Fluorescent quantitative PCR (Polymerase Chain Reaction) detection method and kit for giardia duodenalis

Abstract

The invention provides a fluorescent quantitative PCR (Polymerase Chain Reaction) detection method and kit for giardia duodenalis, the giardia duodenalis is accurately and quantitatively detected by utilizing a TaqMan fluorescent quantitative technology, and by constructing a BG gene plasmid with high purity and good stability as a standard substance and combining with an optimized TaqMan fluorescent quantitative PCR reaction system and conditions, the fluorescent quantitative PCR detection method for the giardia duodenalis can be used for accurately and quantitatively detecting the giardia duodenalis. High sensitivity, high specificity and accurate quantitative detection on the giardia duodenalis are realized, so that the requirements on giardia detection in the fields of clinical diagnosis, environmental monitoring, epidemiological research and the like are met.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular provides a method for the quantitative PCR detection of Giardia lamblia, which utilizes TaqMan quantitative PCR technology for accurate quantitative detection of Giardia lamblia. The invention further provides a kit for implementing this method. Background Technology

[0002] Giardia lamblia ( Giardia duodenalis Giardia lamblia is a widespread intestinal parasite that can cause diarrhea and other intestinal diseases in humans and various animals, seriously threatening public and animal health. It is estimated that approximately 280 million people worldwide are infected with Giardia lamblia. Currently, there is still no effective vaccine available to prevent Giardiasis. While some drugs are effective in treating Giardiasis, drug resistance has become a worrying issue. Individuals who frequently come into contact with animals, whether pets or farm animals, have been reported to exhibit higher rates of infection. Therefore, developing an effective method for detecting Giardia lamblia to reduce economic losses is of public health and veterinary significance.

[0003] Traditional methods for detecting Giardia lamblia, such as microscopic examination, require highly experienced operators, have low sensitivity, and are prone to false negatives. While immunological detection methods possess some specificity, cross-reactivity can affect the accuracy of results. Molecular biological detection techniques based on nucleic acid amplification, such as conventional PCR, can only provide qualitative or semi-quantitative detection and cannot precisely determine the Giardia lamblia content. Real-time quantitative PCR (qPCR) overcomes some of these shortcomings, enabling quantitative detection of pathogen nucleic acids with advantages such as high sensitivity, strong specificity, and speed. Specifically, TaqMan qPCR utilizes the 5' exonuclease activity of Taq polymerase. During PCR amplification, when the primer extends to the probe binding site, the Taq polymerase cleaves the fluorescent reporter group at the 5' end of the probe, separating it from the quencher group at the 3' end, thus generating a fluorescence signal. The intensity of the fluorescence signal is proportional to the amount of amplified product. Real-time monitoring of the fluorescence signal allows for quantitative analysis of the template nucleic acid. However, existing TaqMan qPCR methods for detecting Giardia lamblia still require further improvement in terms of standard construction, accuracy, and stability. Therefore, developing an efficient, accurate, and stable TaqMan fluorescence quantitative detection method based on the Giardia lamblia BG gene is of great practical significance. Summary of the Invention

[0004] The present application aims to provide a duodenal Giardia lamblia fluorescent quantitative PCR detection method and kit, specifically to construct a plasmid containing the BG gene of duodenal Giardia lamblia as a standard for fluorescent quantitative detection, and the related technology for detecting duodenal Giardia lamblia has the characteristics of high sensitivity, strong specificity and good repeatability.

[0005] The present application provides a BG gene for identifying the duodenal Giardia lamblia in the above technical solution, and the conservative sequence of the BG gene is shown in SEQ ID NO: 1.

[0006] The present application provides a duodenal Giardia lamblia fluorescent quantitative PCR detection method, which comprises a primer pair for amplifying the BG gene in the above technical solution, and the primer pair is: The upstream primer BG - F is shown in SEQ ID NO: 2; The downstream primer BG - R is shown in SEQ ID NO: 3.

[0007] The present application provides a primer set for duodenal Giardia lamblia fluorescent quantitative PCR detection, which comprises: The upstream primer BG - qF is shown in SEQ ID NO: 4 The downstream primer BG - qR is shown in SEQ ID NO: 5 The probe primer BG - Probe is shown in SEQ ID NO: 6.

[0008] The present application provides a duodenal Giardia lamblia fluorescent quantitative PCR detection kit, which comprises the primer set, the upstream primer BG - qF, the downstream primer BG - qR, the probe primer BG - Probe and the TaqMan fluorescent quantitative PCR detection reagent.

[0009] The present application provides a preparation method of a duodenal Giardia lamblia fluorescent quantitative PCR detection kit, comprising the following steps 1. Extracting sample genome; 2. Using the detection reagent and the primer set to perform fluorescent quantitative PCR detection on the genome of step 1); 3. Obtaining the amplification curve and Ct value of step 2); 4. The result can be judged by Ct. When the Ct value of the sample to be tested is < 35, the sample detection result is positive, and the sample contains duodenal Giardia lamblia; when the Ct value is ≥ 35, the sample detection result is negative, and the sample does not contain duodenal Giardia lamblia.

[0010] The positive effect of the present application is that: the TaqMan fluorescence quantitative technology is used for accurate quantitative detection of duodenal Giardia, a high-purity and stable BG gene plasmid is constructed as a standard product, combined with an optimized TaqMan fluorescence quantitative PCR reaction system and conditions, high sensitivity, high specificity and accurate quantitative detection of duodenal Giardia are realized, so as to meet the needs of clinical diagnosis, environmental monitoring and epidemiological research and other fields for Giardia detection. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 It is the PCR amplification result of duodenal Giardia BG gene (from left to right, DL1000 Marker, BG gene PCR amplification product); Figure 2 It is the standard curve drawn by the method of the present application (the horizontal coordinate is the logarithm of the concentration of the standard product, the vertical coordinate is the Ct value, the curve has a good linear relationship, and the correlation coefficient R 2 is greater than 0.99, etc.); Figure 3 It is the sensitivity detection result of the method of the present application (from left to right, 1 × 10 9 copies / μL, 1 × 10 8 copies / μL, 1 × 10 7 copies / μL, 1 × 10 6 copies / μL, 1 × 10 5 copies / μL, 1 × 10 4 copies / μL as template amplification curve); Figure 4 It is the specificity detection result of the method of the present application. DETAILED DESCRIPTION

[0012] In order to facilitate the technical personnel in the field to better understand the present application scheme, the following will combine the drawings and examples to explain and introduce the present application scheme in detail. However, the described examples are only a part of the embodiments of the present application, and therefore the present application is not limited in the described examples.

[0013] Example 1

[0014] Genomic DNA was extracted from in vitro pure cultured duodenal Giardia trophozoites as a PCR amplification template, and according to the published BG gene sequence, the amplification primers BG-F and BG-R were designed by Shenguo Bioengineering (Shanghai) Co., Ltd. Select BamH I and EcoR I as the enzyme cutting site, clone into pET28a prokaryotic expression vector, and transform into E. coli DH5α competent cells. Pick single colony for expansion culture, identify positive clones by bacterial liquid PCR, and send to Shenguo Bioengineering (Shanghai) Co., Ltd. for sequencing of bacterial liquid.

[0015] The plasmid extracted from the correct bacterial liquid was sequenced and named pET28a - BG, which was used as the duodenal Giardia positive plasmid standard. The concentration and purity of the plasmid were determined using a microspectrophotometer, and the OD 260 / OD 280 The ratio was between 1.8 and 2.0, and the concentration was 120 ng / μL, indicating that the extracted plasmid was of good quality and could be used to draw a standard curve.

[0016] The total volume of the PCR reaction system for amplifying the duodenal Giardia BG gene was 20 μL: 2 × PCR Mix 10 μL, BG - F 1 μL, BG - R 1 μL, template 2 μL, ddH2O 6 μL.

[0017] The reaction conditions were as follows: 95 ℃ pre-denaturation for 5 min; 95 ℃ for 30 s, 58 ℃ for 30 s, 72 ℃ for 1 min, 35 cycles.

[0018] The results of agarose gel electrophoresis showed that the BG gene with a single band was obtained by PCR amplification, and gene sequencing proved that the constructed pET28a - BG standard plasmid had no mutation, insertion or deletion, and was consistent with the published BG gene sequence (such as Figure 1 ).

[0019] The primer sequence and the full-length sequence of BG amplification are as follows: the conserved sequence of the BG gene is SEQ ID NO: 1; The primer pair of the BG gene is as follows: The upstream primer BG - F is as shown in SEQ ID NO: 2; The downstream primer BG - R is as shown in SEQ ID NO: 3.

[0020] Experimental Example 2, a kit for detecting duodenal Giardia by fluorescence quantitative PCR The kit comprises the following steps: 1. Dilution of plasmid standard The recombinant pET28a - BG plasmid correctly sequenced in Example 1 was gradient diluted, and sterilized ddH2O was used for 10-fold dilution in sequence to prepare standard solution of 1 × 10 9 copies / μL, 1 × 10 8 copies / μL, 1 × 10 7 copies / μL, 1 × 10 6 copies / μL, 1 × 10 5 copies / μL, 1 × 10 4 copies / μL.

[0021] 2. Design of detection primer and probe The BG gene sequence of duodenal Giardia was retrieved from the nucleic acid database GenBank of NCBI (http: / / www.ncbi.nlm.nih.gov) (GenBank sequence number: AY655702, EU014394, AY072723, EU642897, AY072727, AY545646, MT713327), the gene sequence was analyzed by using MEGA11 software, the conserved nucleotide sequence was selected, and the related sequence was designed according to the principle of primer and TaqMan probe design: The upstream primer BG - qF is shown in SEQ ID NO: 4; The downstream primer BG - qR is shown in SEQ ID NO: 5; The probe primer BG - Probe is shown in SEQ ID NO: 6.

[0022] 3. Establishing a standard curve 1 × 10 9 copies / μL, 1 × 10 8 copies / μL, 1 × 10 7 copies / μL, 1 × 10 6 copies / μL, 1 × 10 5 copies / μL of the standard solution were used as templates to perform TaqMan fluorescent quantitative PCR reaction.

[0023] The fluorescent quantitative PCR reaction system was 50 μL: 2 × SGExcel GoldStar TaqMan Mixture 25 μL, BG - qF (10 μM) 1 μL, BG - qR (10 μM) 1 μL, BG - Probe (10 μM) 1 μL, 2 μL of different dilution concentrations of plasmid standard, and 30 μL of ddH2O.

[0024] The reaction conditions were as follows: 95 ℃ pre-denaturation for 10 min; 95 ℃ for 15 s, 60 ℃ for 1 min, and 72 ℃ for 1 min for 40 cycles. The obtained Ct value was calculated to obtain the standard curve equation, and the standard curve was drawn.

[0025] The results showed that the standard curve equation and the amplification curve drawn are shown in the figure, the correlation coefficient R 2 > 0.99, indicating that the standard plasmid copy number of the TaqMan fluorescent quantitative PCR method has a good linear relationship with the Ct value (as Figure 2 , Figure 3 ).

[0026] 4. Specific detection Using the genomes of Giardia duodenalis trophozoites, Cryptosporidium microsporum, Toxoplasma gondii, Eimeria spp., and Escherichia coli as templates, the specificity of the method was verified by TaqMan real-time PCR.

[0027] The results showed that the established TaqMan real-time PCR detection method had good specificity, exhibiting good amplification only for the Giardia duodenalis trophozoite genome. No amplification curves or Ct values ​​were observed for the genomes of Cryptosporidium microsporum, Toxoplasma gondii, Eimeria tenella, and Escherichia coli. (e.g.) Figure 4 ).

[0028] Example 3: Application of the Giardia lamblia detection kit 1. Sample collection

[0029] In 2023, fecal samples were collected from raccoon farmer farms in Mudanjiang City, Heilongjiang Province. The samples were immediately transported to the laboratory, where nucleic acids were extracted using a fecal DNA extraction kit. The samples were then aliquoted and stored at -80°C.

[0030] 2. Sample testing The extracted nucleic acid was used as a template and amplified and detected according to the TaqMan quantitative PCR reaction system and conditions described in Example 2. Nested PCR was also used for detection. The results showed that the positive detection rate of the TaqMan quantitative PCR method of this invention was slightly higher than that of the microscopic examination method, further verifying the superiority of the method of this invention.

[0031] Table 1: Clinical Sample Test Results

[0032] Conclusion: This invention enables highly sensitive, specific, and accurate quantitative detection of Giardia lamblia, and can be used for Giardia detection in fields such as clinical diagnosis, environmental monitoring, and epidemiological research.

Claims

1. A primer set for detecting Giardia duodenalis by fluorescent quantitative PCR, characterized in that: the primer set comprises an upstream primer BG - qF as shown in SEQ ID NO: 4; a downstream primer BG - qR as shown in SEQ ID NO: 5; and a probe primer BG - Probe as shown in SEQ ID NO:

6. The kit comprises the primer set of claim 1, the upstream primer BG - qF, the downstream primer BG - qR, the probe primer BG - Probe and TaqMan fluorescent quantitative PCR detection reagent. 3.A method for preparing the kit for detecting Giardia duodenalis by fluorescent quantitative PCR according to claim 2, comprising the following steps: 1) dilution of plasmid standard 2. A kit for the detection of Giardia duodenale by fluorescent quantitative PCR, characterized in that it comprises: 2) design of detection primer and probe The BG gene sequences of Giardia duodenalis were retrieved from the nucleic acid database GenBank of NCBI: AY655702, EU014394, AY072723, EU642897, AY072727, AY545646, MT713327, the gene sequences were analyzed by using MEGA11 software, the conservative nucleotide sequences were selected, and the related sequences were designed according to the design principles of primer and TaqMan probe: the upstream primer BG - qF as shown in SEQ ID NO: 4; The recombinant pET28a-BG plasmid was gradient diluted, 10-fold dilution was performed successively using sterilized ddH2O, and the standard solution of 1 × 10 9 copies / μL, 1 × 10 8 copies / μL, 1 × 10 7 copies / μL, 1 × 10 6 copies / μL, 1 × 10 5 copies / μL, 1 × 10 4 copies / μL, 1 × 10 copies / μL the downstream primer BG - qR as shown in SEQ ID NO: 5; the probe primer BG - Probe as shown in SEQ ID NO: 6; 3) establishment of standard curve The fluorescent quantitative PCR reaction system is 50 μL: 2 × SGExcel GoldStar TaqMan Mixture 25 μL, BG - qF (10 μM) 1 μL, BG - qR (10 μM) 1 μL, BG - Probe (10 μM) 1 μL, different dilution concentrations of plasmid standard 2 μL, and ddH2O 30 μL; The reaction conditions are as follows: 95 ℃ pre-denaturation for 10 min; 95 ℃ for 15 s, 60 ℃ for 1 min, and 72 ℃ for 1 min for 40 cycles; the obtained Ct value is calculated to obtain the standard curve equation, and the standard curve is drawn; 4) specific detection 1 x 10 9 copies / μL, 1 x 10 8 copies / μL, 1 x 10 7 copies / μL, 1 x 10 6 copies / μL, 1 x 10 5 copies / μL of standard solution as template, TaqMan fluorescent quantitative PCR reaction was carried out; The trophozoite genome of Giardia duodenalis, the genome of Cryptosporidium parvum, the genome of Toxoplasma gondii, the genome of Eimeria stiedoni and the genome of Escherichia coli were used as templates, and the established TaqMan fluorescent quantitative PCR was used for detection to verify the specificity of the method. ​ ​ ​

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