Composition, kit, method and application for joint detection of leishmania and co-infection pathogens of leishmania

Abstract

The invention belongs to the field of molecular biology detection, particularly relates to detection of leishmania and co-infection pathogens thereof, and more particularly relates to detection and distinguishing of at least four pathogens in leishmania, HIV, plasmodium, schistosome, leprosy bacillus and mycobacterium tuberculosis. According to the combined detection composition provided by the invention, different pathogens are detected by mainly utilizing a multiple fluorescent PCR analysis method and detecting target spots on the different pathogens, so that detection and distinguishing of at least four pathogens in leishmania, HIV, plasmodium, schistosome, leprosy bacillus and mycobacterium tuberculosis are simultaneously realized in a single-tube reaction system; the detection efficiency is improved. The composition disclosed by the invention is high in detection sensitivity which reaches 400 copies / mL, and also has good specificity.

Description

Technical Field

[0001] The present invention relates to the field of molecular biological detection, in particular, to the joint detection of Leishmania and co-infecting pathogens thereof, and more particularly, to the detection and differentiation of Leishmania, human immunodeficiency virus (HIV), Mycobacterium leprae, Mycobacterium tuberculosis, Schistosoma japonicum and Plasmodium. Background Art

[0002] Leishmania spp., a parasite transmitted by the bite of sand flies, is the main pathogen causing visceral leishmaniasis (also known as kala-azar) and cutaneous leishmaniasis. It belongs to the genus Leishmania of the class Trypanosoma of the phylum Protozoa, and can be divided into visceral leishmaniasis, cutaneous leishmaniasis and mucocutaneous leishmaniasis according to the different parasitic sites and symptoms. The symptoms of visceral leishmaniasis include long-term irregular fever, hepatosplenomegaly and lymphadenopathy, especially splenomegaly, which is the most obvious sign of kala-azar. Cutaneous leishmaniasis often manifests as skin ulcers, with pus often flowing out of the ulcers. In severe cases, the patient may lose some of his ability to work. In terms of epidemiology, Leishmania infection is prevalent in more than 80 countries and regions around the world, with an estimated number of patients exceeding 15 million. Metronidazole, chloroquine, albendazole and other drugs are usually used to treat Leishmania infection. These drugs, used under the guidance of professional doctors, can effectively interfere with the DNA synthesis of Leishmania and inhibit its growth and reproduction.

[0003] It is worth noting that there is a risk of co-infection between Leishmania infection and pathogens such as human immunodeficiency virus (HIV), Mycobacterium leprae, Mycobacterium tuberculosis, Schistosoma japonicum and Plasmodium. Although these six pathogens have different biological characteristics and transmission routes, they all have characteristics such as immune escape and chronic infection. When co-infected, these pathogens may cooperate with each other to aggravate damage to the immune system and lead to more serious clinical consequences. For example, when AIDS patients are infected with Leishmania, they may be more likely to develop severe leishmaniasis due to their impaired immune system. According to research reports, this co-infection may not only aggravate the original condition, but also increase the complexity and difficulty of treatment. HIV-infected people are more likely to be infected with Leishmania due to their impaired immune system, resulting in more severe illness, and Leishmania infection may also aggravate the progression of HIV. Therefore, for patients at risk of co-infection, a more cautious and comprehensive treatment strategy is needed.

[0004] Co-infection with Leishmania and other infectious pathogens is particularly common in tropical and subtropical regions, which are often high-incidence areas for multiple infectious diseases. For example, some areas in Africa and Asia are both endemic areas for AIDS and tuberculosis, as well as high-incidence areas for Leishmaniasis and malaria. The occurrence of co-infection not only increases the complexity of the disease, but may also lead to more serious clinical consequences.

[0005] In addition, the treatment of co-infected patients faces many challenges. Different pathogens have different sensitivities to drugs, and there may be interactions between drugs. For example, when AIDS patients are infected with Mycobacterium tuberculosis, the choice and dosage of anti-tuberculosis drugs may need to be adjusted to avoid interactions with antiretroviral drugs. Therefore, accurate diagnosis of co-infection is crucial for developing a reasonable treatment plan.

[0006] In summary, Leishmania infection is a global public health problem, and its wide geographical distribution makes prevention and control work particularly complicated. At the same time, the co-infection of Leishmania with other pathogens further increases the challenge of treatment, and professional doctors are required to develop personalized treatment plans based on the specific conditions of the patients. The development of a six-in-one nucleic acid detection kit for the above six pathogens is of great clinical significance. It can improve the speed and accuracy of diagnosis of co-infected patients, thereby providing patients with timely and effective treatment options. Early identification of multiple pathogens can prevent potential complications and reduce the risk of drug interactions during treatment. At the same time, many of these diseases have no radical cure and are mainly prevented. Early screening helps in the timely diagnosis of the disease. Detection of these pathogens helps to comprehensively assess the health status of patients and develop comprehensive treatment strategies. Due to the wide variety of pathogenic pathogens, it poses a huge challenge to the diagnosis and prevention of diseases.

[0007] Therefore, there is a need in the art for a product that can simply and quickly detect the above-mentioned pathogens with high sensitivity and good specificity, which is conducive to providing prevention policies and medication guidance in a timely manner. Summary of the invention

[0008] The existing detection methods of Leishmania and its co-infected pathogens are complex, slow in reaction speed, and cost-effective. In view of this, the present invention, in a first aspect, provides a composition for the joint detection of Leishmania and its co-infected pathogens, comprising primer pairs and probes for detecting at least four pathogens of Leishmania, HIV, Plasmodium, Schistosoma, Mycobacterium leprae, and Mycobacterium tuberculosis:

[0009] Primer pairs and probes for detecting Leishmania as shown in SEQ ID NOs: 1 to 3;

[0010] Primer pairs and probes for detecting HIV as shown in SEQ ID NOs: 4-6 and SEQ ID NOs: 7-9;

[0011] Primer pairs and probes for detecting Plasmodium as shown in SEQ ID NOs: 10 to 12;

[0012] A primer pair and a probe for detecting Schistosoma japonicum as shown in SEQ ID NOs: 13 to 15;

[0013] A primer pair and a probe for detecting Mycobacterium leprae as shown in SEQ ID NOs: 16 to 18; and

[0014] The primer pair and probe for detecting Mycobacterium tuberculosis are shown in SEQ ID NOs: 19-21.

[0015] The present invention provides a combined detection composition, which mainly uses a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously realizing the detection and identification of at least four pathogens of Leishmania, HIV, Plasmodium, Schistosoma, Mycobacterium leprae and Mycobacterium tuberculosis in a single-tube reaction system. The composition of the present invention has a higher detection sensitivity of 400 copies / mL, good specificity, and more accurate detection.

[0016] The most common method for pathogen detection is fluorescent quantitative PCR (polymerase chain reaction). The PCR reaction system contains a pair of specific primers and a Taqman probe. The probe is a specific oligonucleotide sequence with a reporter fluorescent group and a quencher fluorescent group labeled at both ends. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; if there is a target sequence in the reaction system, the probe binds to the template during the PCR reaction, and the DNA polymerase uses the exonuclease activity of the enzyme to enzymatically degrade the probe along the template, and the reporter group separates from the quencher group and emits fluorescence. For each amplified DNA chain, a fluorescent molecule is generated. The fluorescent quantitative PCR instrument can monitor the number of cycles (Ct value) at which the fluorescence reaches a preset threshold value, which is related to the concentration of pathogen nucleic acid. The higher the concentration of pathogen nucleic acid, the smaller the Ct value. In the end, the infection can be determined based on the Ct value.

[0017] In some embodiments, the composition of the present invention may include one or more sets of the above primer pairs and probes. In this article, "set" refers to upstream primers, downstream primers and probes that match each other to detect a target.

[0018] Furthermore, the composition of the present invention can be arbitrarily combined into any combination form for detecting corresponding targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe sets corresponding to the targets. These combinations are all included in the present invention.

[0019] Furthermore, the components in the composition do not interfere with each other and their fluorescence does not crosstalk with each other.

[0020] In this article, "no interference and no fluorescence crosstalk" means that the components in the composition will not affect each other's detection, and different channels can be used for detection. For example, ATTO 425, FAM, HEX, ROX, CY5 and Quasar705 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other. And the fluorescence of each component does not crosstalk with each other.

[0021] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0022] In some specific embodiments, the probes in the compositions of the present invention are labeled with a fluorescent reporter group at the 5' end.

[0023] Furthermore, the fluorescent reporter group of the probe for detecting Leishmania as shown in SEQ ID NO:3 is FAM; the fluorescent reporter group of the probe for detecting HIV as shown in SEQ ID NO:6 and SEQ ID NO:9 is HEX; the fluorescent reporter group of the probe for detecting Plasmodium as shown in SEQ ID NO:12 is ROX; the fluorescent reporter group of the probe for detecting Schistosoma as shown in SEQ ID NO:15 is CY5; the fluorescent reporter group of the probe for detecting Mycobacterium leprae as shown in SEQ ID NO:18 is ATTO 425; the fluorescent reporter group of the probe for detecting Mycobacterium tuberculosis as shown in SEQ ID NO:21 is Quasar705.

[0024] In some specific embodiments, the 3' end of the probe further has a fluorescence quenching group, such as MGB, BHQ1 or BHQ2.

[0025] Furthermore, the components of the composition exist in a mixed form.

[0026] In a second aspect, the invention provides a method for preparing a reagent or a kit for the joint detection of Leishmania and co-infecting pathogens thereof, wherein the pathogens include Leishmania, HIV, Mycobacterium leprae, Mycobacterium tuberculosis, Schistosoma japonicum and Plasmodium falciparum.

[0027] In a third aspect, a kit for the joint detection of Leishmania and co-infecting pathogens thereof is provided, wherein the kit comprises the composition of the present invention.

[0028] Furthermore, in each reaction system, the amount of primer used is 0.05 to 5 μM, and the amount of probe used is 0.05 to 5 μM.

[0029] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0030] In a specific embodiment, the negative control is physiological saline, but is not limited to physiological saline, and can also be at least one of DEPC H2O and internal standard gene.

[0031] Commonly used internal standards include exogenous non-human genome internal standards, human genome internal standards, artificially synthesized internal standards, etc.

[0032] In a specific embodiment, the positive control is at least one of a fragment plasmid or a fragment DNA of Leishmania, HIV, Mycobacterium leprae, Mycobacterium tuberculosis, Schistosoma japonicum, and Plasmodium.

[0033] Furthermore, the kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a PCR buffer, an RNase inhibitor, a reverse transcriptase, a DNA polymerase, dNTPs (T) and Mg 2+ At least one of .

[0034] In some specific embodiments, the RNase inhibitor is Rnasin.

[0035] In some specific embodiments, the reverse transcriptase is Neoscript RT enzyme.

[0036] In some specific embodiments, the DNA polymerase is H-Taq enzyme.

[0037] In a specific embodiment, the kit of the present invention comprises PCR buffer, Rnasin, Neoscript RT enzyme, H-Taq enzyme, dNTPs (T), primers, probes and Mg 2+ .

[0038] Common PCR buffers are composed of Tris-HCl, KCl, MgCl2 and other buffer systems. The total volume in a single PCR reaction tube is generally 20 to 200 μL.

[0039] In a fourth aspect, a method for joint detection of Leishmania and co-infecting pathogens thereof for non-diagnostic purposes is provided, the method comprising the following steps:

[0040] (1) extracting or releasing nucleic acid from the sample to be tested;

[0041] (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the composition of the present invention or the kit of the present invention;

[0042] (3) Obtain and analyze the results.

[0043] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0044] Pre-denaturation and enzyme activation, temperature: 95℃, time: 1 min, 1 cycle; reverse transcription, temperature: 50℃, time: 30 min, 1 cycle; cDNA pre-denaturation, temperature: 95℃, time: 1 min, 1 cycle; denaturation, temperature: 95℃, time: 15 s, annealing, extension and fluorescence acquisition, temperature: 58℃, time: 30 s, 45 cycles; instrument cooling, temperature: 25℃, time: 10 s, 1 cycle.

[0045] In a specific embodiment, the present invention provides a composition for preparing a reagent for joint detection of Leishmania and co-infecting pathogens thereof, the use comprising the following steps:

[0046] (1) Extracting or releasing nucleic acid from the sample to be tested;

[0047] (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the composition of the present invention or the kit of the present invention;

[0048] (3) Obtain and analyze the results.

[0049] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0050] Pre-denaturation and enzyme activation, temperature: 95℃, time: 1 min, 1 cycle; reverse transcription, temperature: 50℃, time: 30 min, 1 cycle; cDNA pre-denaturation, temperature: 95℃, time: 1 min, 1 cycle; denaturation, temperature: 95℃, time: 15 s, annealing, extension and fluorescence acquisition, temperature: 58℃, time: 30 s, 45 cycles; instrument cooling, temperature: 25℃, time: 10 s, 1 cycle.

[0051] In this article, the term "non-diagnostic purpose" refers to not being intended to obtain information on whether an individual is infected with the above pathogens and related diseases. For example, the presence of the above pathogens can be detected in a test culture.

[0052] In the present invention, the term "primer pair" refers to two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target gene, and the other primer is complementary to another DNA template strand at the other end of the target gene. Alternatively, it refers to two oligonucleotide chains used to specifically amplify the target DNA fragment in the polymerase chain reaction (PCR). This pair of primers corresponds to the two ends of the target DNA sequence, one is complementary to the 5' end of the template strand, called the upstream primer or forward primer; the other is complementary to the 3' end of the template strand, called the downstream primer or reverse primer.

[0053] In the present invention, the term "probe" refers to a nucleic acid sequence (DNA or RNA) with a detection label, a known sequence, and complementary to the target gene.

[0054] In the present invention, the term "nucleotide sequence" refers to the order of bases in DNA or RNA.

[0055] The above scheme of the present invention has the following beneficial effects:

[0056] The present invention designs a tube of reaction solution, and only needs to perform one nucleic acid extraction and one PCR test, and can simultaneously perform joint gene detection of Leishmania and its five co-infected infectious pathogens. It is possible to effectively detect the infection and co-infection of six common infectious pathogens such as Leishmania in a one-tube reaction system, and perform qualitative analysis on them, and there is no mutual interference between primers and probes of multiple pathogens. The method is simple to operate, short in time, can reduce the cost of clinical testing, detect the co-interference of each pathogen, help to early detect co-infection, guide patients to take medicine, evaluate prognosis risks, and monitor changes in the disease, which will provide an auxiliary means for clinical diagnosis or treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0058] Figure 1 It is an amplification curve diagram of the combined test of Leishmania, HIV, Plasmodium, Schistosoma, Mycobacterium leprae and Mycobacterium tuberculosis by the composition of the embodiment of the present invention;

[0059] Figure 2 This is an amplification curve diagram of the combined test of Leishmania, HIV, Plasmodium, and Schistosoma japonicum by the composition of the embodiment of the present invention;

[0060] Figure 3 This is an amplification curve diagram of the combined test of Leishmania, HIV, Plasmodium, Schistosoma, and Mycobacterium leprae by the composition of the embodiment of the present invention;

[0061] Figure 4 This is a sensitivity result diagram of the combined test of the composition of the embodiment of the present invention for Leishmania, HIV, Plasmodium, Schistosoma, Mycobacterium leprae and Mycobacterium tuberculosis;

[0062] Figure 5 is the specific detection result of the composition of the embodiment of the present invention;

[0063] Figure 6 It is a comparison diagram of amplification curves of single detection of Plasmodium in the embodiment of the present invention and the comparative example;

[0064] Figure 7It is an amplification curve diagram of the comparative example composition of the present invention for joint detection of Leishmania, HIV, Plasmodium, Schistosoma, and Mycobacterium leprae. DETAILED DESCRIPTION

[0065] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0066] The disclosed embodiments are described to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

[0067] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0068] The target "LEIS" in this article refers to "Leishmania", "HIV-1" refers to "HIV", "PLA" refers to "Plasmodium", "SCH" refers to "Schistosome", "ML" refers to "Molecular Bacillus leprae", and "MTB" refers to "Molecular Bacillus tuberculosis".

[0069] Example 1: Primer pairs and probes used in the present invention

[0070] The primer pairs and probes used in the present invention are shown in Table 1 below.

[0071] Table 1

[0072]

[0073] Note: "+" specifically refers to LNA modification, for example: "T+C" means locked nucleic acid modification on base "C".

[0074] Among them, the fluorescent reporter group of the probe for detecting Leishmania as shown in SEQ ID NO:3 is FAM; the fluorescent reporter group of the probe for detecting HIV as shown in SEQ ID NO:6 and SEQ ID NO:9 is HEX; the fluorescent reporter group of the probe for detecting Plasmodium as shown in SEQ ID NO:12 is ROX; the fluorescent reporter group of the probe for detecting Schistosoma as shown in SEQ ID NO:15 is CY5; the fluorescent reporter group of the probe for detecting Mycobacterium leprae as shown in SEQ ID NO:18 is ATTO 425; the fluorescent reporter group of the probe for detecting Mycobacterium tuberculosis as shown in SEQ ID NO:21 is Quasar 705.

[0075] Example 2: Method for detecting pathogens

[0076] (1) Reagent preparation:

[0077] Take the corresponding amount of PCR reaction solution and enzyme mixture solution (27 μL / person of PCR reaction solution + 3 μL / person of enzyme mixture solution) in proportion, and mix them thoroughly to form a PCR mixture solution.

[0078] Preparation of enzyme mixture:

[0079] The enzyme mixture was prepared by mixing Neoscript RT enzyme (10U / μL) and H-Taq enzyme (5U / μL) in a certain ratio (1μL Neoscript RT enzyme and 2μL Taq enzyme per person).

[0080] In addition, a negative control using sterile physiological saline as the matrix and a mixed plasmid containing the specific gene target sequences listed in Table 1 were used as the positive control.

[0081] (2) Sample processing and sample addition

[0082] Take 300 μL of the sample to be tested, negative control, and positive control into a 1.5 mL centrifuge tube, and use conventional nucleic acid extraction or purification reagents according to their instructions to extract nucleic acids.

[0083] 20 μL of the above-treated samples, negative controls and positive controls were pipetted and added to reaction tubes respectively. 30 μL of PCR mixture was added to each reaction tube and the tube cap was covered.

[0084] The real-time fluorescence PCR reaction system is configured according to Table 2 below:

[0085] Table 2

[0086] Components The volume of each reaction PCR Buffer 21.77μL Primer (50pmol / μL) 0.2μL / strip Probe (50pmol / μL) 0.1μL / strip dNTPs(T) 1.2μL <![CDATA[MgCl2]]> 0.4μL Rnasin 0.13μL Enzyme mixture 3μL Total 30μL

[0087] Note: 0.2 μL / strip means that the volume of each primer is 0.2 μL, and the present invention has 14 primers in total; 0.1 μL / strip means that the volume of each probe is 0.1 μL, and the present invention has 7 probes in total.

[0088] (3) On a fully automatic medical PCR analysis system, PCR amplification is performed according to a certain temperature and time setting program. The amplification reaction program is shown in Table 3.

[0089] Table 3

[0090]

[0091] (4) Testing, result analysis and judgment

[0092] If the FAM, HEX, ROX, CY5, ATTO 425 and Quasar 705 channels have obvious S-shaped amplification curves and Ct values ​​≤ 40, they are judged as positive; if the FAM, HEX, ROX, CY5, ATTO425 and Quasar 705 channels have no amplification curve (NoCt) or Ct values ​​> 40, they are judged as negative; the details are shown in Table 4 below:

[0093] Table 4

[0094]

[0095] Example 3: Test results of the test samples of the composition of the present invention

[0096] The primer pairs and probes shown in Example 1 were verified on samples of Leishmania, HIV, Plasmodium, Schistosoma, Mycobacterium leprae and Mycobacterium tuberculosis according to the method of Example 2. The results showed that Leishmania, HIV, Plasmodium, Schistosoma, Mycobacterium leprae and Mycobacterium tuberculosis could be jointly detected and distinguished.

[0097] Among them, the results of the joint test for Leishmania, HIV, Plasmodium, Schistosoma, Leprosy and Mycobacterium tuberculosis are as follows: Figure 1 shown.

[0098] The results of the joint test for Leishmania, HIV, Plasmodium, and Schistosoma are as follows Figure 2 shown.

[0099] The results of the joint test for Leishmania, HIV, Plasmodium, Schistosoma, and Mycobacterium leprae are as follows Figure 3 As shown. Figures 1 to 3 It can be seen that the composition of the present invention can perform joint detection and differentiation on four, five or six of the above pathogens.

[0100] Example 4: Sensitivity of the composition of the present invention

[0101] The composition in Example 1 of the present invention was used to perform LOD (sensitivity) detection on various pathogens. The detection rate results are shown in Table 5, and the sensitivity results are shown in Table 5. Figure 4 As shown, the test results show that the composition of the present invention can still accurately detect each channel of samples as low as 400 copies / mL, indicating that the sensitivity of the composition of the present invention is 400 copies / mL.

[0102] Table 5

[0103]

[0104] Example 5. Specificity of the composition of the present invention

[0105] Using the fluorescent PCR reaction system in Example 2 of the present invention, according to the detection method in Example 2, specificity tests were performed in reaction systems containing other relevant pathogens such as Babesia, Trypanosoma, dengue virus, Japanese encephalitis virus, West Nile virus, yellow fever virus, human T-lymphotropic virus (HTLV), and hepatitis C virus (HCV). The test results are as follows: Figure 5 As shown, each target channel has no non-specific amplification and no cross reaction, which shows that the composition provided by the present invention has good specificity.

[0106] Comparative Example 1: Other primers and probes designed by the present invention with poor effects

[0107] Due to the principle of complementary base pairing, dimers will form between primers and (or) probes, but this probability is very small and can be excluded at the beginning of the design. However, when multiple pathogens are detected together, there are many primers and probes, and dimers are likely to occur between primers and primers, probes and probes, or primers and probes. It is necessary to ensure the conservatism of the design (conservatism is crucial to the accuracy of the detection) and consider the mutual interference between different primers and probes, so the primers and probes need to be carefully designed. Therefore, the inventors designed some other primers and probes.

[0108] This comparative example refers to the reaction system and detection method of Example 2, except that the primer pair probe sequence of Plasmodium is different (as shown in Table 6 below). In addition to the above differences, the other materials and process operations used in this comparative example are strictly consistent with those of Example 2.

[0109] The primer pair and probe (SEQ ID NO: 10-12) in Example 1 and the primer pair and probe (SEQ ID NO: 22-24) in Comparative Example 1 were respectively used to perform single detection of Plasmodium pathogens, and the detection results are as follows: Figure 6 As shown, from Figure 6 It can be seen from the amplification curve that when the malarial pathogen is tested alone, there is little difference between the test of the embodiment and the comparative example, and the amplification curve shows an S-shaped amplification and the detection Ct is at the front.

[0110] The primer pair and probe (SEQ ID NO: 22-24) in Comparative Example 1 were used to replace the primer pair and probe (SEQ ID NO: 10-12) in Example 1, and combined with other primer pairs and probes in Example 1 to form a composition (SEQ ID NO: 1-9 and SEQ ID NO: 13-24) to perform specific gene detection of six pathogens. The results are as follows: Figure 7 As shown. Figure 7 It can be seen that when the six pathogen targets were jointly detected, the target detection performance of the comparative malarial parasite and the other five pathogens was reduced, and the detection Ct was seriously delayed.

[0111] In summary, the results of the two tests are similar, but the results of the combined tests are poor. This fully demonstrates the superiority of the composition of the present invention, and the mutual interference between different primer pairs and probes is small. In other words, the combination of primers and probes in the present invention is irreplaceable and substitutable.

[0112] Table 6

[0113]

[0114] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition for joint detection of Leishmania and co-infecting pathogens thereof, characterized in that: Includes primer pairs and probes for detection of at least 4 pathogens including Leishmania, HIV, Plasmodium, Schistosoma, Leprosy, and Mycobacterium tuberculosis: Primer pairs and probes for detecting Leishmania as shown in SEQ ID NOs: 1 to 3; Primer pairs and probes for detecting HIV as shown in SEQ ID NOs: 4-6 and SEQ ID NOs: 7-9; Primer pairs and probes for detecting Plasmodium as shown in SEQ ID NOs: 10 to 12; A primer pair and a probe for detecting Schistosoma japonicum as shown in SEQ ID NOs: 13 to 15; A primer pair and a probe for detecting Mycobacterium leprae as shown in SEQ ID NOs: 16 to 18; and The primer pair and probe for detecting Mycobacterium tuberculosis are shown in SEQ ID NOs: 19-21.

2. The composition according to claim 1, characterized in that The components in the composition do not interfere with each other and their fluorescence does not crosstalk with each other.

3. The composition according to claim 1, characterized in that The fluorescent reporter group of the probe for detecting Leishmania as shown in SEQ ID NO:3 is FAM; the fluorescent reporter group of the probe for detecting HIV as shown in SEQ ID NO:6 and SEQ ID NO:9 is HEX; the fluorescent reporter group of the probe for detecting Plasmodium as shown in SEQ ID NO:12 is ROX; the fluorescent reporter group of the probe for detecting Schistosoma as shown in SEQ ID NO:15 is CY5; the fluorescent reporter group of the probe for detecting Mycobacterium leprae as shown in SEQ ID NO:18 is ATTO 425; the fluorescent reporter group of the probe for detecting Mycobacterium tuberculosis as shown in SEQ ID NO:21 is Quasar 705.

4. The composition according to any one of claims 1 to 3, characterized in that The components of the composition are present in admixture.

5. Use of the composition according to any one of claims 1 to 4 in the preparation of a reagent or kit for the joint detection of Leishmania and co-infecting pathogens thereof, wherein: The pathogens include Leishmania, HIV, Mycobacterium leprae, Mycobacterium tuberculosis, Schistosoma japonicum and Plasmodium.

6. A kit for the joint detection of Leishmania and its co-infecting pathogens, characterized in that: The kit comprises the composition according to any one of claims 1 to 4.

7. The kit according to claim 6, characterized in that In each reaction system, the amount of primer used is 0.05-5 μM, and the amount of probe used is 0.05-5 μM.

8. The kit according to claim 6 or 7, characterized in that The kit also includes a negative quality control product and a positive quality control product.

9. The kit according to any one of claims 6 to 8, characterized in that The kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a PCR buffer, an RNase inhibitor, a reverse transcriptase, a DNA polymerase, dNTPs (T) and Mg 2+ At least one of .

10. A method for the joint detection of Leishmania and co-infecting pathogens for non-diagnostic purposes, characterized in that: The method comprises the following steps: (1) extracting or releasing nucleic acid from the sample to be tested; (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the composition according to any one of claims 1 to 4 or the kit according to any one of claims 6 to 9; (3) Obtain and analyze the results.

Images