Specific molecular target of shigella flexneri and detection method thereof

Abstract

The invention discloses a specific molecular target of shigella flexneri and a detection method of the specific molecular target. The nucleotide sequence of the specific molecular target is as shown in SEQ ID NO. 1. The nucleotide sequences of an upstream primer of the specific molecular target SEQ ID NO.1 are as shown in SEQ ID NO.2 and SEQ ID NO.5, and the nucleotide sequences of a downstream primer of the specific molecular target SEQ ID NO.1 are as shown in SEQ ID NO.3 and SEQ ID NO.6. And the nucleotide sequences of the TaqMan probes are as shown in SEQ ID NO. 4 and SEQ ID NO. 7. According to the invention, the detection sensitivity of the shigella flexneri can reach 3.9 * 10 < 2 > CFU / mL, meanwhile, the method has better specificity and repeatability, and a simple, convenient, rapid, efficient and low-cost method is provided for the detection of the shigella flexneri.

Description

Technical Field

[0001] The invention relates to the technical field of bacteria detection, and in particular to a specific molecular target of Shigella flexneri and a fluorescence quantitative PCR detection method thereof. Background Art

[0002] Shigella is a Gram-negative, facultative anaerobic, non-spore-forming, non-motile and usually lactose-free rod-shaped bacterium. First discovered by Japanese scientists 100 years ago, Shigellosis is a highly contagious and severe inflammatory diarrhea caused by infection through the fecal-oral route. Its high morbidity and mortality in developing countries have attracted global attention. Shigella is divided into four types: Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei, and Shigella flexneri is the most moderate serotype. In developing and developed countries, about 60% of bacterial dysentery is found to be caused by Shigella flexneri. Shigella flexneri mainly grows in acidic conditions and saline-alkali environments. In addition, Shigella flexneri is highly toxic to various foods such as vegetables, meat, bread, milk, etc. Shigella flexneri strains invade the colon and rectal epithelium of the human body and induce a strong inflammatory response, ultimately leading to severe tissue damage, which manifests as a range of clinical symptoms, from watery diarrhea to fever, abdominal cramps, and bloody mucoid stools. Shigella flexneri is highly contagious, with only 10-100 bacterial cells capable of causing infection. In order to maintain global health and food safety, early detection of Shigella flexneri is critical for accurate diagnosis of infectious diseases and rapid control of outbreaks.

[0003] At present, the conventional national standard method is bacterial enrichment and selective culture, followed by biochemical and serological methods for detection and typing. However, this process often takes several days, which makes the detection efficiency extremely low. With the development of molecular biology technology, especially fluorescent qPCR technology, it is increasingly widely used in the detection and identification of Shigella pathogens.

[0004] The accuracy of the results of nucleic acid amplification methods based on molecular biology mainly depends on the high quality of the detection target. However, fluorescent qPCR is the gold standard for molecular detection, and the design of its primers is crucial to the accuracy of the specificity and sensitivity of its detection method. ipaH The gene sequence was used as a fluorescent qPCR target gene to detect Shigella flexneri, but Shigella and Escherichia coli showed 80%-90% similarity at the nucleotide level, and both Shigella and enteroinvasive Escherichia coli (EIEC) were present. ipaH Genes make it difficult to distinguish EIEC from Shigella flexneri. Therefore, there is an urgent need to systematically and comprehensively explore new targets for specific detection to improve the accuracy of fluorescent qPCR detection of Shigella flexneri. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and further explore the specific targets of Shigella flexneri through pan-genomic analysis, thereby providing a TaqMan fluorescent quantitative PCR detection method based on a new target.

[0006] The present invention is experimentally performed through the following technical solutions: A specific molecular target for detecting Shigella flexneri, wherein the nucleotide sequence of the specific molecular target is shown as SEQ ID NO.1.

[0007] A set of primers for detecting specific molecular targets of Shigella flexneri, the nucleotide sequences of the upstream primers of the specific molecular target SEQ ID NO.1 are shown in SEQ ID NO.2 and SEQ ID NO.5, and the nucleotide sequences of the downstream primers are shown in SEQ ID NO.3 and SEQ ID NO.6.

[0008] A set of TaqMan probes for detecting specific molecular targets of Shigella flexneri, the nucleotide sequences of the TaqMan probes are shown in SEQ ID NO. 4 and SEQ ID NO. 7. The 5' end of the TaqMan probe is labeled with a reporter fluorescent dye FAM, and the 3' end is labeled with a fluorescent quencher group BHQ1.

[0009] Application of the above-mentioned specific molecular target for detecting Shigella flexneri in preparing a reagent for detecting Shigella flexneri.

[0010] A kit for detecting Shigella flexneri, comprising all nucleotide sequences described in SEQ ID NO.1-7.

[0011] A method for detecting Shigella flexneri for non-disease diagnosis and treatment purposes, comprising the following steps: (1) Obtain the DNA of the microorganism to be tested, and use the extracted DNA as a template and the specific molecular target SEQ ID NO.1 to design primers and probes; (2) Perform qPCR amplification using primers and probes designed based on the specific molecular target SEQ ID NO.1, and collect the fluorescence signal; (3) Result determination: Determine whether the sample contains Shigella flexneri based on the fluorescence signal and Ct value collected in step (2). When the Ct value is greater than 35 or the system determines it to be negative, the result is determined to be negative.

[0012] Preferably, in the above-mentioned method for detecting Shigella flexneri, the reaction system for qPCR amplification is 10 mL 2×Pro Taq HS Probe Premix, 0.7 μL of each upstream and downstream primer with an initial concentration of 10 μM, 1 μL of the probe with an initial concentration of 10 μM, the total volume of the template and ddH2O is 7.6 μL, and the total volume of the system is 20 μL.

[0013] Preferably, in the above method for detecting Shigella flexneri, the reaction program of the qPCR amplification is: the fluorescence channel is set to temperature control, the program is set to 95°C 5min; 40 cycles include 95°C 15s, 60°C 30s; the qPCR instrument fluorescence channel is set to: FAM, and the fluorescence signal is collected by the qPCR instrument at the same time.

[0014] The beneficial effects of the present invention are as follows: The present invention discloses a specific molecular sequence for identifying Shigella flexneri, and provides relevant specific primers and probe sequences, as well as a corresponding qPCR detection method. The detection sensitivity of the present invention for Shigella flexneri can reach 3.9×10 2 CFU / mL, with good specificity and repeatability, provides a simple, fast, efficient and low-cost method for the detection of Shigella flexneri. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a graph verifying the results of the Shigella flexneri SF-1 primer and probe qPCR detection method in Example 1.

[0016] Figure 2 This is a graph showing the experimental results of Example 1 that the primers and probes for Shigella flexneri SF-1 are most suitable for the Taqman qPCR detection system.

[0017] Note: A is the optimization of primer concentration in the Taqman qPCR detection method for Shigella flexneri; B is the optimization of probe concentration in the Taqman qPCR detection method for Shigella flexneri.

[0018] Figure 3 This is a validation diagram of the sensitivity test results of the Shigella flexneri Taqman qPCR detection method of Example 1 and its standard curve.

[0019] Figure 4 This is a verification diagram of the specificity test results of the Shigella flexneri Taqman qPCR detection method in Example 1. DETAILED DESCRIPTION Example 1

[0020] (1) Analysis of Shigella flexneri-specific genes and primers A total of 498 Shigella and non-Shigella genome sequences were downloaded from the NCBI (https: / / www.ncbi.nlm.nih.gov / genome) database. The genome sequences that met the requirements were annotated using the annotation software prokka, and then the annotation results were analyzed using Roary. Genes that were present in more than 95% of all Shigella flexneri genome sequences and not present (or present in less than 5%) in other non-target strain genome sequences were selected as candidate target genes. After obtaining these target genes, BLAST comparison was performed on the NCBI database for further screening, and finally the specific target genes of Shigella flexneri were obtained.

[0021] (2) Primers and probes were designed based on the specific target sequences of Shigella flexneri screened in the early stage, and the designed primers were compared by BLAST on the NCBI website to ensure primer specificity (Table 1).

[0022]

[0023] (3) Bacterial culture and DNA extraction The Shigella flexneri strain ATCC12022 in the cryopreserved tube was selected as the target strain, inoculated into sterile Luria-Bertani broth for activation culture, and gradient dilution and plate count were performed with sterile PBS. Shigella flexneri, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Vibrio fluvii, Escherichia coli, Cronobacter sakazakii, Salmonella and other strains were cultured and their DNA was extracted for subsequent target gene specificity verification. Shigella flexneri and non-Shigella were inoculated into liquid culture medium and cultured at 37°C until the logarithmic phase of growth. DNA was extracted from the bacterial solution using a DNA extraction kit and stored in a -20°C refrigerator as a template for subsequent amplification.

[0024] (4) Amplification system and amplification conditions The qPCR system was 20 μL, including 10 mL 2×Pro Taq HS Probe Premix, 0.7 mL each of upstream and downstream primers with an initial concentration of 10 mM, 1 mL of probe with an initial concentration of 10 mM, the total volume of the template and ddH2O was 7.6 mL, and the total volume of the system was 20 mL.

[0025] The reaction program of the qPCR amplification is: the fluorescence channel is set to temperature control, the program is set to 95°C for 5 min; 40 cycles include 95°C for 15 s and 60°C for 30 s; the fluorescence channel of the qPCR instrument is set to: FAM, and the fluorescence signal is collected by the qPCR instrument at the same time.

[0026] (5) Exploration of the optimal reaction system The selected SF-1 primers and probes were added to the same reaction system. Different primer and probe addition amounts were set, with the Shigella flexneri concentration of 3.9×10 6 CFU / mL was used to explore the optimal reaction conditions, as shown in Table 2 below.

[0027]

[0028] Table 2 and Figure 2 It can be seen that when the amount of primer added is 0.7 mL and the amount of probe added is 1 mL, the qPCR reaction has a lower Ct value and a stronger fluorescence intensity for the Shigella flexneri template. The optimal reaction system of the Shigella flexneri Taqman qPCR detection method is shown in Table 3 below.

[0029]

[0030] (6) Primer sensitivity verification and standard curve establishment The Shigella flexneri bacterial solution was diluted with sterile PBS to obtain bacterial solutions of different concentrations (3.9×10 6 –3.9×10 1 ), DNA was extracted. 2mL of DNA from each gradient was used as a template for qPCR reaction, and double distilled water was used as a negative control to explore the sensitivity of the detection method. At the same time, in order to confirm the reliability of the sensitivity, 3 replicates were set for each concentration using this detection method.

[0031]

[0032] Table 4 is the genomic DNA of Shigella flexneri (3.9×10 6 - 3.9×10 2 Ct value of the qPCR test result. Figure 3 Shigella flexneri (3.9×10 6 - 3.9×10 2 CFU / mL) qPCR amplification curve. Figure 3 It can be seen that the sensitivity of the qPCR detection method established in the present invention can reach 3.9×10 2 CFU / mL.

[0033] (7) Primer specificity verification Shigella flexneri, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Vibrio fluvii, 5 strains of Escherichia coli, 2 strains of Pseudomonas aeruginosa, Cronobacter sakazakii, and Salmonella were cultured at 37°C until the logarithmic phase of growth. DNA from different bacterial cultures was extracted by dilution with sterile PBS and used as template for qPCR amplification to investigate the specificity of SF-1 primers.

[0034]

[0035] The specificity of the SF-1 primer for Shigella flexneri was verified, and it was found that only the target pathogen was detected normally, while non-Shigella were not detected, indicating that the primer and its detection method have good specificity.

[0036] (8) Stability test Fluorescence quantitative qPCR detection method was used to detect Shigella flexneri, with the concentration ranging from 3.9×10 6 _ 3.9×10 2 CFU / mL, 3 groups (group 1, group 2 and group 3) were tested at different times, and each group was repeated 3 times (repeat 1, repeat 2 and repeat 3). Then the CV% values ​​between and within the groups were calculated, and the repeatability of the method was tested based on the CV%.

[0037]

[0038] The stability of the detection method was determined by comparing the CV% values ​​between and within the groups of the multiple detection method for detecting a single pathogen. As shown in Table 5, most of the coefficients of variation between and within the groups of each concentration gradient were less than 1%, and a few were between 1% and 5%. The results showed that the detection method had excellent stability.

Claims

1. A specific molecular target for detecting Shigella flexneri, characterized in that The nucleotide sequence of the specific molecular target is shown in SEQ ID NO.

1.

2. A set of primers for detecting specific molecular targets of Shigella flexneri as claimed in claim 1, characterized in that: The nucleotide sequences of the upstream primers of the specific molecular target SEQ ID NO.1 are shown in SEQ ID NO.2 and SEQ ID NO.5, and the nucleotide sequences of the downstream primers are shown in SEQ ID NO.3 and SEQ ID NO.

6.

3. A set of TaqMan probes for detecting specific molecular targets of Shigella flexneri as claimed in claim 1, characterized in that: The nucleotide sequences of the TaqMan probes are shown in SEQ ID NO.4 and SEQ ID NO.

7.

4. The TaqMan probe for detecting a specific molecular target of Shigella flexneri according to claim 3, characterized in that: The 5' end of the TaqMan probe is labeled with a reporter fluorescent dye FAM, and the 3' end is labeled with a fluorescent quencher group BHQ1.

5. Use of the specific molecular target for detecting Shigella flexneri according to claim 1 in preparing a reagent for detecting Shigella flexneri.

6. A kit for detecting Shigella flexneri, characterized in that: Including all nucleotide sequences described in SEQ ID NO.1-7.

7. A method for detecting Shigella flexneri for non-disease diagnosis and treatment purposes, characterized in that: The following steps are involved: (1) Obtain the DNA of the microorganism to be tested, and use the extracted DNA as a template and the specific molecular target SEQ ID NO.1 to design primers and probes; (2) Perform qPCR amplification using primers and probes designed based on the specific molecular target SEQ ID NO.1, and collect the fluorescence signal; (3) Result determination: Determine whether the sample contains Shigella flexneri based on the fluorescence signal and Ct value collected in step (2). When the Ct value is greater than 35 or the system determines it to be negative, the result is determined to be negative.

8. The method for detecting Shigella flexneri according to claim 7, characterized in that: The reaction system of the qPCR amplification is 10mL2×Pro Taq HS Probe Premix, 0.7μL of each upstream and downstream primer with an initial concentration of 10μM, 1μL of the probe with an initial concentration of 10μM, the total volume of the template and ddH2O is 7.6μL, and the total volume of the system is 20μL.

9. The method for detecting Shigella flexneri according to claim 7, characterized in that: The reaction program of the qPCR amplification is: the fluorescence channel is set to temperature control, the program is set to 95°C for 5 min; 40 cycles include 95°C for 15 s and 60°C for 30 s; the fluorescence channel of the qPCR instrument is set to: FAM, and the fluorescence signal is collected by the qPCR instrument at the same time.

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