LAMP primer combination for detecting chlamydia psittaci and application of LAMP primer combination

Abstract

The invention belongs to the field of prevention and control of psittaci diseases, and relates to an LAMP primer combination for detecting chlamydia psittaci and application of the LAMP primer combination. The invention provides an LAMP (loop-mediated isothermal amplification) primer combination for detecting chlamydia psittaci, and the LAMP primer combination comprises the following primers: FIP: 5 '-GTACATGTTTCAAGTTTATTGCCGATGCTAACAAATTTCAGAACAATTACAACAACAATTACAACAACGATTCCGC-3', BIP: 5 '-ACTTGGAAGCTATCTGTAGGTAAACAAGGAGA-3', F3: 5 '-GTATCTGCAACGGATCTG-3' and B3: 5 '-TCCTGTTAACGATTCCGC-3'. The primer combination can be used for LAMP detection of chlamydia psittaci, has the advantages of high sensitivity, strong specificity and

Description

Technical Field

[0001] This invention belongs to the field of psittacosis disease prevention and control, specifically relating to LAMP primer combinations for detecting Chlamydia psittaci and their applications. Background Technology

[0002] Psittacosis is a zoonotic bacterial infectious disease primarily caused by *Chlamydia psittaci* (C. psittaci). C. psittaci is a Gram-negative, obligate intracellular parasite with multi-host disease characteristics. There are two main routes of transmission for C. psittaci: contact transmission (through direct contact with infected poultry) and aerosol transmission (through inhalation of bioaerosols formed from the feces, urine, respiratory secretions, and eye secretions of infected poultry). C. psittaci is highly contagious, widely infecting humans, poultry, wild birds, and livestock, and can cause various related diseases, posing a significant threat to public health and the livestock industry. In avian hosts, C. psittaci causes severe respiratory and digestive diseases and reduced productivity, with some infections remaining asymptomatic, resulting in long-term harm and losses to poultry farming. The probability of infection in the population is significantly correlated with occupational exposure, and the disease typically presents as atypical pneumonia of the lower respiratory tract. Therefore, developing efficient and convenient C. psittaci screening technologies is crucial for the prevention and control of psittaci.

[0003] Diagnostic methods for Chlamydia psittaci mainly include isolation and culture detection, serological detection, and molecular biological detection. Isolation and culture detection is the most specific and accurate method for diagnosing psittaci, but it carries the risk of transmission to laboratory personnel, and the experimental procedures must be performed in a biosafety level 3 laboratory. Many conserved sequences of C. psittaci have been developed and used for molecular biological detection, with detection targets generally being the 16S-23S rRNA, ompA, and cpsit sequences of C. psittaci. However, the high conservatism of the Chlamydia genus makes species-specific detection very difficult, and C. psittaci is also constantly evolving; therefore, it is necessary to develop new targets and detection technologies.

[0004] Currently, many detection methods are limited by pathogen isolation and culture, as well as laboratory conditions, making them difficult to fully utilize in practical applications. Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification method that is simple to operate and requires no special equipment. Commonly used closed-tube detection methods include visual turbidity, turbidimetric detection, and dye colorimetric methods, with dye colorimetric methods being the current research focus for LAMP closed-tube detection. Calcein colorimetric methods reduce reaction sensitivity, while SYBR Green dye colorimetric methods require special treatment or adding the dye after the reaction; the former inhibits the LAMP reaction, and the latter easily causes contamination. Hydroxynaphthol blue (HNB) colorimetric methods have unique advantages: hydroxynaphthol blue (HNB) is a metal ion indicator that reacts with Mg... 2+ The combination initially gives the system a violet color; as the reaction proceeds, Mg... 2+ The precipitated pyrophosphate ions react with the precipitated magnesium pyrophosphate ions to form magnesium pyrophosphate precipitate, and hydroxynaphthol blue loses its Mg content. 2+ The reaction system turns sky blue, while the unreacted system remains violet. Furthermore, HNB is low-cost, does not inhibit reaction efficiency, and can be directly added to the reaction system. Adding HNB to the LAMP system and interpreting the results based on the color change before and after the reaction avoids aerosol contamination caused by opening the container after the reaction, making it more suitable for on-site pathogen screening. Summary of the Invention

[0005] To rapidly and accurately control psittacosis, this invention establishes a rapid detection method for psittacosis based on loop-mediated isothermal amplification (LAMP) technology, targeting a specific region of the Chlamydia psittaci IncA gene. This method is highly specific, sensitive, easy to operate, and provides visualized results.

[0006] In a first aspect, the present invention provides a LAMP primer set for detecting Chlamydia psittaci, the primer set comprising the following primers:

[0007] FIP: 5'-GTACATGTTTCAAGTTTATTGCCGATGCTAACCAAATTTCAGAACT-3',

[0008] BIP: 5'-ACTTGGAAGCTTTCAAATCTGTAGGTAAACGCTCAAAAGGAGAGA-3',

[0009] F3: 5'-GTATCTGCAAACAGGGATCT-3', and

[0010] B3: 5'-TCCTGTTAACGATTCCGC-3'.

[0011] Secondly, the present invention provides a kit for detecting Chlamydia psittaci, the kit comprising the above-described LAMP primer combination.

[0012] In some embodiments, the primers FIP, BIP, F3, and B3 are packaged separately.

[0013] In some embodiments, the primers FIP, BIP, F3, and B3 are packaged together, wherein the molar ratio of FIP, BIP, F3, and B3 is 4:4:3:3.

[0014] In some embodiments, the kit further includes universal reagents for loop-mediated isothermal amplification.

[0015] In some embodiments, the universal reagents include isothermal amplification buffer, MgSO4 solution, and DNA polymerase.

[0016] In some embodiments, the working concentrations of primers FIP, BIP, F3, and B3 are 16 μM, 16 μM, 12 μM, and 12 μM, respectively, and the working concentration of MgSO4 is 6 mM.

[0017] In some embodiments, the kit further includes hydroxynaphthol blue (HNB).

[0018] In some embodiments, the kit further includes universal reagents for gel electrophoresis.

[0019] In some embodiments, common reagents for gel electrophoresis include DNA loading buffer, nucleic acid dyes, and DNA markers.

[0020] Thirdly, this invention also provides the application of the above-described LAMP primer combination in the preparation of products for detecting Chlamydia psittaci.

[0021] Fourthly, the present invention also provides the application of the above-mentioned LAMP primer combination in the detection of Chlamydia psittaci.

[0022] In some embodiments, the application includes: using a sample suspected of containing Chlamydia psittaci as a template, performing a LAMP reaction using the above-described LAMP primer combination; if specific amplification occurs, then the sample contains Chlamydia psittaci.

[0023] In some embodiments, the LAMP reaction system is as follows: 2.5 μL of 10× Isothermal Amplification Buffer, 1.5 μL of MgSO4 (100 mM), 3.5 μL of dNTPs (10 mM), 4 μL each of inner primers FIP / BIP (100 μM), 3 μL each of outer primers F3 / B3 (100 μM), 1 μL of Bst 2.0 WarmStart DNA polymerase (8000 U / mL), 1 μL of template, and water to make up the remaining 25 μL. The reaction conditions are: incubation at 65 °C for 1 h.

[0024] In some embodiments, the presence of *Chlamydia psittaci* in the sample is determined by detecting the LAMP reaction products using agarose gel electrophoresis. The presence of clear, stepped bands indicates a positive result for *Chlamydia psittaci*, while the absence of bands indicates a negative result.

[0025] In some embodiments, hydroxynaphthol blue (HNB) is added to the LAMP reaction system, and the presence of *Chlamydia psittaci* in the sample is determined by LAMP-HNB color development. If the reaction solution color changes from violet (initial state, Mg...) 2+ -HNB complex) turns sky blue (Mg 2+ If the reaction solution is exhausted, it is considered positive for Chlamydia psittaci; if there is no color change in the reaction solution, it is considered negative for Chlamydia psittaci.

[0026] The present invention has the following beneficial effects:

[0027] High sensitivity Using the above LAMP primer combination, LAMP reactions can detect copy number concentrations as low as 10. 2 The IncA gene of Chlamydia psittaci was detected in copies / μL, and the agarose gel electrophoresis results of the conventional LAMP reaction were completely consistent with the color development results of the LAMP-HNB reaction. Figure 3 and Figure 4 ).

[0028] High specificity The above LAMP primer combination only specifically amplifies samples containing the Chlamydia psittaci IncA gene (IncA gene recombinant plasmid, Chlamydia psittaci type B genomic DNA, Chlamydia psittaci CP3 genomic DNA), and does not specifically amplify genomic DNA of other pathogens (Campylobacter jejuni genomic DNA, Escherichia coli genomic DNA, Salmonella genomic DNA). Furthermore, the agarose gel electrophoresis results of the conventional LAMP reaction are completely consistent with the colorimetric results of the LAMP-HNB reaction. Figure 5 and Figure 6Therefore, this primer combination has high specificity and can accurately distinguish Chlamydia psittaci from other pathogens.

[0029] Easy to operate Using the above LAMP primer combination for LAMP-HNB detection of Chlamydia psittaci, no complicated instruments are required, and the results can be directly interpreted by the naked eye during on-site screening, enabling rapid diagnosis and precise prevention and control of psittaci.

[0030] Safe and reliable The hydroxyl blue (HNB) colorimetric method was used for the closed-tube detection of Chlamydia psittaci in LAMP, which avoided aerosol contamination caused by opening the tube after the reaction.

[0031] The LAMP primer combination and LAMP-HNB detection method provided by this invention have high sensitivity, strong specificity and simple operation. They can quickly and accurately detect whether a sample contains Chlamydia psittaci without complicated instruments. They are suitable for primary medical institutions and on-site screening scenarios, and provide reliable technical support for the early diagnosis and epidemiological monitoring of psittacosis. Attached Figure Description

[0032] Figure 1 The results are shown in the agarose gel electrophoresis analysis of the LAMP reaction products. A, B, C, D, E, and F represent different reaction systems, each with two lanes. Lane 1 is the positive control (1.7 × 10⁻⁶). 10 The positive plasmid pUC57-IncA (copy / μL) was used as the template for the LAMP reaction product, and lane 2 was used as the negative control (LAMP reaction product using nucleic acid-free water as the template).

[0033] Figure 2 The colorimetric results are for the LAMP-HNB system established in Example 1.

[0034] Figure 3 The results of sensitivity analysis of the LAMP detection method for Chlamydia psittaci (agarose gel electrophoresis images of LAMP reaction products from positive plasmid samples at different concentrations). Lanes 1-7 are 1.7 × 10⁻⁶. 6 copies / μL, 1.7×10 5 copies / μL, 1.7×10 4 copies / μL, 1.7×10 3 copies / μL, 1.7×10 2 copies / μL, 1.7×10 1 copies / μL, 1.7×10 0LAMP reaction product of pUC57-IncA plasmid sample (copies / μL); lane 8 is negative control (LAMP reaction product using nucleic acid-free water as template).

[0035] Figure 4 The results of sensitivity analysis of the LAMP-HNB detection method for Chlamydia psittaci (color change of the LAMP-HNB reaction solution) are shown. The seven tubes on the left, from left to right, show values ​​of 1.7 × 10⁻⁶. 6 copies / μL, 1.7×10 5 copies / μL, 1.7×10 4 copies / μL, 1.7×10 3 copies / μL, 1.7×10 2 copies / μL, 1.7×10 1 copies / μL, 1.7×10 0 LAMP-HNB reaction solution for pUC57-IncA plasmid sample (copies / μL); the rightmost tube is the negative control (LAMP-HNB reaction solution with nucleic acid-free water as template).

[0036] Figure 5 This is the specificity analysis result of the LAMP detection method for Chlamydia psittaci (agarose gel electrophoresis image of LAMP reaction products). Lanes 1-7 show, in order, the positive plasmid pUC57-IncA, Chlamydia psittaci type B genomic DNA, Chlamydia psittaci CP3 genomic DNA, Campylobacter jejuni genomic DNA, Escherichia coli genomic DNA, Salmonella genomic DNA, and LAMP reaction products of nucleic acid-free water (negative control).

[0037] Figure 6 The results of the specificity analysis of the LAMP-HNB detection method for Chlamydia psittaci (color change of the LAMP-HNB reaction solution) are shown. Tubes 1-7 contain, in order, positive plasmid pUC57-IncA, Chlamydia psittaci type B genomic DNA, Chlamydia psittaci CP3 genomic DNA, Campylobacter jejuni genomic DNA, Escherichia coli genomic DNA, Salmonella genomic DNA, and nucleic acid-free water (negative control) LAMP-HNB reaction solution.

[0038] Sequence Description

[0039] In the accompanying sequence listing, the nucleotide sequences follow the standard convention of starting from the 5' end and proceeding towards the 3' end.

[0040] SEQ ID NO:1-4 Nucleotide sequences of LAMP primers used for detecting Chlamydia psittaci.

[0041] SEQ ID NO:5 Nucleotide sequence of IncA gene of Chlamydia psittaci strain 6BC. Detailed Implementation

[0042] The technical solution of the present invention will be described in detail below with reference to embodiments. The following embodiments are for explanation and illustration only, and are not intended to limit the scope of the invention. In the following embodiments, reagents not specifically described are conventional reagents in the art, commercially available or prepared according to conventional methods in the art, and of analytical purity. Experimental methods and conditions not specifically described are conventional experimental methods and conditions in the art, and can be found in relevant experimental manuals, public literature, or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0043] The main reagents used in the following examples:

[0044] Plasmid miniprep kits and Trans10 competent cells were purchased from TransGenBiotech Co., Ltd. Bst 2.0 WarmStart DNA polymerase and 10× Isothermal Amplification Buffer were purchased from New England Biolabs, catalog number M0402L. Hydroxynaphthol Blue (HNB) was purchased from Solarbio Science & Technology Co., Ltd., catalog number G1218. DL2000 Premix DNA Marker was purchased from Takara.

[0045] Example 1: Establishment of a rapid detection method for Chlamydia psittaci

[0046] 1. Primer design and synthesis

[0047] Nine representative *Chlamydia psittaci* strains (WC, 6BC, WS / RT / E30, VS225, NJ1, M56, 84 / 85, CP3, and MN) were selected. Multiple sequence alignment was performed on their IncA gene sequences (GenBank accession numbers: CP003796.1, CP002549.1, CP003794.1, CP003793.1, CP003798.1, CP003795.1, CP003790.1, CP003797.1, and CP003796.1, respectively) to identify specific regions. Using Primer Explorer V5 software, LAMP primers were designed using the identified specific regions as templates, resulting in primer combinations consisting of four primers: FIP, BIP, F3, and B3 (Table 1). Their nucleotide sequences are shown in SEQ ID NO: 1-4. The primers shown in Table 1 were synthesized by Sangon Biotech Shanghai Co., Ltd.

[0048]

[0049] 2. Construction of positive plasmids

[0050] Using the pUC57-Kan plasmid as a framework, the IncA gene sequence of Chlamydia psittaci strain 6BC (SEQ ID NO:5) was inserted between the KpnI and SalI restriction sites of the pUC57-Kan plasmid to obtain the sequence of the positive plasmid pUC57-IncA. The positive plasmid pUC57-IncA was synthesized by Sangon Biotech Shanghai Co., Ltd., and transformed into Trans10 competent cells (Beijing TransGold) to obtain the recombinant strain Trans10-pUC57-IncA.

[0051] Activate the recombinant bacteria Trans10-pUC57-IncA in a kanamycin-containing culture dish. Pick a single colony and inoculate it into 5 mL of LB liquid medium, then incubate overnight at 37°C on a shaker. Remove the bacterial culture and transfer it to a 1.5 mL EP tube for centrifugation. Collect the culture by multiple centrifugations at 1000 g for 1 min. Discard the supernatant and collect the remaining supernatant with filter paper. Extract the positive plasmid pUC57-IncA from Trans10-pUC57-IncA using a plasmid miniprep kit (Beijing TransGen, catalog number EM101-02) according to the product instructions.

[0052] The concentration of the extracted positive plasmid pUC57-IncA was determined to be 70 ng / μL using an ultra-micro spectrophotometer. Based on the classical molecular copy number calculation formula: [copy number = (DNA / ng × 6.022 × 10⁻⁶)], the concentration was calculated as follows: 23) / fragment length / bp×660×10 9 The copy number concentration of the original pUC57-IncA plasmid solution was calculated to be 1.7 × 10⁻⁶. 10 copies / μL.

[0053] 3. Establishment of the LAMP system

[0054] Using the primers shown in Table 1, multiple 25 μL reaction systems were prepared for loop-mediated isothermal amplification (LAMP) to screen for the optimal LAMP reaction system.

[0055] Reaction system A: 10× Isothermal Amplification Buffer 2.5 μL, MgSO4 (100 mM) 1.5 μL, dNTPs (10 mM) 3.5 μL, inner primer FIP / BIP (100 μM) 1 μL each, outer primer F3 / B3 (100 μM) 1 μL each, Bst 2.0 WarmStart DNA polymerase (8000 U / mL) 1 μL, template 1 μL, and water to make up the remaining 25 μL.

[0056] Reaction system B: 10× Isothermal Amplification Buffer 2.5 μL, MgSO4 (100 mM) 1.5 μL, dNTPs (10 mM) 3.5 μL, inner primers FIP / BIP (100 μM) 4 μL each, outer primers F3 / B3 (100 μM) 1 μL each, Bst 2.0 WarmStart DNA polymerase (8000 U / mL) 1 μL, template 1 μL, and water to make up the remaining 25 μL.

[0057] Reaction system C: 10× Isothermal Amplification Buffer 2.5 μL, MgSO4 (100 mM) 1.5 μL, dNTPs (10 mM) 3.5 μL, inner primers FIP / BIP (100 μM) 4 μL each, outer primers F3 / B3 (100 μM) 2 μL each, Bst 2.0 WarmStart DNA polymerase (8000 U / mL) 1 μL, template 1 μL, and water to make up the remaining 25 μL.

[0058] Reaction system D: 10× Isothermal Amplification Buffer 2.5 μL, MgSO4 (100 mM) 0.7 μL, dNTPs (10 mM) 3.5 μL, inner primers FIP / BIP (100 μM) 4 μL each, outer primers F3 / B3 (100 μM) 2 μL each, Bst 2.0 WarmStart DNA polymerase (8000 U / mL) 1 μL, template 1 μL, and water to make up the remainder to 25 μL.

[0059] Reaction system E: 10× Isothermal Amplification Buffer 2.5 μL, MgSO4 (100 mM) 1.5 μL, dNTPs (10 mM) 3.5 μL, inner primers FIP / BIP (100 μM) 4 μL each, outer primers F3 / B3 (100 μM) 2.5 μL each, Bst 2.0 WarmStart DNA polymerase (8000 U / mL) 1 μL, template 1 μL, and water to make up the remainder to 25 μL.

[0060] Reaction system F: 10× Isothermal Amplification Buffer 2.5 μL, MgSO4 (100 mM) 1.5 μL, dNTPs (10 mM) 3.5 μL, inner primers FIP / BIP (100 μM) 4 μL each, outer primers F3 / B3 (100 μM) 3 μL each, Bst 2.0 WarmStart DNA polymerase (8000 U / mL) 1 μL, template 1 μL, and water to make up the remaining 25 μL.

[0061] Positive control: with a copy number concentration of 1.7 × 10⁻⁶. 10 The positive plasmid pUC57-IncA (copies / μL) was used as a template.

[0062] Negative control: using nucleic acid-free water as a template.

[0063] Reaction conditions: After mixing the reaction system, place it in a 65 ℃ metal bath and react at a constant temperature for 1 h.

[0064] After the reaction, the reaction products were subjected to 1.5% agarose gel electrophoresis (70 V, 90 min), and the electrophoretic bands were observed using a gel imaging system. The presence of clear step-like bands indicated a positive result for Chlamydia psittaci, while the absence of bands indicated a negative result for Chlamydia psittaci.

[0065] like Figure 1 As shown, reaction system F is optimal. LAMP products from reaction system F were analyzed by 1.5% agarose gel electrophoresis. The positive control showed typical step-like bands, while the negative control showed no amplification bands, indicating stable and reliable performance.

[0066] 4. Establishment of the LAMP-HNB system

[0067] Introducing hydroxynaphthol blue (HNB) as a colorimetric indicator into the loop-mediated isothermal amplification (LAMP) system enables visualization of the amplification products, allowing direct interpretation of detection results through color changes. First, 100× HNB indicator (Beijing Solarbio) was diluted to 10× HNB dye with nucleic acid-free water. Then, 2.5 μL of 10× HNB dye was added to the above reaction system F beforehand, resulting in a total volume of 25 μL of LAMP-HNB system. A positive control was prepared with a copy number concentration of 1.7×10⁻⁶. 10 The positive plasmid pUC57-IncA (copies / μL) was used as a template. Nucleic acid-free water was used as a negative control template. After mixing the reaction mixture, it was incubated in a 65 °C metal bath for 1 h. After the reaction, if the color of the reaction solution changed from violet (initial state, Mg...) to... 2+ -HNB complex) turns sky blue (positive, Mg 2+ If the reaction solution is exhausted, it is considered positive for Chlamydia psittaci; if there is no color change in the reaction solution, it is considered negative for Chlamydia psittaci.

[0068] like Figure 2 As shown, the LAMP-HNB system exhibits significant color differences under natural light: the reaction solution of the positive control appears sky blue, while the reaction solution of the negative control retains its initial violet color. The HNB color development results are completely consistent with the electrophoretic detection results of the LAMP amplification products.

[0069] 5. Sensitivity Analysis

[0070] For a copy number concentration of 1.7 × 10⁻⁶ 10 The pUC57-IncA plasmid solution was serially diluted 10-fold to prepare 7 concentrations (1.7 × 10⁻⁶ copies / μL). 6 1.7×10 5 1.7×10 4 1.7×10 3 1.7×10 2 1.7×10 1 1.7×10 0 Plasmid samples (copies / μL).

[0071] Seven plasmid samples of varying concentrations were used as templates for LAMP reactions in reaction system F described above. Nucleic acid-free water was used as a negative control template. After mixing the reaction system, the mixtures were incubated at 65 °C in a metal bath for 1 h. The products were then subjected to 1.5% agarose gel electrophoresis (70 V, 90 min), and the bands were observed using a gel imaging system to interpret the results. The presence of clear, stepped bands indicated a positive result for *Chlamydia psittaci*, while the absence of bands indicated a negative result.

[0072] Simultaneously, seven plasmid samples of different concentrations were used as templates, and 2.5 μL of 10× HNB dye was added to reaction system F beforehand to obtain a total volume of 25 μL of LAMP-HNB system. Nucleic acid-free water was used as a negative control template. After mixing the LAMP-HNB system, it was incubated in a 65 ℃ metal bath for 1 h. After the reaction, the color change of the reaction solution was observed to determine the experimental results. If the color of the reaction solution changed from violet to sky blue, it was considered positive for *Chlamydia psittaci*; if there was no color change, it was considered negative for *Chlamydia psittaci*.

[0073] like Figure 3 As shown, when the plasmid copy number is 1.7 × 10⁻⁶ 6 ~1.7×10 2 Within the copy number range of 1.7 × 10⁻⁶, LAMP reactions can be effectively amplified, and agarose gel electrophoresis analysis shows clear ladder-like bands (lanes 1-5); however, when the copy number is below 1.7 × 10⁻⁶, the amplification is significantly reduced. 2 At copies / μL, no stepwise amplification bands appeared in the LAMP reaction. Therefore, the limit of detection for reaction system F is 10. 2 It has high sensitivity, with copies / μL.

[0074] like Figure 4 As shown, when the plasmid copy number is 1.7 × 10⁻⁶ 6 ~1.7×10 2 When the copy number is within the range of copies / μL, the LAMP-HNB reaction solution appears sky blue; when the copy number is below 1.7 × 10⁻⁶, the reaction solution appears sky blue. 2 At copies / μL, the LAMP-HNB reaction solution showed the same color as the negative control, exhibiting the initial violet hue. Therefore, the color development results of the LAMP-HNB reaction solution are highly consistent with the agarose gel electrophoresis results of the LAMP reaction products.

[0075] 6. Specificity analysis

[0076] To verify the specificity of the LAMP primer combination, genomic DNA from Campylobacter jejuni (C. jejuni) (Cultural Heritage No.: NCTC11168), Escherichia coli (E. coli) (Cultural Heritage No.: ATCC25922), Salmonella enterica (S. enterica) (Cultural Heritage No.: CVCC1800), Chlamydia psittaci CP3 (positive clinical sample), Chlamydia psittaci type B (NCBI accession number: PV868337), and the aforementioned positive plasmid pUC57-IncA were used to test the reaction system. All genomic DNA was provided by the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences.

[0077] The genomic DNA described above was used as a template for LAMP reaction in reaction system F. Nucleic acid-free water was used as a negative control template. After mixing the reaction system, it was incubated in a 65 °C metal bath for 1 h. The products were then subjected to 1.5% agarose gel electrophoresis (70 V, 90 min), and the bands were observed using a gel imaging system to determine the experimental results. The appearance of clear, stepped bands indicated a positive result for *Chlamydia psittaci*, while the absence of bands indicated a negative result.

[0078] Simultaneously, using the aforementioned genomic DNA as templates, 2.5 μL of 10× HNB dye was added to reaction system F beforehand, resulting in a total volume of 25 μL of LAMP-HNB system. Nucleic acid-free water was used as the negative control template. After mixing the LAMP-HNB system, it was incubated in a 65 ℃ metal bath for 1 h. After the reaction, the color change of the reaction solution was observed to determine the experimental results. If the color of the reaction solution changed from violet to sky blue, it was considered positive for *Chlamydia psittaci*; if there was no color change, it was considered negative for *Chlamydia psittaci*.

[0079] like Figure 5As shown, when the template was the positive plasmid pUC57-IncA, *C. psittaci* type B genomic DNA, or *C. psittaci* CP3 genomic DNA, the LAMP reaction showed specific amplification, and agarose gel electrophoresis showed clear ladder-like bands (lanes 1-3). When the template was *C. jejuni* genomic DNA, *E. coli* genomic DNA, or *S. enterica* genomic DNA, the LAMP reaction did not show specific amplification, and agarose gel electrophoresis showed no typical amplification bands (lanes 4-6). The results indicate that the developed LAMP primer combination and LAMP detection method have good specificity and can accurately distinguish *C. psittaci* from other pathogens.

[0080] like Figure 6 As shown, when the template is the positive plasmid pUC57-IncA, C. psittaci type B genomic DNA, or C. psittaci CP3 genomic DNA (tubes 1-3), the LAMP-HNB reaction solution appears sky blue; when the template is C. jejuni genomic DNA, E. coli genomic DNA, or S. enterica genomic DNA (tubes 4-6), the LAMP-HNB reaction solution is consistent with the negative control, appearing violet. Therefore, the colorimetric interpretation results of the LAMP-HNB reaction solution are highly consistent with the agarose gel electrophoresis detection results of the LAMP reaction products.

[0081] Example 2: Preparation of a kit for detecting Chlamydia psittaci

[0082] 1. Reagent kit assembly

[0083] Synthesize the nucleotide sequences developed in Example 1, as shown in SEQ ID NO:1-4, using primers F3, B3, FIP, and BIP. Load the solid primers directly into centrifuge tubes, or prepare primer solutions at 10-100 times working concentration and load them into centrifuge tubes. Then, load these solutions into a kit along with the universal loop-mediated isothermal amplification (LAMP) reagent, hydroxynaphthol blue (HNB), and the kit instructions. Label the kit casing.

[0084] 2. Reagent Kit Instructions

[0085] Using the sample as a template, LAMP reaction was performed using primers FIP, BIP, F3, and B3. The reaction system consisted of: 2.5 μL 10×Buffer, 1.5 μL MgSO4 (100 mM), 3.5 μL dNTPs (10 mM), 4 μL each of inner primers FIP and BIP (100 μM), 3 μL each of outer primers F3 and B3 (100 μM), 1 μL DNA polymerase (8000 U / mL), 2.5 μL 10× HNB dye, 1 μL template, and water to a final volume of 25 μL. The reaction was carried out at 65 ℃ for 1 h. After the reaction, the color change of the reaction solution was observed. If the reaction solution changed from violet to sky blue, the sample was considered positive for Chlamydia psittaci; if there was no color change, the sample was considered negative for Chlamydia psittaci.

Claims

1. A LAMP primer combination for detecting Chlamydia psittaci, characterized in that, The primer combination includes the following primers: FIP: 5'-GTACATGTTTCAAGTTTATTGCCGATGCTAACCAAATTTCAGAACT-3', BIP: 5'-ACTTGGAAGCTTTCAAATCTGTAGGTAAACGCTCAAAAGGAGAGA-3', F3: 5'-GTATCTGCAAACAGGGATCT-3', and B3: 5'-TCCTGTTAACGATTCCGC-3'.

2. A kit for detecting Chlamydia psittaci, characterized in that, Includes the LAMP primer combination as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The primers FIP, BIP, F3, and B3 are packaged separately.

4. The reagent kit according to claim 2, characterized in that, The primers FIP, BIP, F3 and B3 are packaged together, wherein the molar ratio of FIP, BIP, F3 and B3 is 4:4:3:

3.

5. The reagent kit according to claim 2, characterized in that, It also includes universal reagents for loop-mediated isothermal amplification.

6. The reagent kit according to claim 5, characterized in that, The general reagents include isothermal amplification buffer, MgSO4 solution, and DNA polymerase.

7. The reagent kit according to claim 6, characterized in that, The working concentrations of primers FIP, BIP, F3, and B3 are 16 μM, 16 μM, 12 μM, and 12 μM, respectively, and the working concentration of MgSO4 is 6 mM.

8. The kit according to any one of claims 2-7, characterized in that, It also includes hydroxynaphthol blue or a general reagent for gel electrophoresis.

9. The use of the LAMP primer combination of claim 1 in the preparation of products for detecting Chlamydia psittaci.

10. The application of the LAMP primer combination according to claim 1 in the detection of Chlamydia psittaci.

Images