therefore, this region can be selected as a universal detection area.

[0032] To ensure specific identification, a conserved region of the ompA gene in *Chlamydia abortus* that differs from that in *Chlamydia psittaci* was selected. The 650-850 bp segment (*Chlamydia abortus* sequence number: CR848038.1) was found to be highly conserved, but also showed a deletion of 5 consecutive bases compared to *Chlamydia psittaci*. Figure 2 Therefore, this area was selected as the identification and detection area.

[0033] The selected universal and specific detection target sequences were sent to Sangon Biotech (Shanghai) Co., Ltd. to synthesize plasmids: pUC57-ompA-universal and pUC57-ompA-identification.

[0034] II. Primer / Probe Design and Synthesis

[0035] 1. Universal primers / probes

[0036] Primers and probes were designed using oligo 7 software based on the target sequence. Two sets of universal primers / probes were designed by adjusting the probe length, GC content, and free energy value (Table 2). The region where the P2 probe is located contains three base mutation sites, so a degenerate base synthesis probe sequence was adopted. At the same time, in order to improve the specificity and annealing temperature of the short sequence probe, both probes were labeled with FAM fluorescent groups.

[0037] One point that needs to be emphasized is that, Figure 1 As shown in the black box, the most conserved region of *Chlamydia psittaci* can be divided into two categories, one of which is the same as *Chlamydia abortus*. Therefore, when designing universal primers and probes for this region, in order to simultaneously detect these two types of chlamydia, the universal primers and probes are designed with degenerate bases targeting the three specific bases discovered (for example, the site selected in the first box has both T and C bases; to simultaneously detect T and C bases, the probe for this site is designed as R). This is an original invention.

[0038] 2. Identify primers / probes.

[0039] To ensure no cross-reaction with Chlamydia psittaci, upstream primers were positioned in regions with consecutive base deletions to target specific sequences. Downstream primers and probes were designed by adjusting probe length, GC content, and free energy (Table 2), with the probes labeled with the VIC fluorescent group.

[0040] The designed primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0041] Table 2 Primer / Probe Sequences

[0042]

[0043] III. Primer / Probe Screening

[0044] To screen the effectiveness of universal and differential primers / probes, pure cultures of *Chlamydia psittaci* and *Chlamydia abortus* were used as templates for amplification. Universal primer / probe combinations F1 / R1 / P1 and F2 / R2 / P2, and differential primer / probe combination F / R / P were used for amplification, respectively. The ability of the two universal primer / probe combinations to simultaneously detect *Chlamydia psittaci* and *Chlamydia abortus* was verified by comparing Ct values; the combination with the smaller Ct value was considered the optimal universal primer / probe combination. The differential primers were also verified to show a distinct "S"-shaped amplification curve and a small Ct value when detecting *Chlamydia abortus* nucleic acid, while *Chlamydia psittaci* showed no amplification curve or Ct value.

[0045] The components of the qPCR reaction system used for screening primers / probes were: 10 μL Premix Ex Taq (2X), 0.4 μL upstream primer (10 pmol / μL), 0.4 μL downstream primer, 0.4 μL probe (10 pmol / μL), 6.8 μL nuclease-free water, and 2 μL template.

[0046] The qPCR reaction conditions used for primer screening were: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 55℃ annealing extension for 30s, for 40 cycles.

[0047] Universal primer / probe screening results indicate that ( Figure 3 A) Both the F1 / R1 / P1 and F2 / R2 / P2 primer / probe combinations can detect the nucleic acids of *Chlamydia psittaci* and *Chlamydia abortus*. However, the Ct values ​​of the F2 / R2 / P2 combination (23.91 for *Chlamydia psittaci* and 29.00 for *Chlamydia abortus*) are lower than those of the F1 / R1 / P1 combination (25.44 for *Chlamydia psittaci* and 30.33 for *Chlamydia abortus*), and its fluorescence intensity is also higher than that of the F1 / R1 / P1 combination. Therefore, the F2 / R2 / P2 primer / probe combination is selected as the universal primer / probe combination.

[0048] The results of primer / probe screening indicate that ( Figure 3 B), the F / R / P combination showed a distinct "S"-shaped amplification curve and a small Ct value when detecting Chlamydia abortus, and no amplification curve when detecting Chlamydia psittaci.

[0049] IV. Annealing Temperature Optimization

[0050] At a concentration of 3.10 × 10 3 Amplification was performed using copies / μL of pUC57-ompA-universal and pUC57-ompA-identifier plasmids as templates. Annealing temperatures were set at 51.1℃, 51.8℃, 53.1℃, 55.8℃, 57℃, 59.4℃, 60.5℃, and 61.1℃. The Ct values ​​and fluorescence intensities at different annealing temperatures were compared to determine the optimal annealing temperature. The results (Table 3) show that the universal and identification primer / probe combinations exhibited the lowest Ct value and relatively high fluorescence intensity when annealed at 57℃ for detecting the two Chlamydia species. Therefore, the optimal annealing temperature for this invention is 57℃.

[0051] Table 3 Optimization of Annealing Temperature

[0052]

[0053] V. Primer / Probe Concentration Optimization

[0054] To achieve the best detection results, the concentrations of general and identification primers / probes were first optimized to determine the optimal concentrations of primers and probes in each combination; then the primer and probe concentrations of the two combinations were optimized.

[0055] With a concentration of 3.10 × 10 3 Amplification was performed using pUC57-ompA-universal and pUC57-ompA-discriminative plasmids (copies / μL) as templates. Orthogonal experiments were conducted with primer concentrations of 0.075 μM, 0.15 μM, 0.3 μM, 0.45 μM, and 0.9 μM, and PC.psittaci probe concentrations of 0.05 μM, 0.1 μM, 0.15 μM, 0.2 μM, and 0.25 μM, respectively. Based on the Ct value and relative fluorescence intensity, the optimal upstream and downstream primer and probe concentrations for the universal and discriminative primer / probe combinations were determined. The results showed that for both the universal and discriminative primer / probe combinations, the Ct value was minimized and the relative fluorescence intensity was highest when the primer concentration was 0.45 μM and the probe concentration was 0.2 μM.

[0056] With a concentration of 3.10 × 10 3 Amplification was performed using pUC57-ompA-u / μL of universal and differential pUC57-ompA-identifier plasmids as templates. The two primer / probe combinations were tested at ratios of 1:1, 1:0.8, 1:0.6; 0.8:1, 0.8:0.8, 0.8:0.6; 0.6:1, 0.6:0.8, 0.6:0.6. The optimal ratio of the universal and differential primer / probe combinations was determined based on Ct values ​​and relative fluorescence intensity. The results showed that a 1:1 ratio of the universal and differential primer / probe combinations yielded the best detection performance.

[0057] Therefore, taking into account both the Ct value and the relative fluorescence intensity, the concentrations of the universal primer / probe combination and the identification primer / probe combination in the reaction system of this invention are: a final primer concentration of 0.45 μM and a final probe concentration of 0.2 μM.

[0058] VII. Sensitivity Verification

[0059] pUC57-ompA-General (3.10×10 9 (copies / μL) and pUC57-ompA-distinguishing function (3.14×10) 9 The samples were serially diluted 10-fold (copies / μL) at 3.10 × 10⁻⁶ ppm. 8 copies / μL - 3.10×10 0 copies / μL and 3.14×108 copies / μL - 3.14 × 10 0 Amplification was performed using plasmid copies / μL as templates, a standard curve was constructed, and the limit of detection was verified.

[0060] Reaction system: 10 μL Premix Ex Taq (2X), 0.9 μL F-general (10 pmol / μL), 0.9 μL R-general (10 pmol / μL), 0.4 μL P-general (10 pmol / μL), 0.9 μL F-identification (10 pmol / μL), 0.9 μL R-identification (10 pmol / μL), 0.4 μL P-identification (10 pmol / μL), 3.6 μL nuclease-free water, 2 μL template.

[0061] The qPCR reaction conditions used were: 95℃ pre-denaturation for 20s; 95℃ denaturation for 5s; 57℃ annealing extension for 20s; 40 cycles.

[0062] result( Figure 4 This indicates that the universal detection sensitivity for Chlamydia psittaci and Chlamydia abortus is 3.10 copies / μL. Figure 4 -A1), R 2 The value was 0.995, which is greater than 0.99, indicating a good linear relationship in the standard curve. The amplification efficiency was 95.237%, falling between 90-110%. Figure 4 -A2); the detection sensitivity was 3.14 copies / μL ( Figure 4 -B1), R 2 The value was 0.998, greater than 0.99, indicating a good linear relationship in the standard curve. The amplification efficiency was 101.11%, falling between 90-110%. Figure 4 -B2).

[0063] VIII. Specificity Verification

[0064] The method of this invention was used to detect Chlamydia psittaci (types A, B, and C), Chlamydia abortus (C. abortus), Brucella, Newcastle disease virus (NDV), Avian Influenza Virus H9N2 subtype, Foot-and-Mouth Disease Virus serotype O, Peste des petitsruminants virus, Escherichia coli, and Avian Salmonella, and the specificity of this invention was evaluated.

[0065] result( Figure 5 This indicates that the present invention can specifically detect and effectively distinguish Chlamydia psittaci and Chlamydia abortus, while other related bacteria and viruses are detected negatively, indicating good specificity.

[0066] IX. Repeatability Validation

[0067] Using pUC57-ompA-universal plasmid (concentration 3.10 × 10⁻⁶) 4 copies / μL - 3.10×10 1 (copies / μL) and pUC57-ompA-discriminative plasmid (concentration of 3.14 × 10⁻⁶ copies ...). 4 copies / μL - 3.14 × 10 1 Using copies / μL as a template, three replicates were performed for each concentration, and the results were measured three times at different time periods. The coefficient of variation of Ct values ​​was calculated to verify the intra-group and inter-group repeatability of the present invention.

[0068] The results (Table 4) show that the coefficients of variation within and between the general and differential detection groups are all less than 5%, indicating that the method has good repeatability within groups and components.

[0069] Table 4 Repeatability Validation

[0070]

[0071] X. Reagent Kit Composition and Application

[0072] The kit includes: real-time PCR detection reagent, positive control, and negative control.

[0073] The quantitative real-time PCR (qPCR) detection reagent contains a qPCR premix, universal primers / probes, and identification primers / probes. The qPCR premix includes at least a DNA polymerase mixture, dNTPs, and MgCl2. In this invention, the qPCR premix was purchased from TAKARA (catalog number RR390Q). The detection reagent also contains a universal primer / probe with a primer concentration of 0.5 μM and a identification primer / probe combination with a probe concentration of 0.22 μM (the concentrations here refer to the primer / probe concentrations contained in the reagent, while the final primer / probe concentration mentioned above refers to the concentration in the entire reaction system after adding nucleic acid (i.e., the 20 μL reaction system in the reaction tube), for example: 0.5 μM * 18 μL detection reagent / (18 μL detection reagent + 2 μL nucleic acid) = 0.45 μM final concentration).

[0074] The positive control is a mixed solution containing pUC57-ompA-universal plasmid and pUC57-ompA-discrimination plasmid.

[0075] The negative control was RNase-free water, purchased from TAKARA, catalog number 9012.

[0076] application:

[0077] (1) Nucleic acid extraction: Nucleic acid was extracted from the sample according to the instructions of the commercial nucleic acid extraction kit used.

[0078] (2) Preparation of fluorescence quantitative PCR reaction solution: Based on the total number of samples to be tested, add 18 μL of fluorescence quantitative PCR detection reagent directly to each PCR reaction tube.

[0079] (3) Sample addition: Take 2 μL of each of the positive control, negative control and nucleic acid extracted in step (1), add them to the corresponding reaction tubes, cap the tubes, mix well, and centrifuge briefly, avoiding air bubbles.

[0080] (4) PCR amplification: Place the reaction tube from step (3) into a real-time PCR instrument for amplification. The reaction conditions are: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 57℃ annealing extension for 30s, 40 cycles.

[0081] (5) Result determination:

[0082] 1) Test conditions: The positive control has Ct values ​​of <30 in both FAM and VIC channels and shows an "S"-shaped amplification curve, while the negative control has no Ct value and no amplification curve. The test is valid when the above requirements are met.

[0083] 2) Positive for Chlamydia psittaci: If the sample shows an "S"-shaped amplification curve in the FAM channel corresponding to the fluorescent reporter group labeled at the 5' end of the P-general terminus (i.e., Ct value ≤ 40) within 40 cycles, while the VIC channel corresponding to the fluorescent reporter group labeled at the 5' end of the P-identification terminus shows no Ct value or "S"-shaped amplification curve, then it is considered positive for Chlamydia psittaci.

[0084] 3) Positive for Chlamydia abortion: If the sample shows an "S"-shaped amplification curve in both the FAM channel corresponding to the 5'-labeled fluorescent reporter group of the P-general type (Ct value ≤ 40) and the VIC channel corresponding to the 5'-labeled fluorescent reporter group of the P-identifier type (Ct value ≤ 40) within 40 cycles, then it is considered positive for Chlamydia abortion.

[0085] 4) Negative: If the sample has no Ct value or "S"-shaped amplification curve in the FAM and VIC channels, it is considered negative.

[0086] XI. Clinical Sample Validation

[0087] Clinical samples were collected, including 30 parrot swabs, 30 pigeon swabs, 200 wild bird droppings, and 30 cattle and sheep swabs. Nucleic acid was extracted using a commercial nucleic acid extraction kit, and the nucleic acid of the clinical samples was detected using the method described in this invention. The results were compared with the parrot thermofluorescent quantitative PCR detection method (Parrot Fever Quarantine Technical Specification, SN / T 2846-2011) and the Chlamydia abortus PCR method.

[0088] The results (Table 5) show that the positive and negative counts of Chlamydia psittaci detected by this invention are consistent with the detection standard, with a compliance rate of 100%. However, the standard detection method had some suspected positive results (2 suspected positive results from parrot swabs and 1 suspected positive result from pigeon swabs), while the detection method of this invention can clearly determine the positive result. The detection method of this invention detected 10 positive samples of Chlamydia abortus, which is more accurate than the ordinary PCR method, which only detected 8 samples.

[0089] In summary, the detection method of the present invention has higher sensitivity than existing detection methods.

[0090] Table 5 Clinical Sample Testing

[0091]

[0092] * indicates that an individual sample tested suspected to be positive.

[0093] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer-probe combination, characterized in that, It includes a universal primer-probe combination and a distinguishing primer-probe combination. The universal primer-probe combination sequence is shown in SEQ ID NO.1~SEQ ID NO.3 or in SEQ ID NO.4~SEQ ID NO.6, and the distinguishing primer-probe combination sequence is shown in SEQ ID NO.7~SEQ ID NO.

9.

2. The primer-probe combination according to claim 1, characterized in that, The primer-probe combination also includes fluorescent groups, including FAM fluorescent groups, VIC fluorescent groups, and BHQ1 fluorescent groups.

3. The primer-probe combination according to claim 2, characterized in that, The universal primer-probe combination contains FAM fluorescent groups and BHQ1 fluorescent groups, and the identification primer-probe combination contains VIC fluorescent groups and BHQ1 fluorescent groups.

4. The primer-probe combination according to claim 3, characterized in that, The universal primer-probe combination sequence is shown in SEQ ID NO.4~SEQ ID NO.

6.

5. The use of the primer-probe combination according to any one of claims 1 to 4 in the preparation of products capable of simultaneously identifying Chlamydia psittaci and Chlamydia abortus.

6. The application according to claim 5, characterized in that, The primer-probe combination is annealed at 57°C in this application.

7. The application according to claim 5, characterized in that, The final concentration of the primers in both the universal primer-probe combination and the identification primer-probe combination is 0.45 μM, and the final concentration of the probes is 0.2 μM.

8. The application according to claim 5, characterized in that, The mixing ratio of the universal primer-probe combination to the identification primer-probe combination is 1:

1.

9. A product capable of simultaneously identifying Chlamydia psittaci and Chlamydia abortus, characterized in that, It includes the primer-probe combination as described in any one of claims 1 to 4.

10. The product according to claim 9, characterized in that, The product forms include reagent kits.

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