Primers, method and kit for multiple tandem PCR gene disc detection of Leishmania

Abstract

The present invention discloses a primer, method and kit for multiple tandem PCR gene disc detection of Leishmania. It comprises at least two sets of primers, each set of primers comprises a pair of outer primers and a pair of inner primers. The specific primers are shown in SEQ ID NO.1-16. The present application can simultaneously carry out high-throughput detection of Leishmania for four Leishmania-specific DNA fragments, with a sensitivity of 100%, a specificity of 99.42%, and an accuracy of up to 99.55%, which can shorten the detection time and improve the detection efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to a primer, a method and a kit for performing multiple tandem PCR gene disc detection on Leishmania. Background Art

[0002] Leishmaniasis is listed as one of the five major parasitic diseases by the World Health Organization. It is caused by Leishmania spp., has complex pathogenicity, and is widely distributed in Asia, Europe, Africa, Latin America, etc. Every year, 1.5 million to 2 million people are infected with Leishmaniasis worldwide. About 80 countries have reported Leishmaniasis, posing a threat to 350 million healthy people. The incidence of Leishmaniasis in my country is relatively low, maintaining at a level of 0.0372 / 100,000, mainly sporadic cases. There is little research on the rapid detection of Leishmania in China, and there is a lack of commercial PCR reagents for rapid detection. Leishmaniasis outbreaks continue to occur in the main destinations of technical and labor personnel abroad in my country. my country has monitored returning labor personnel suspected of being infected with Leishmania at multiple entry ports. The difference in epidemic situation at home and abroad has resulted in foreign laborers infected with Leishmaniasis facing a situation where there is no Leishmania detection reagent when entering the country, which has caused great difficulties in the later diagnosis and treatment of cases. Therefore, the establishment of high-throughput detection technology for Leishmaniasis and the identification of the infected Leishmania species in the first place are the fundamental guarantee for improving the efficiency of port prevention and control of Leishmaniasis. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the present invention provides a primer, method and kit for multiple tandem PCR gene disc detection of Leishmania. The present application can simultaneously carry out high-throughput detection of Leishmania for four Leishmania-specific DNA fragments, which can shorten the detection time and improve the detection efficiency.

[0004] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0005] The invention discloses a primer for multiple tandem PCR gene disc detection of Leishmania, comprising at least two groups of primers, each group of primers comprising a pair of outer primers and a pair of inner primers.

[0006] Furthermore, the primers include at least four sets of primers, each set of primers includes a pair of outer primers and a pair of inner primers.

[0007] Furthermore, the primers are shown in SEQ ID NOs. 1 to 16.

[0008] Further, the Leishmania is Leishmania donovani, Leishmania infantum, Leishmania tropicalis and Leishmania major.

[0009] A method for detecting Leishmania based on multiple tandem PCR gene discs comprises the following steps:

[0010] (1) Extracting DNA from samples to be tested;

[0011] (2) using the inner primers among the above primers to prepare a gene disc for later use;

[0012] (3) Use the outer primers among the above primers to perform the first round of PCR amplification, and then use the first round amplification product as a template and use the gene disc to perform the second round of PCR amplification.

[0013] Furthermore, the sample to be tested is a plasmid standard containing at least one gene of Leishmania donovani, Leishmania infantum, Leishmania tropicalis and Leishmania major.

[0014] Furthermore, the preparation process of the gene disc is as follows:

[0015] Take 11 μL of inner primer and add it to the amplification reaction plate, adjust the concentration to make the final concentration reach 0.4 μmol / L. Then place the amplification reaction plate at -50°C for 20 minutes for drying, seal the amplification reaction plate with sealing film, and finally place the amplification reaction plate at 4°C for standby use.

[0016] Furthermore, the first round of PCR amplification reaction system included: 0.3 μL Mix1, 20 ng nucleic acid template, 0.2 μmol / L outer primer mixture, 25 μL Mix2, and finally supplemented to 50 μL with ddH2O;

[0017] The amplification process was as follows: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 30 s; annealing at 58°C for 60 s; extension at 72°C for 60 s, for a total of 20 cycles; and finally extension at 72°C for 1 min.

[0018] Furthermore, the second round PCR amplification reaction system includes: 56 μL of detection reagent, 4.5 μL of the first round PCR product dilution, and 52 μL of ddH2O. After uniform mixing, the mixture is added to the amplification reaction plate of the gene disc, 25 μL per well;

[0019] The amplification program was as follows: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 10 s, annealing at 54°C for 10 s, extension at 72°C for 30 s, for a total of 35 cycles, and a final extension at 72°C for 1 min.

[0020] A kit for detecting Leishmania, comprising the above primers.

[0021] Beneficial effects of the present invention:

[0022] This application constructs a recombinant plasmid template of ITS-1, ITS-2, P0, and RACK target gene fragments contained in four species of Leishmania, designs primers, and directly distinguishes the four gene groups by the difference in melting temperature. The amplification curves of the four gene groups are significantly different, and the four Leishmania species can be specifically detected with strong specificity. The detection limits of ITS-1, P0, ITS-2, and RACK4 genes can all reach 103 copies / μL, which is consistent with the detection limit of GeXP multiple gene detection technology in the detection of common highly lethal pathogens in pigs, and the test results are clear and accurate. The sensitivity of the detection of four Leishmania species is 100%, the specificity is 99.42%, and the accuracy is 99.55%.

[0023] This application has established a high-throughput detection technology for Leishmania donovani, Leishmania infantum, Leishmania tropicalis and Leishmania major based on MT-PCR technology, which can detect four types of Leishmania at one time with high specificity and sensitivity. The detection technology established in this study can be applied to the rapid detection of Leishmania in inbound passengers at the port, which can identify the pathogen or narrow the screening scope at the first time, and is of great significance for timely treatment of cases and rapid control of the epidemic. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Amplification and melting curves for detecting Leishmania donovani, Leishmania infantum, Leishmania tropicalis, and Leishmania major;

[0025] Figure 2 Amplification curves and melting curves of ITS-1, P0, ITS-2, and RACK genes;

[0026] Figure 3 The sensitivity and repeatability test results of the mixed infection model. DETAILED DESCRIPTION

[0027] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0028] Main reagents

[0029] Deoxyribonucleic acid extraction reagent: Wizard Genomic DNA purification Kit (Promega, A1120), gene amplification detection reagent: SYBR Premix Ex Taq (Takara, DRR041A), multi-gene amplification detection reagent: MμLtiplexPCR Assay Kit (Takara, RR062B). Main instruments: vacuum freeze dryer: Eppendorf, concentrator plus, nucleic acid extractor: Thermo Fisher FLEX, fluorescent quantitative PCR instrument: Roche 480. Primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0030] Example 1 Primer Design

[0031] Four positive recombinant plasmid templates containing ITS-1, ITS-2, P0, and RACK target gene fragments were constructed for four different types of Shimania, and the plasmid DNA was extracted and stored in an ultra-low temperature refrigerator at -80°C. The gene sequences were retrieved (GenBank accession no: AF306777, JN673394, JN673389, JN673385, AB175625, AY297458, AF499109, and M12691), and primers were designed and evaluated using Primer Premier V5.0 and Oligo V6.22 to establish nested primer pairs. The primer information is shown in Table 1.

[0032] Table 1. Primer information

[0033]

[0034]

[0035] Example 2 Multiple tandem PCR

[0036] 1. Making gene discs

[0037] Take 11 μL of inner primer and add it to the amplification reaction plate, adjust the concentration to make the final concentration reach 0.4 μmol / L. Then place the amplification reaction plate at -50°C for 20 minutes for drying, seal the amplification reaction plate with sealing film, and finally place the amplification reaction plate at 4°C for standby use.

[0038] 2. Multiplex tandem PCR

[0039] The first round of PCR amplification reaction system included: 0.3 μL Mix1, 20 ng nucleic acid template, 0.2 μmol / l external primer mixture (each set of external primers was added in equal amounts), 25 μL Mix2, and finally supplemented to 50 μL with ddH2O;

[0040] The amplification process was as follows: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 30 s; annealing at 58°C for 60 s; extension at 72°C for 60 s, for a total of 20 cycles; and finally extension at 72°C for 1 min.

[0041] Then the product of the first round of PCR amplification reaction was diluted 25 times as the template for the second round of PCR amplification reaction. The second round of PCR amplification reaction was carried out using the gene amplification detection reagent (gene disc). The second round of PCR amplification reaction system included: SYBR amplification reagent 56μL of SYBR Premix Ex Taq, 4.5μL of the first round of PCR product dilution, and 52μL of ddH2O. After uniform mixing, the mixture was added to the amplification reaction plate of the gene disc, 25μL per well;

[0042] The amplification program was as follows: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 10 s, annealing at 54°C for 10 s, extension at 72°C for 30 s, for a total of 35 cycles, and a final extension at 72°C for 1 min.

[0043] Example 3 MT-PCR system specificity verification

[0044] According to the template DNA preparation method, positive recombinant plasmid template DNA containing the target fragments of ITS-1, ITS-2, P0, and RACK genes were extracted respectively, and then the established system was used to detect the positive control DNA, adenovirus (type 1 and type 4), influenza A viruses (H1N1 pdm09, H3N2 and H5N1), influenza B virus (influenza B virus), human rhinovirus A and dengue virus reverse transcribed DNA to verify the specificity of the system.

[0045] The experimental results show that the positive and negative coincidence rates of the system in detecting positive control DNA, adenovirus, influenza A virus, influenza B virus, rhinovirus A, and dengue virus are all greater than 95%, which can exclude the interference of other viral nucleic acids. The accuracy is reliable and the specificity meets the requirements.

[0046] After multiple amplification, the melting curves were analyzed. The amplified products of the four gene groups all formed obvious specific melting peaks. According to the melting curves, the amplified products were all target products. The experimental results of the four genes are shown in Figure 1 and Figure 2 .

[0047] Figure 1In the figure, A is the amplification curve and melting curve of ITS-1 gene, which is used to detect Leishmania donovani; B is the amplification curve and melting curve of P0 gene, which is used to detect Leishmania infantum; C is the amplification curve and melting curve of ITS-2 gene, which is used to detect Leishmania tropicalis; D is the amplification curve and melting curve of RACK gene, which is used to detect Leishmania major.

[0048] A large number of experimental data were statistically analyzed to determine that the Tm values ​​of the amplified products of ITS-1, P0, ITS-2, and RACK genes were 84±0.33℃, 89±0.11℃, 86±0.37℃, and 88.5±0.16℃, respectively. No obvious specific peaks were produced in the negative control group. In addition, the amplification curves of the four gene groups were significantly different, indicating that this set of primers can be used for multiple detection studies of Leishmania donovani, Leishmania infantum, Leishmania tropicalis, and Leishmania major.

[0049] Example 4 Verification of sensitivity and quantitative ability of MT-PCR system

[0050] According to the template DNA preparation method, the positive recombinant plasmid template DNA containing the target fragments of ITS-1, ITS-2, P0, and RACK genes was extracted respectively, and the DNA concentration was determined by fluorescence spectrophotometry. The copy number was calculated using the DNA concentration formula: copy number (copies / μL) = [(DNA concentration (ng / μL) × 10 -9 )×(6.02×10 23 )]÷(number of bases in the plasmid × 345) to calculate the number of copies of the template DNA. Use TE buffer to dilute the template DNA to a concentration of 1×10 6 The sample was diluted with a 10-fold concentration gradient, and each dilution gradient was repeated 3 times to determine the minimum detection limit.

[0051] In order to simulate the infection of the virus under natural conditions, a mixed infection model was constructed. The test results of the mixed infection model are shown in Figure 3 .

[0052] In the ITS-1 mixed model, the concentration of ITS-1 standard template was diluted to 10 5 , 10 4 , 10 3 , 10 2 When the template concentration of P0, ITS-2, and RACK standard is fixed at 10 copies / μL, 6 copies / μL.

[0053] In the P0 mixed model, the concentration of the P0 standard template was diluted to 10 5 , 10 4 , 103 , 10 2 When the template concentration of ITS-1, ITS-2, and RACK standard samples is fixed at 10 copies / μL, 6 copies / μL.

[0054] In the ITS-2 mixed model, the concentration of ITS-2 standard template was diluted to 10 5 , 10 4 , 10 3 , 10 2 When the template concentration of ITS-1, P0, and RACK standard samples is fixed at 10 copies / μL, 6 copies / μL.

[0055] In the RACK mixed model, the RACK standard template concentration was diluted to 10 5 , 10 4 , 10 3 , 10 2 When the template concentration of ITS-1, P0, and ITS-2 standards is fixed at 10 copies / μL, 6 copies / μL.

[0056] It was finally determined that the detection limits of ITS-1, P0, ITS-2, and RACK can all reach 10 3 The intra-batch and inter-batch coefficients of variation of the three concentration gradients were all less than 5%, indicating good experimental repeatability.

[0057] Example 5 Simulated clinical sample detection

[0058] ITS-1, P0, ITS-2, RACK positive plasmids were randomly mixed with influenza A virus positive specimens, norovirus positive specimens, rotavirus positive specimens and negative specimens obtained from clinical tests to produce simulated clinical samples. The various Leishmania methods established in this study were used for detection. The positive coincidence rate, negative coincidence rate, total coincidence rate, sensitivity, specificity and method efficiency of the verification method in clinical sample detection were shown in Table 2. The results showed that the sensitivity of the detection of the four Leishmania species was 100%, the specificity was 99.42%, and the accuracy was 99.55%. Among them, the specificity of Leishmania donovani was 97.62% and the accuracy was 98.46%; the specificity and accuracy of Leishmania infantum, Leishmania tropicalis and Leishmania major were all 100%.

[0059] Table 2 Simulated clinical sample test results

[0060] Sequence Listing <110> Sichuan International Travel Health Care Center <120> Primers, method and kit for multiple tandem PCR gene disc detection of Leishmania <160> 16 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 acataacgtg tcgcgatgga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ttgcaagcac cagagaggag 20 <210> 3 <211> twenty two <212> DNA <213> Artificial Sequence <400> 3 cgcagtaaag tgcgataagt gg 22 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <400> 4 aatatggcat gcacgggga 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 gtacgagttc gaggagcaca 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 tcgtctcctt cgttgttggt 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <400> 7 cgagttcgag gagcacaac 19 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <400> 8 tagaagagac cgcccatgc 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 aggcgtgtgt ttgtgttgtg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 aaaaatggcc aacgcgaagt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 gtgcacacgt agacaagtca 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 tgctttttgc aagcaccaga 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 ctcaagggcc acagcaacta 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 attactcggc gtcggagatg 20 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <400> 15 ggagaggtct ctgtccgtg 19 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <400> 16 gtacagagtg ttgccgtcg 19

Claims

1. A primer for multiplex tandem PCR gene disc detection of Leishmania, characterized in that: The primers include four groups of primers, each group of primers includes a pair of outer primers and a pair of inner primers, the sequences of the outer primers are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.13 and SEQ ID NO.14; the sequences of the inner primers are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.8, SEQID NO.11, SEQ ID NO.12, SEQ ID NO.15 and SEQ ID NO.

16.

2. A method for detecting Leishmania based on a multiplex tandem PCR gene disc for non-diagnostic / therapeutic purposes, characterized in that: The Leishmania is Leishmania donovani, Leishmania infantum, Leishmania tropicalis and Leishmania major; comprising the following steps: (1) Extract DNA from samples to be tested; (2) preparing a gene disc using the inner primer among the primers described in claim 1; (3) The first round of PCR amplification is performed using the outer primers in the primers described in claim 1, and then the second round of PCR amplification is performed using the gene disc with the first round of PCR amplification product as a template.

3. The method according to claim 2, characterized in that The preparation method of the gene disc is as follows: Each inner primer was added in pairs to the amplification reaction plate, and then the concentration was adjusted to 0.4 μmol / L with ddH2O, and then dried at -50°C for 20 minutes, the amplification reaction plate was sealed, and finally the amplification reaction plate was placed at 4°C.

4. The method according to claim 2, characterized in that: The amplification program of the first round of PCR amplification is as follows: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 30 s; annealing at 58°C for 60 s; extension at 72°C for 60 s, for a total of 20 cycles; and finally extension at 72°C for 1 min.

5. The method according to claim 2, characterized in that: The process of carrying out the second round of PCR amplification using the gene disk is as follows: 56 μL of SYBR amplification reagent of SYBR Premix Ex Taq, 4.5 μL of the first round PCR product dilution, and 52 μL of ddH2O are evenly mixed, and then the above mixture is added to the amplification reaction plate of the gene disk, 25 μL per well; The second round of PCR amplification program was as follows: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 10 s, annealing at 54°C for 10 s, extension at 72°C for 30 s, for a total of 35 cycles, and finally extension at 72°C for 1 min.

6. A kit for detecting Leishmania, characterized in that: Comprising the primer of claim 1.

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