Nucleic acid detection kit for typhoid fever, paratyphoid fever, cholera, shiga and dysentery and application

Abstract

The invention discloses a nucleic acid detection kit for typhoid fever, paratyphoid fever, cholera, shigella and dysentery. The nucleic acid detection kit comprises a multiple fluorescent PCR detection system, and the multiple fluorescent PCR detection system comprises a typhoid fever salmonella primer probe, a paratyphoid fever salmonella primer probe, a vibrio cholera primer probe, a dysentery amoeba primer probe and a shigella primer probe; according to the kit, specific and conservative characteristic genes of different bacteria species are selected for primer probe design, so that the specificity, the accuracy and the sensitivity of detection are ensured.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a nucleic acid detection kit for typhoid, paratyphoid, cholera, Shigella and dysentery and an application thereof. Background Art

[0002] Existing technologies are generally designed based on the internal transcribed spacer (ITS) region of bacteria, which is sometimes not specific enough and is generally single-plex detection. It generally uses ordinary taq enzymes, has more non-specific amplification, and is prone to false positives.

[0003] The detection of diarrheal bacteria is currently mainly monitored through culture, multiplex PCR and single fluorescent PCR, which is time-consuming, labor-intensive, costly and lacks specificity. Summary of the invention

[0004] The purpose of the present invention is to provide a nucleic acid detection kit for typhoid, paratyphoid, cholera, Shigella, and dysentery based on the prior art. The present invention selects species-specific gene fragments for different pathogens by comparing genomes and gene evolution trees, and uses Taq enzymes that remove Escherichia coli nucleic acid for amplification, thereby ensuring the specificity of amplification and detection, and uses 2 tubes for multiple joint detection to improve detection efficiency.

[0005] Another object of the present invention is to provide a multiplex fluorescence PCR detection system that can simultaneously detect typhoid, paratyphoid, cholera, Shigella, and dysentery.

[0006] The third object of the present invention is to provide a specific use of the above-mentioned nucleic acid detection kit.

[0007] The purpose of the present invention can be achieved by the following measures: A nucleic acid detection kit for typhoid, paratyphoid, cholera, Shigella and dysentery, comprising a multiplex fluorescence PCR detection system, wherein the multiplex fluorescence PCR detection system comprises a primer probe for Salmonella typhi, a primer probe for Salmonella paratyphi, a primer probe for Vibrio cholerae, a primer probe for Entamoeba histolytica and a primer probe for Shigella; wherein The sequences of the Salmonella typhi primer probe include SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3; The sequences of the primer probes for Salmonella paratyphi include SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12; The sequences of the Vibrio cholerae primer probe include SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15; The sequences of the primer probes for Entamoeba histolytica include SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18; The sequences of Shigella primer probes include SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21.

[0008] The present invention also includes group modification of the probe sequence, wherein the 5' end and 3' end of SEQ ID NO.3 and SEQ ID NO.18 are respectively labeled with FAM / BHQ1, the 5' end and 3' end of SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.12 are respectively labeled with VIC / BHQ1, and the 5' end and 3' end of SEQ ID NO.15 and SEQ ID NO.21 are respectively labeled with CY5 and BHQ3.

[0009] The kit of the present invention comprises various probe mixtures, enzyme and buffer mixtures and positive quality control. Further, the kit comprises a primer probe mixture of group A and a primer probe mixture of group B, wherein the primer probe mixture of group A contains primer probes for Salmonella typhi, primer probes for Salmonella paratyphi and primer probes for Vibrio cholerae, and the primer probe mixture of group B contains primer probes for Entamoeba histolytica and primer probes for Shigella.

[0010] The invention discloses a multiplex fluorescent PCR detection system capable of simultaneously detecting typhoid, paratyphoid, cholera, Shigella and dysentery. The multiplex fluorescent PCR detection system comprises a typhoid Salmonella primer probe, a paratyphoid Salmonella primer probe, a cholerae Vibrio primer probe, a histolytica primer probe and a Shigella primer probe; wherein the sequence of the typhoid Salmonella primer probe comprises SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; the sequence of the paratyphoid Salmonella primer probe comprises SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12; the sequence of the cholerae Vibrio primer probe comprises SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15; the sequence of the histolytica primer probe comprises SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO.19; NO.18; the sequences of Shigella primer probes include SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21.

[0011] The probe sequences were modified with groups, wherein the 5' and 3' ends of SEQ ID NO.3 and SEQ ID NO.18 were labeled with FAM / BHQ1, respectively; the 5' and 3' ends of SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.12 were labeled with VIC / BHQ1, respectively; and the 5' and 3' ends of SEQ ID NO.15 and SEQ ID NO.21 were labeled with CY5 and BHQ3, respectively.

[0012] The nucleic acid detection kit of the present invention can be used in detecting diarrhea pathogens, in particular, in detecting typhoid, paratyphoid, cholera, Shigella, and dysentery pathogens.

[0013] Beneficial effects of the present invention: The detection of diarrheal bacteria is currently mainly monitored by culture, multiplex PCR and single fluorescent PCR, which is time-consuming, labor-intensive, and costly, and lacks specificity. Through comparative genomics and evolutionary tree analysis, this kit selects several species-specific and conservative characteristic genes of different bacteria for primer probe design to ensure the specificity, accuracy and sensitivity of the detection. Among them, the typhoid Salmonella uses species-specific deaminase domain-containing protein genes, the three types of paratyphoid Salmonella A, B and C are respectively inferred to have unique genes, AAA family ATPase genes and THiF family adenosyltransferase genes, Vibrio cholerae selects transcriptional regulator genes, Entamoeba histolytica uses cysteine ​​protease genes, and Shigella selects the specific gene ipaH gene.

[0014] Instruction Manual Figure 1 It is the test result of the sample to be tested in tube A; Figure 2 It is the test result of the sample in tube B. DETAILED DESCRIPTION

[0015] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0016] Example 1 This kit selects the characteristic target genes of pathogen species for multiplex fluorescence PCR detection, and performs nucleic acid detection of 5 diarrheal pathogens in 2 tubes, including detection and identification of Salmonella typhi, Salmonella paratyphi, Vibrio cholerae, Shigella and Entamoeba histolytica, and can obtain accurate, sensitive and specific detection results in a short time. This kit includes primer probe mixture, enzyme and buffer mixture and positive quality control.

[0017] During operation, prepare the premix according to the proportion, add the extracted and purified nucleic acid template, and then perform amplification detection. The reaction conditions are 95 degrees pre-denaturation for 2 minutes, 95 degrees denaturation for 5 seconds, 60 degrees extension for 30 seconds and collect FAM / VIC / CY5 fluorescence, and perform 40 cycles. The result is judged by the Ct value, and Ct<35 is positive.

[0018] The sequences of SEQ ID NO.1-21 were sent to Shanghai Bio-Technology Co., Ltd. for synthesis. TE (pH 8.0) was used as solvent to dissolve the dry powder, and the concentration was 100 μM. The primer-probe mixture was prepared according to the following table: Tube A: 0.04 μl of upstream and downstream primers for Salmonella typhi, Salmonella paratyphi, and Vibrio cholerae, 0.02 μl of probes, and finally 1.6 μl of TE (pH 8.0), a total of 2.0 μl; Tube B: 0.04 μl of upstream and downstream primers for Entamoeba histolytica and Shigella, 0.02 μl of probes, and finally 1.6 μl of TE (pH 8.0), a total of 2.0 μl; Preparation of premix: 10XqPCR reaction solution: 2.5μl; 25mM MgCl2: 1μl; 2mM dNTP: 1μl; Taq enzyme: 0.5μl (5Μ / μl); Primer mixture: 2 μl (prepared separately in tubes A and B); Enzyme-free PCR water: 13 μl.

[0019] 1. Primer probe for Salmonella typhi: SEQ ID NO.1:GGAAGTCGAAAGTTCACTACC, SEQ ID NO.2: CGTTGTTGGAAATTTATTGGCTATA, SEQ ID NO.3: AGAATCGAAAGCCCTGTCATGCCC 2. Primer probe for Salmonella paratyphi: SEQ ID NO.4:ATCTGGCATTGTTGGTTGT, SEQ ID NO.5:AATGTCGAAGCTATTAGTGTTTAG, SEQ ID NO.6:CAACCGAACCTGGCAACATACCAT, SEQ ID NO.7:GCACTTTTAGGAAGCGCTG, SEQ ID NO.8:CCATCTTTAACCCTCGAACG, SEQ ID NO.9: TGCTATTCCGCCAACCCGTAAGG, SEQ ID NO.10: CCTTCATGATGTCGGGATG, SEQ ID NO.11:TCGCGTTTGTCCGTAGTA, SEQ ID NO.12:AATGCCACCAAGACTACCGTTCACT, 3. Primer probe for Vibrio cholerae: SEQ ID NO.13:GGAAGTAAGACCGCTATCAGA, SEQ ID NO.14:GCAGTGTGCCTTCATCAG, SEQ ID NO.15: CCGCAGCCAGCCAATGTCGT, 4. Primer probe for Entamoeba histolytica: SEQ ID NO.16:CCTGAAACAGTAGATTGGAGAAA, SEQ ID NO.17:CTTCCTTCAAGTGCTGCAA, SEQ ID NO.18: ACACCCTCCACACTCTGCTTGATCTCT, 5. Shigella primer probe: SEQ ID NO.19:TGTGGATGAGATAGAAGTCTAC, SEQ ID NO.20:CCGACACGCCATAGAA, SEQ ID NO.21: CTTCCAGACATGCTCGCAGA The probe sequences were modified with groups, wherein the 5' and 3' ends of SEQ ID NO.3 and SEQ ID NO.18 were labeled with FAM / BHQ1, respectively; the 5' and 3' ends of SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.12 were labeled with VIC / BHQ1, respectively; and the 5' and 3' ends of SEQ ID NO.15 and SEQ ID NO.21 were labeled with CY5 and BHQ3, respectively.

[0020] During the experiment, take 20 μl of the above premix, add 5 μl of extracted and purified nucleic acid template, and then perform amplification detection.

[0021] The reaction conditions were 95°C pre-denaturation for 10 minutes, 95°C denaturation for 5 seconds, 60°C extension for 30 seconds and FAM / VIC / CY5 fluorescence collection, and 40 cycles. The results were judged by the Ct value, Ct<35 was positive, and Ct≥35 was negative.

[0022] Accordingly, the nucleic acid detection kit for the five diarrhea-causing pathogens consists of: 10XqPCR reaction solution, 25mM MgCl2, 2mMdNTP, Taq enzyme, group A primer-probe mixture, group B primer-probe mixture, and positive quality control products containing specific target gene fragments of the above five pathogens.

[0023] Embodiment 2, detection The kit obtained in Example 1 was used, and the standard strains / clinical isolates / food isolates of Salmonella typhi and Salmonella paratyphi (A / B / C), Vibrio cholerae, Shigella and feces of confirmed cases of clinical etiology of Entamoeba histolytica were used as samples to be tested. A fluorescent quantitative PCR instrument (Bio-Rad CFX96) was used to detect the characteristic genes of the five pathogens through two qPCR reaction systems on the same sample. Tube A was used for nucleic acid detection of Salmonella typhi, Salmonella paratyphi (A / B / C), and Vibrio cholerae, and tube B was used for nucleic acid detection of Shigella and Entamoeba histolytica.

[0024] The fluorescence signal curve (typical S-shaped curve) and Ct value (<35) are used to determine whether the target pathogen nucleic acid is detected in the sample to be tested. The reference method adopts the target bacteria identification method specified in the national food safety standard. The results are shown in Figure 1 and 2 .

[0025] The results showed that the reporter groups FAM (target bacteria: Salmonella typhi), CY5 (target bacteria: Vibrio cholerae), and VIC (target bacteria: Salmonella paratyphi) in tube A all had S-type fluorescence signal curves, and the Ct value was <35; the reporter groups FAM (target bacteria: Entamoeba histolytica) and CY5 (target bacteria: Shigella) in tube B all had S-type fluorescence signal curves, and the Ct value was <35.

[0026] The test results were reported as positive for Salmonella typhi, Salmonella paratyphi (A / B / C), Vibrio cholerae, Shigella and Entamoeba histolytica. The Kappa value of the fluorescence PCR test results compared with the reference method was 1, and the test results of the two detection methods were consistent.

[0027] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that it is still possible to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nucleic acid detection kit for typhoid, paratyphoid, cholera, Shigella, and dysentery, characterized in that The method comprises a multiplex fluorescent PCR detection system, wherein the multiplex fluorescent PCR detection system comprises a primer probe for Salmonella typhi, a primer probe for Salmonella paratyphi, a primer probe for Vibrio cholerae, a primer probe for Entamoeba histolytica and a primer probe for Shigella; The sequences of the primer probes for Salmonella typhi include SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; the sequences of the primer probes for Salmonella paratyphi include SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12; The sequences of the Vibrio cholerae primer probe include SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15; the sequences of the Entamoeba histolytica primer probe include SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18; The sequences of the Shigella primer probe include SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.

21.

2. The kit according to claim 1, characterized in that The probe sequences were modified with groups, wherein the 5' and 3' ends of SEQ ID NO.3 and SEQ ID NO.18 were labeled with FAM / BHQ1, respectively; the 5' and 3' ends of SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.12 were labeled with VIC / BHQ1, respectively; and the 5' and 3' ends of SEQ ID NO.15 and SEQ ID NO.21 were labeled with CY5 and BHQ3, respectively.

3. The kit according to claim 1, characterized in that The kit includes a probe mix, an enzyme and buffer mix, and a positive quality control.

4. The kit according to claim 3, characterized in that The kit comprises a primer probe mixture of group A and a primer probe mixture of group B, wherein the primer probe mixture of group A contains the primer probe for Salmonella typhi, the primer probe for Salmonella paratyphi and the primer probe for Vibrio cholerae, and the primer probe for group B contains the primer probe for Entamoeba histolytica and the primer probe for Shigella.

5. A multiplex fluorescent PCR detection system capable of simultaneously detecting typhoid, paratyphoid, cholera, Shigella, and dysentery, characterized in that It includes primer probes for Salmonella typhi, primer probes for Salmonella paratyphi, primer probes for Vibrio cholerae, primer probes for Entamoeba histolytica, and primer probes for Shigella; The sequences of the Salmonella typhi primer probe include SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; the sequences of the Salmonella paratyphi primer probe include SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12; The sequences of the Vibrio cholerae primer probe include SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15; the sequences of the Entamoeba histolytica primer probe include SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18; The sequences of the Shigella primer probe include SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.

21.

6. The multiplex fluorescence PCR detection system according to claim 1, characterized in that The probe sequences were modified with groups, wherein the 5' and 3' ends of SEQ ID NO.3 and SEQ ID NO.18 were labeled with FAM / BHQ1, respectively; the 5' and 3' ends of SEQ ID NO.6, SEQ ID NO.9 and SEQ ID NO.12 were labeled with VIC / BHQ1, respectively; and the 5' and 3' ends of SEQ ID NO.15 and SEQ ID NO.21 were labeled with CY5 and BHQ3, respectively.

7. Use of the nucleic acid detection kit according to claim 1 in detecting diarrheal pathogens.

8. Use of the nucleic acid detection kit according to claim 1 in detecting pathogens of typhoid, paratyphoid, cholera, Shigella, and dysentery.

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