CN117385105A - PCR detection reagent, method and application - Google Patents
PCR detection reagent, method and application Download PDFInfo
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- CN117385105A CN117385105A CN202311657462.6A CN202311657462A CN117385105A CN 117385105 A CN117385105 A CN 117385105A CN 202311657462 A CN202311657462 A CN 202311657462A CN 117385105 A CN117385105 A CN 117385105A
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Abstract
The invention relates to a PCR detection reagent, a method and application thereof, belongs to the technical field of bioscience, and aims to solve the problems that the existing laboratory PCR technology only aims at single pathogen for detection, wastes time and labor, delays treatment progress and has low detection efficiency. A PCR detection reagent, comprising one or more of EBV DNA amplification reagent, HCMV DNA amplification reagent, HSV DNA amplification reagent, TB DNA amplification reagent, JCV DNA amplification reagent and TG DNA amplification reagent; the EBV DNA amplification reagent, the HCMV DNA amplification reagent, the HSV DNA amplification reagent, the TB DNA amplification reagent, the JCV DNA amplification reagent and the TG DNA amplification reagent respectively comprise corresponding forward primers and reverse primers, and the sequences of the forward primers and the reverse primers are shown in a sequence table. The detection reagent and the method have comprehensive detection and can realize rapid diagnosis.
Description
Technical Field
The invention relates to the technical field of bioscience, in particular to a PCR detection reagent, a PCR detection method and application.
Background
The tissue polymerase chain reaction (Polymerase Chain Reaction, PCR) is a commonly used molecular biology technique for detecting and amplifying specific DNA or RNA sequences in tissue samples in recent years, and the method is convenient to operate and has low risk of laboratory contamination. In clinical diagnosis of central nervous system infectious diseases, PCR is widely used for etiology detection of samples such as cerebrospinal fluid. For intracranial space-occupying lesions (BML), PCR is currently not used for routine detection, especially for HIV infection-related diagnosis.
AIDS (Acquired Immunodeficiency Syndrome, abbreviated as AIDS) is a serious immune system disease caused by the human immunodeficiency virus (Human Immunodeficiency Virus, abbreviated as HIV). Aids patients are susceptible to infectious lesions due to compromised immune systems, and multiple intracranial space occupying lesions (brain mass lesions, BML) may form in the central nervous system (central nervous system, CNS); infection inflammation stimulus or space occupying effect causes the intracranial pressure of a patient to increase, possibly causes headache, epileptic seizure, disturbance of consciousness or coma and the like, easily causes higher fatality rate and bad prognosis, and early diagnosis and timely treatment are vital to improving the prognosis of intracranial infectious space occupying lesions of the patient. Conventional imaging, such as electronic Computed Tomography (CT), magnetic Resonance Imaging (MRI) and laboratory examinations (e.g., biochemical and pathogenic detection of blood and cerebrospinal fluid) are of assistance in identifying lesion locations, assessing surrounding tissue involvement and the extent of inflammatory response, and stereotactic intracranial lesion biopsy pathology provides support for further defining the nature and etiology of lesions. However, according to the previous clinical experience, the diagnosis of the etiology of the intracranial space occupying lesions related to AIDS is difficult, which also leads to the failure to timely and accurately implement targeted treatment on the related pathogens in the clinical diagnosis and treatment process.
The existing research results prove that the PCR technology can remarkably improve the diagnosis efficiency of the Central Nervous System (CNS) infectious diseases of HIV infected persons, and the PCR detection technology for obtaining the tissue specimens at first has higher specificity and sensitivity for diagnosing the diseases. The PCR technology can be used for simultaneous detection or identification of various pathogenic microorganisms, parting identification of certain genetic diseases and oncogenes, and the like, and has the characteristics of high efficiency, systemicity, economy and simplicity. However, in terms of diagnosis of Central Nervous System (CNS) infectious diseases of HIV-infected persons, the current laboratory PCR technique is time-consuming and laborious for single pathogen detection, and delays treatment progress, so that it is highly desirable to apply the PCR technique to intracranial space occupying lesions (BMLs) of the Central Nervous System (CNS) of HIV-infected persons, to improve detection efficiency of Central Nervous System (CNS) infectious diseases of HIV-infected persons, to rapidly distinguish whether the Central Nervous System (CNS) of HIV-infected persons is infected with intracranial pathogen, to make a correct laboratory diagnosis for suspected cases as early as possible, and to have great significance in improving prognosis of intracranial infectious space occupying lesions of patients.
Disclosure of Invention
In view of the above analysis, the present invention aims to provide a PCR detection reagent, a method and an application thereof, so as to solve the problems of time and effort consuming, delay of treatment progress, and low detection efficiency of central nervous system infection pathogens of aids caused by the fact that the existing laboratory PCR technology is only used for detecting single pathogens.
The aim of the invention is mainly realized by the following technical scheme:
the invention provides a PCR detection reagent, which comprises one or more of an EBV DNA amplification reagent, an HCMV DNA amplification reagent, an HSV DNA amplification reagent, a TB DNA amplification reagent, a JCV DNA amplification reagent and a TG DNA amplification reagent;
the EBV DNA amplification reagent comprises an EBV forward primer and an EBV reverse primer; the sequence of the EBV forward primer is: CAGCTTTGACGATGGAGTAG; the sequence of the EBV reverse primer is: CAYCTCCDTCATCTCCGTCAT;
the HCMV DNA amplification reagent comprises a forward primer of HCMV and a reverse primer of HCMV; the sequence of the HCMV forward primer is: CTTCTTATCACCATCAGCG; the sequence of the HCMV reverse primer is: GACGCTGGTGGGGGTCGGC;
the HSV DNA amplification reagent comprises a forward primer and a reverse primer of HSV; the sequence of HSV forward primer is: GGYCTGYCCCATCCGAACG; the sequence of the HSV reverse primer is: CAGGTACGTRCCSGCGGTCT;
the TB DNA amplification reagent comprises a TB forward primer and a TB reverse primer; the sequence of the TB forward primer is: CTCCGTACCCGGAGCGCCAA; the sequences of the TB reverse primer were: GAATCGGTCTGGACGTCAAGG;
the JCV DNA amplification reagent comprises a JCV forward primer and a JCV reverse primer; the sequence of the JCV forward primer is: GCTCATACCTAGGGAGCC; the JCV reverse primer sequences were: AGGGGTTTCACTATAACTG;
the TG DNA amplification reagent comprises a forward primer and a TG reverse primer of TG; the sequence of the TG forward primer is: GAGCACGCAACCCTGGACC; the sequence of the TG reverse primer is: CACCTCAGTCTCTTGCCTGC.
Further, the EBV DNA amplification reagent further comprises an EBV fluorescent probe, and the sequence of the EBV fluorescent probe is as follows: FAM-CGGCAGCCCCTTCCACCATAGG-BHQ1; and/or the number of the groups of groups,
the HCMV DNA amplification reagent also comprises a HCMV fluorescent probe, and the sequence of the HCMV fluorescent probe is as follows: FAM-CCACGCTGACAACCCACTCTT-BHQ1; and/or the number of the groups of groups,
the HSV DNA amplification reagent also comprises an HSV fluorescent probe, and the sequence of the HSV fluorescent probe is as follows: AGCGCCGTCAGCGAGGAT; and/or the number of the groups of groups,
the TB DNA amplification reagent also comprises a TB fluorescent probe, and the sequence of the TB fluorescent probe is as follows: FAM-TCTCCTTCGCTAAGCTGCGC-BHQ1; and/or the number of the groups of groups,
the JCV DNA amplification reagent also comprises JCV fluorescent probes, and the sequences of the JCV fluorescent probes are as follows: FAM-CAGCCAGAGGGAGCCCT-BHQ1; and/or the number of the groups of groups,
the TG DNA amplification reagent also comprises a TG fluorescent probe, and the sequence of the TG fluorescent probe is FAM-TAGCGGAATGAGCAATTGTGC-BHQ1.
The invention also provides application of the PCR detection reagent, wherein the application comprises any one of the following steps:
detecting pathogens causing infection of the central nervous system of AIDS;
preparing a kit for detecting pathogens causing infection of the central nervous system of AIDS;
detecting whether a sample to be detected contains pathogens causing infection of the central nervous system of AIDS;
preparing a kit for detecting whether a sample to be tested contains pathogens causing infection of the central nervous system of AIDS.
The invention also provides a PCR detection kit for the infection of the central nervous system of the AIDS, which comprises six amplification reagents in the PCR detection reagents.
Further, in the above PCR detection kit, the original concentration of the EBV forward primer is 50. Mu.M, and the final concentration in a 25. Mu.L reaction system is 300nM; the original concentration of the EBV reverse primer was 50. Mu.M, and the final concentration in the 25. Mu.L reaction system was 300nM; the original concentration of the EBV fluorescent probe was 50. Mu.M, and the final concentration in the 25. Mu.L reaction system was 150nM.
Further, the EBV DNA amplification reagent, the HCMV DNA amplification reagent, the HSV DNA amplification reagent, the TB DNA amplification reagent, the JCV DNA amplification reagent and the TG DNA amplification reagent further comprise Taq DNA polymerase, magnesium chloride solution, potassium chloride solution, dNTPs and Tris-HCL buffer solution respectively, wherein the pH value of the Tris-HCL buffer solution is 8.0.
Further, the initial concentration of Taq DNA polymerase is 5U/. Mu.L, and the final concentration in a 25. Mu.L reaction system is 1U/. Mu.L;
the initial concentration of the magnesium chloride solution was 1M, and the final concentration in the 25. Mu.L reaction system was 1mM;
the original concentration of the potassium chloride solution was 1M, and the final concentration in the 25. Mu.L reaction system was 30mM;
the original concentration of dNTPs was 10mM, and the final concentration in the 25. Mu.L reaction system was 0.4mM;
the original concentration of the Tris-HCl buffer (pH 8.0) was 200mM, and the final concentration in the 25. Mu.L reaction system was 20mM.
Further, the kit is more than 6 wells, and each well contains an amplification reagent.
The invention also provides a PCR detection method of the HIV infection pathogen, which uses the PCR detection kit to detect, and comprises the following steps:
s1: extracting DNA of a sample to be detected;
s2: preparing an amplification system to complete a PCR amplification reaction;
s3: and collecting FAM detection channel fluorescence signals to obtain detection results.
Further, the conditions of the PCR amplification reaction are as follows: predenaturation temperature: 95 ℃; pre-denaturation time: 5min; fluorescence detection was then performed at 95℃for 15s and at 60℃for 35s, with 50 cycles alternating.
Compared with the prior art, the invention has at least one of the following beneficial effects:
1. the invention develops a PCR detection reagent by designing primers and fluorescent labeling probes of human intracranial EBV (EBV), human Cytomegalovirus (HCMV), herpes Simplex Virus (HSV), mycobacterium Tuberculosis (TB), JC polyoma virus (JCV) and Toxoplasma (TG), and can be used for detecting whether a sample to be detected is infected with single or multiple pathogens or preparing a kit for detecting the infection with the single or multiple pathogens, thereby saving cost and having high detection efficiency.
2. The PCR detection kit and the detection method for the infection of the central nervous system of the AIDS can detect whether the sample has EBV (EBV), human Cytomegalovirus (HCMV), herpes Simplex Virus (HSV), mycobacterium Tuberculosis (TB), JC polyoma virus (JCV) and Toxoplasma Gondii (TG) simultaneously from the nucleic acid sample through one PCR reaction, can save cost and have good repeatability compared with the PCR technology aiming at single pathogen detection, can rapidly and effectively judge intracranial space occupying lesions (BML) of the Central Nervous System (CNS) of an HIV infected person, and improve the detection efficiency of the infection diseases of the Central Nervous System (CNS) of the HIV infected person.
3. The design of the PCR detection kit for the HIV infection basically covers related pathogens of common HIV secondary infection, the detection is relatively comprehensive, the diagnosis can be realized quickly, a patient can obtain the targeted diagnosis at an early stage, the next targeted treatment can be carried out as early as possible, the course of the disease is shortened, and more damages to the central nervous system caused by the infection are reduced.
In the invention, the technical schemes can be mutually combined to realize more preferable combination schemes. Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention may be realized and attained by the structure particularly pointed out in the written description and drawings.
Drawings
The drawings are only for purposes of illustrating particular embodiments and are not to be construed as limiting the invention, like reference numerals being used to designate like parts throughout the drawings;
FIG. 1a is a sensitivity test amplification curve of an EBV DNA amplification reagent (50 copies/reaction) of the present invention;
FIG. 1b is a sensitivity test amplification curve of the EBV DNA amplification reagent (25 copies/reaction) of the present invention;
FIG. 1c is a sensitivity test amplification curve of the EBV DNA amplification reagent (10 copies/reaction) of the present invention;
FIG. 2a is a sensitivity test amplification curve of the HCMV DNA amplification reagent (50 copies/reaction) of the present invention;
FIG. 2b is a sensitivity test amplification curve of the HCMV DNA amplification reagent (25 copies/reaction) of the present invention;
FIG. 2c is a sensitivity test amplification curve of the HCMV DNA amplification reagent (10 copies/reaction) of the present invention;
FIG. 3a is a plot of the sensitivity test amplification of the HSV DNA amplification reagent (50 copies/reaction) of the present invention;
FIG. 3b is a plot of the sensitivity test amplification of the HSV DNA amplification reagent (25 copies/reaction) of the present invention;
FIG. 3c is a plot of the sensitivity test amplification of the HSV DNA amplification reagent (10 copies/reaction) of the present invention;
FIG. 4a is a sensitivity test amplification curve of a TB DNA amplification reagent (50 copies/reaction) according to the invention;
FIG. 4b is a sensitivity test amplification curve of the TB DNA amplification reagent (25 copies/reaction) of the invention;
FIG. 4c is a sensitivity test amplification curve of the TB DNA amplification reagent (10 copies/reaction) of the invention;
FIG. 5a is a sensitivity test amplification curve of a JCV DNA amplification reagent (50 copies/reaction) of the present invention;
FIG. 5b is a sensitivity test amplification curve of the JCV DNA amplification reagent (25 copies/reaction) of the present invention;
FIG. 5c is a sensitivity test amplification curve of the JCV DNA amplification reagent (10 copies/reaction) of the present invention;
FIG. 6a is a sensitivity test amplification curve of a TG DNA amplification reagent (50 copies/reaction) of the present invention;
FIG. 6b shows the sensitivity test amplification curve of the TG DNA amplification reagent (25 copies/reaction);
FIG. 6c is a sensitivity test amplification curve of a TG DNA amplification reagent (10 copies/reaction);
FIG. 7a is a graph showing the amplification curves of the EBV DNA amplification reagent and specific reference P1 specificity test of the present invention;
FIG. 7b is a graph showing the amplification curves of the HCMV DNA amplification reagent and the specific reference P2 specificity test of the present invention;
FIG. 7c is a graph showing the amplification curves of the TB DNA amplification reagent and the specific reference P3 specificity test of the invention;
FIG. 7d is a graph showing the amplification curves of the JCV DNA amplification reagent and specific reference P4 specificity test of the present invention;
FIG. 7e is a plot of the HSV DNA amplification reagents and specific reference P5 specific test amplification of the present invention;
FIG. 7f is a graph showing the amplification curves of the TG DNA amplification reagent and specific reference P6 specificity test of the present invention;
FIG. 8 is a PCR amplification curve of a sample 66295 according to the present invention;
FIG. 9 is a PCR amplification curve of a sample 75156 according to the present invention;
FIG. 10 is a PCR amplification curve of a sample of example 53968 according to the present invention;
FIG. 11 is a PCR amplification curve of a sample 281039 according to the present invention;
FIG. 12 is a PCR amplification curve of a sample 291121 according to the present invention;
FIG. 13 is a PCR amplification curve of sample 1046 according to the present invention;
FIG. 14 is a PCR amplification curve of sample 1048 of the present invention;
FIG. 15 is a PCR amplification curve of a sample of example 7736 of the present invention;
FIG. 16 is a PCR amplification curve of a sample 66307 according to the present invention;
FIG. 17 shows the PCR amplification curve of a sample of example 60164 of the present invention.
Detailed Description
Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form a part hereof, and together with the description serve to explain the principles of the invention, and are not intended to limit the scope of the invention.
The invention provides a PCR detection reagent, which is used for detecting six pathogens, wherein the six pathogens are respectively: human intracranial EB virus (EBV), human cytomegalovirus (human cytomegalovirus, HCMV), herpes simplex virus (herpes simplex virus, HSV), mycobacterium tuberculosis (Mycobacterium tuberculosis, TB), JC polyomavirus (JC polyomairus, JCV) and toxoplasma (Toxoplasma gondii, TG).
The detection reagent comprises one or more of an EBV DNA amplification reagent, an HCMV DNA amplification reagent, an HSV DNA amplification reagent, a TB DNA amplification reagent, a JCV DNA amplification reagent and a TG DNA amplification reagent;
the EBV DNA amplification reagent comprises an EBV forward primer and an EBV reverse primer; the sequence of the EBV forward primer is: CAGCTTTGACGATGGAGTAG (SEQ ID NO: 1); the sequence of the EBV reverse primer is: CAYCTCCDTCATCTCCGTCAT (SEQ ID NO: 2);
the HCMV DNA amplification reagent comprises a forward primer of HCMV and a reverse primer of HCMV; the sequence of the HCMV forward primer is: CTTCTTATCACCATCAGCG (SEQ ID NO: 4); the sequence of the HCMV reverse primer is: GACGCTGGTGGGGGTCGGC (SEQ ID NO: 5);
the HSV DNA amplification reagent comprises a forward primer and a reverse primer of HSV; the sequence of HSV forward primer is: GGYCTGYCCCATCCGAACG (SEQ ID NO: 7); the sequence of the HSV reverse primer is: CAGGTACGTRCCSGCGGTCT (SEQ ID NO: 8);
the TB DNA amplification reagent comprises a TB forward primer and a TB reverse primer; the sequence of the TB forward primer is: CTCCGTACCCGGAGCGCCAA (SEQ ID NO: 10); the sequences of the TB reverse primer were: GAATCGGTCTGGACGTCAAGG (SEQ ID NO: 11);
the JCV DNA amplification reagent comprises a JCV forward primer and a JCV reverse primer; the sequence of the JCV forward primer is: GCTCATACCTAGGGAGCC (SEQ ID NO: 13); the JCV reverse primer sequences were: AGGGGTTTCACTATAACTG (SEQ ID NO: 14);
the TG DNA amplification reagent comprises a forward primer and a TG reverse primer of TG; the sequence of the TG forward primer is: GAGCACGCAACCCTGGACC (SEQ ID NO: 16); the sequence of the TG reverse primer is: CACCTCAGTCTCTTGCCTGC (SEQ ID NO: 17).
The EBV DNA amplification reagent can be used for detecting whether a sample to be detected is infected with EBV virus or preparing a kit for detecting the pathogen infected with the EBV virus, and the minimum detection limit of the EBV virus is 25 copies/reaction. The HCMV DNA amplification reagent can be used for detecting whether a sample to be tested is infected with HCMV virus or preparing a kit for detecting pathogen infected with HCMV, and the minimum detection limit of the HCMV virus is 25 copies/reaction. The HSV DNA amplification reagent can be used for detecting whether a sample to be detected is infected with HSV virus or preparing a kit for detecting the pathogen infected with HSV, and the minimum detection limit of the HSV virus is 25 copies/reaction. The TB DNA amplification reagent can be used for detecting whether a sample to be tested is infected with TB virus or preparing a kit for detecting a pathogen infected with TB, and the minimum detection limit of the TB virus is 50 copies/reaction. The JSV DNA amplification reagent can be used for detecting whether a sample to be detected is infected with JSV virus or preparing a kit for detecting the pathogen infected with JSV, and the minimum detection limit of the JSV virus is 25 copies/reaction. The TG DNA amplification reagent can be used for detecting whether a sample to be detected is infected with TG virus or preparing a kit for detecting the pathogen infected with HSV, and the lowest detection limit of the TG virus is 25 copies/reaction.
When the PCR detection reagent comprises several of EBV DNA amplification reagent, HCMV DNA amplification reagent, HSV DNA amplification reagent, TB DNA amplification reagent, JCV DNA amplification reagent and TG DNA amplification reagent, the reagent is used for detecting whether a sample to be detected is infected with related pathogens, and PCR non-specific amplification of any single pathogen does not occur in other 5 pathogens, and no cross reaction exists.
The EBV DNA amplification reagent also comprises an EBV fluorescent probe, and the sequence of the EBV fluorescent probe is as follows: FAM-CGGCAGCCCCTTCCACCATAGG-BHQ1 (SEQ ID NO: 3); and/or the number of the groups of groups,
the HCMV DNA amplification reagent also comprises a HCMV fluorescent probe, and the sequence of the HCMV fluorescent probe is as follows: FAM-CCACGCTGACAACCCACTCTT-BHQ1 (SEQ ID NO: 6); and/or the number of the groups of groups,
the HSV DNA amplification reagent also comprises an HSV fluorescent probe, and the sequence of the HSV fluorescent probe is as follows: AGCGCCGTCAGCGAGGAT (SEQ ID NO: 9); and/or the number of the groups of groups,
the TB DNA amplification reagent further comprises; the sequence of the TB fluorescent probe is: FAM-TCTCCTTCGCTAAGCTGCGC-BHQ1 (SEQ ID NO: 12); and/or the number of the groups of groups,
the JCV DNA amplification reagent also comprises JCV fluorescent probes, and the sequences of the JCV fluorescent probes are as follows: FAM-CAGCCAGAGGGAGCCCT-BHQ1 (SEQ ID NO: 15); and/or the number of the groups of groups,
the TG DNA amplification reagent also comprises a TG fluorescent probe, and the sequence of the TG fluorescent probe is FAM-TAGCGGAATGAGCAATTGTGC-BHQ1 (SEQ ID NO: 18).
The invention also provides application of the PCR detection reagent, and the application of the PCR detection reagent comprises any one of the following steps: detecting pathogens causing infection of the central nervous system of AIDS; preparing a kit for detecting pathogens causing infection of the central nervous system of AIDS; detecting whether a sample to be detected contains pathogens causing infection of the central nervous system of AIDS; preparing a kit for detecting whether a sample to be tested contains pathogens causing infection of the central nervous system of AIDS.
Through the application of the PCR detection reagent, the invention provides a PCR detection kit for HIV infection, which comprises six amplification reagents in the PCR detection reagent, wherein the kit is 6 holes, and each hole contains 20 mu L of the amplification reagent.
Specifically, in the PCR detection kit for HIV infection, the original concentration of the EBV forward primer is 50 mu M, and the final concentration in a 25 mu L reaction system is 300nM; the original concentration of the EBV reverse primer was 50. Mu.M, and the final concentration in the 25. Mu.L reaction system was 300nM; the original concentration of the EBV fluorescent probe was 50. Mu.M, and the final concentration in the 25. Mu.L reaction system was 150nM;
the original concentration of HCMV forward primer was 50. Mu.M, and the final concentration in 25. Mu.L reaction system was 300nM; the original concentration of HCMV reverse primer was 50. Mu.M, and the final concentration in 25. Mu.L reaction system was 300nM; the initial concentration of HCMV fluorescent probe was 50. Mu.M, and the final concentration in 25. Mu.L reaction system was 150nM;
the original concentration of HSV forward primer was 50. Mu.M, and the final concentration in 25. Mu.L reaction system was 300nM; the initial concentration of HSV reverse primer was 50. Mu.M, with a final concentration of 300nM in 25. Mu.L reaction; the initial concentration of HSV fluorescent probe was 50. Mu.M, and the final concentration in 25. Mu.L reaction system was 150nM;
the initial concentration of TB forward primer was 50. Mu.M, and the final concentration in 25. Mu.L reaction system was 300nM; the initial concentration of TB reverse primer was 50. Mu.M, and the final concentration in 25. Mu.L reaction system was 300nM; the initial concentration of TB fluorescent probe was 50. Mu.M, and the final concentration in 25. Mu.L reaction system was 150nM;
the original concentration of the JCV forward primer was 50. Mu.M, and the final concentration in the 25. Mu.L reaction system was 300nM; the original concentration of the JCV reverse primer was 50. Mu.M, and the final concentration in the 25. Mu.L reaction system was 300nM; the initial concentration of JCV fluorescent probe was 50. Mu.M, and the final concentration in 25. Mu.L reaction system was 150nM;
the original concentration of TG forward primer was 50. Mu.M, and the final concentration in 25. Mu.L reaction system was 300nM; the original concentration of the TG reverse primer was 50. Mu.M, and the final concentration in the 25. Mu.L reaction system was 300nM; the initial concentration of TG fluorescent probe was 50. Mu.M, and the final concentration in the 25. Mu.L reaction system was 150nM.
Specifically, in the PCR detection kit for infection of the central nervous system of the AIDS, the EBV DNA amplification reagent, the HCMV DNA amplification reagent, the HSV DNA amplification reagent, the TB DNA amplification reagent, the JCV DNA amplification reagent and the TG DNA amplification reagent also respectively comprise Taq DNA polymerase, magnesium chloride solution, potassium chloride solution, dNTPs and Tris-HCL buffer solution (pH8.0);
wherein the initial concentration of Taq DNA polymerase is 5U/. Mu.L, and the final concentration in a 25. Mu.L reaction system is 1U/. Mu.L; the original concentration of the magnesium chloride solution was 1M, and the final concentration in the 25. Mu.L reaction system was 1mM; the original concentration of the potassium chloride solution was 1M, and the final concentration in the 25. Mu.L reaction system was 30mM; the original concentration of dNTPs was 10mM, and the final concentration in the 25. Mu.L reaction system was 0.4mM; the original concentration of Tris-HCl buffer (pH 8.0) was 200mM and the final concentration in the 25. Mu.L reaction system was 20mM.
The original concentration is the initial concentration of the component; for detecting a sample of one person, 25 mu L (containing 20 mu L of amplification reagent and 5 mu L of sample nucleic acid) of the corresponding PCR amplification reagent is needed, and the concentration of each component in a 25 mu L reaction system is the final concentration.
The invention also provides a PCR detection method of the HIV infection pathogen, which uses the PCR detection kit for the HIV infection, and comprises the following steps:
s1: extracting DNA of a sample to be detected;
s2: preparing an amplification system to complete a PCR amplification reaction;
s3: collecting FAM detection channel fluorescence signals to obtain detection results, and carrying out result analysis;
in step S1, the DNA of the sample to be tested may be extracted according to a conventional method well known to those skilled in the art, and the nucleic acid kit may be selected and extracted according to the kit instructions. After extraction, the sample DNA volume was not less than 40. Mu.L.
In step S2, 6 amplification reagents are taken out in advance, thawed and melted at room temperature by using the kit, and the tube cover is taken down for standby after instantaneous centrifugation.
And taking 8 rows of PCR tubes with corresponding numbers according to the number of the samples to be detected. Each 8 PCR tubes (first 6 wells) were lined with 20. Mu.L and 5. Mu.L of the same sample nucleic acid as a pathogen DNA amplification reagent, respectively, and were loaded onto the machine after transient centrifugation. Illustratively, the EBV DNA amplification reagent 20. Mu.L and 5. Mu.L of the sample nucleic acid to be tested are added to well 1, the HCMV DNA amplification reagent 20. Mu.L and 5. Mu.L of the sample nucleic acid to be tested are added to well 2, the HSV DNA amplification reagent 20. Mu.L and 5. Mu.L of the sample nucleic acid to be tested are added to well 3, the TB DNA amplification reagent 20. Mu.L and 5. Mu.L of the sample nucleic acid to be tested are added to well 4, the JCV DNA amplification reagent 20. Mu.L and 5. Mu.L of the sample nucleic acid to be tested are added to well 5, and the TG DNA amplification reagent 20. Mu.L and 5. Mu.L of the sample nucleic acid to be tested are added to well 6.
The applicable model is ABI 7500, biorad CFX 96 and a macrostone fluorescence quantitative PCR instrument, the fluorescence channel is selected as FAM, and PCR amplification is carried out according to the following amplification program:
predenaturation temperature: pre-denaturation time at 95 ℃): 5min;
then, multiplex fluorescence detection was performed at 95℃for 15s and 60℃for 35s, and 50 cycles were alternately performed at 60 ℃.
In step S3, the determination is performed according to the PCR result of the sample to be tested, and the determination criteria are as follows:
in the corresponding PCR amplification reaction of the detection samples of human intracranial EBV (EBV), human Cytomegalovirus (HCMV), herpes Simplex Virus (HSV), mycobacterium Tuberculosis (TB), JC polyoma virus (JCV) and Toxoplasma Gondii (TG), the FAM channel has obvious S-shaped amplification curve, and the Ct value is less than or equal to 38, and can be judged as positive; the sample with Ct less than 38 and less than or equal to 40 of the detection sample is recommended to be reworked, and the result of the reworking is that the Ct value is less than 40 and is positive, otherwise, the result is that the sample is negative.
The PCR detection kit for the infection of the central nervous system of the AIDS is used for respectively detecting human intracranial EBV (EBV), human Cytomegalovirus (HCMV), herpes Simplex Virus (HSV), mycobacterium Tuberculosis (TB), JC polyoma virus (JCV) and Toxoplasma Gondii (TG) by adopting the PCR detection method for the infection pathogen of the central nervous system of the AIDS so as to detect the specificity and the sensitivity of the method.
Sensitivity test:
sensitivity to human intracranial EBV (EBV), human Cytomegalovirus (HCMV), herpes Simplex Virus (HSV), mycobacterium Tuberculosis (TB), JC polyomavirus (JCV) and Toxoplasma Gondii (TG) was tested and a calibrated copy number (copies/ml) of the EBV virus synthetic fragment (10 12 cobies/ml), HCMV virus synthetic fragment (10) 12 cobies/ml), HSV virus synthetic fragment (10) 12 cobies/ml), TB synthetic fragment (10 12 cobies/ml), JCV virus synthetic fragment (10) 12 cobies/ml), synthetic fragment of TG virus (10 12 cobies/ml) were diluted with a 10-fold gradient, respectively, and a fluorescent quantitative PCR reaction was performed in parallel experiments to verify the sensitivity of the detection method of the present invention.
The results of the sensitivity test of EBV virus are shown in FIGS. 1a-1c, where it can be seen that 1a is 50 copies/reaction and that amplification curves appear steadily for 4 parallel tests; 1b is 25 copies/reaction, and amplification curves appear stably in 4 parallel experiments; 1c is 10 copies/reaction, 3 amplification curves appear in 4 parallel experiments, and the concentration sample is unstable to detection; thus, the EBV virus minimum detection limit is 25 copies/response.
The results of the sensitivity test of HCMV virus are shown in FIGS. 2a-2c, which show that 2a is 50 copies/reaction and that amplification curves appear stably in 4 parallel tests; 2b is 25 copies/reaction, and amplification curves appear stably in 4 parallel experiments; 2c is 10 copies/reaction, 3 amplification curves appear in 4 parallel experiments, and the concentration sample is unstable to detection; thus, the minimum detection limit for HCMV virus is 25 copies/response.
The results of the sensitivity test of HSV virus are shown in FIGS. 3a-3c, where it can be seen that 3a is 50 copies/reaction and that amplification curves appear steadily in 4 parallel tests; 3b is 25 copies/reaction, and amplification curves appear stably in 4 parallel experiments; 3c is 10 copies/reaction, 3 amplification curves appear in 4 parallel experiments, and the concentration sample is unstable to detection; the minimum detection limit for HSV virus was 25 copies/response.
The results of the TB virus sensitivity test are shown in FIGS. 4a-4c, where it can be seen that 4a is 50 copies/reaction and that amplification curves appear steadily for each of the 4 parallel tests; 4b is 25 copies/reaction, 3 amplification curves appear in 4 parallel experiments; the concentration sample is unstable to detect; the minimum detection limit for TB virus was 50 copies/response.
The result of the sensitivity test of the JCV virus is shown in FIGS. 5a-5c, and as can be seen from the figures, 5a is 50 copies/reaction, and amplification curves stably appear in 4 parallel tests; 5b is 25 copies/reaction, and amplification curves appear stably in 4 parallel experiments; 5c is 10 copies/reaction, 3 amplification curves appear in 4 parallel experiments, and the concentration sample is unstable to detection; the minimum detection limit for JCV virus was 25 copies/reaction.
The results of the sensitivity test of TG virus are shown in FIGS. 6a-6c, in which it can be seen that 6a is 50 copies/reaction and that amplification curves appear stably in 4 parallel tests; 6b is 25 copies/reaction, and amplification curves appear stably in 4 parallel experiments; 6c is 10 copies/reaction, 3 amplification curves appear in 4 parallel experiments, and the concentration sample is unstable to detection; the lowest detection limit of TG virus was 25 copies/reaction.
Specificity test:
the specificity of human intracranial EBV (EBV), human Cytomegalovirus (HCMV), herpes Simplex Virus (HSV), mycobacterium Tuberculosis (TB), JC polyoma virus (JCV) and Toxoplasma Gondii (TG) was tested to verify that PCR non-specific amplification of any single pathogen within the group (6 pathogens) does not occur in the other 5 pathogens. A calibrated copy number (copies/ml) of the EBV virus synthesis fragment (10) 12 cobies/ml), HCMV virus synthetic fragment (10) 12 cobies/ml), HSV virus synthetic fragment (10) 12 cobies/ml), TB synthetic fragment (10 12 cobies/ml), JCV virus synthetic fragment (10) 12 cobies/ml), TG virus synthetic tabletSegment (10) 12 cobies/ml) were diluted with a 10-fold gradient, and mixed to 6 specific references P1 to P6, and tables 1 to 6 are the components of the specific references P1 to P6, the original concentrations and the final concentrations in a 25. Mu.L reaction system; fluorescent quantitative PCR reactions were performed in parallel experiments to verify the specificity of the detection method of the present invention.
The specific references P1-P6 are used as templates respectively, and an amplification system is prepared with the DNA amplification reagent of each pathogen in the kit according to the invention, and the details are shown in Table 7. The dosage of the specific reference products P1-P6 is 5 mu L, and the dosage of the DNA amplification reagent of each pathogen is 20 mu L; the fluorescent PCR reaction was performed in parallel for 5 times, the fluorescent channel was "FAM" selected, and PCR amplification was performed according to the following amplification procedure:
predenaturation temperature: pre-denaturation time at 95 ℃): 5min;
then, multiplex fluorescence detection was performed at 95℃for 15s and 60℃for 35s, and 50 cycles were alternately performed at 60 ℃.
FIGS. 7 a-7 f are specific assay-amplification curves from which it can be seen that any single pathogen does not exhibit PCR nonspecific amplification in other 5 pathogens, without cross-reaction.
Examples
Examples by using the kit of the PCR detection reagent for HIV infection of the present invention, PCR test was performed on 10 cases of HIV-infected persons suffering from CNS (CNS) intracranial infection, as well as on nucleic acid samples of unknown infection types, attached to Beijing forum Hospital, university of capital medical science, by the PCR detection method for HIV infection pathogens of the present invention, and the sample information is shown in Table 8.
Preparation of an amplification system:
the kit of the invention is used for taking out 6 amplification reagents in advance, thawing and thawing at room temperature, and taking down a tube cover for standby after instantaneous centrifugation.
Sample adding:
and taking 8 rows of PCR tubes with corresponding numbers according to the number of the samples to be detected. Each 8 PCR tubes (first 6 wells) were lined with 20. Mu.L and 5. Mu.L of the same sample nucleic acid as a pathogen DNA amplification reagent, respectively, and were loaded onto the machine after transient centrifugation. Using a macrostone fluorescent quantitative PCR instrument, the fluorescent channel was selected for "FAM", and PCR amplification was performed according to the following amplification procedure:
predenaturation temperature: pre-denaturation time at 95 ℃): 5min;
then, multiplex fluorescence detection was performed at 95℃for 15s and 60℃for 35s, and 50 cycles were alternately performed at 60 ℃. The amplification system configuration is shown in Table 9. The results of the measurements are shown in Table 10, and the amplification curves of the samples in the examples are shown in FIGS. 8-17. In order to verify the detection accuracy of the kit, the sample is additionally sent to a third party inspection institute, namely Shanghai biological organism, and the molecular detection gold standard sanger generation sequencing is selected for sequencing verification.
As can be seen from Table 10, the detection results of the sample PCR kit are consistent with the gold standard first-generation sequencing results of molecular detection, which indicates that the detection of the kit is accurate and reliable. The invention develops a PCR detection reagent by designing primers and fluorescent labeling probes of human intracranial EBV (EBV), human Cytomegalovirus (HCMV), herpes Simplex Virus (HSV), mycobacterium Tuberculosis (TB), JC polyomavirus (JCV) and Toxoplasma (TG), which can be used for detecting pathogens causing HIV central nervous system infection; preparing a kit for detecting pathogens causing infection of the central nervous system of AIDS; detecting whether a sample to be detected contains pathogens causing infection of the central nervous system of AIDS; preparing a kit for detecting whether a sample to be tested contains pathogens causing infection of the central nervous system of AIDS. Through one PCR reaction, whether the sample has EBV (EBV), human Cytomegalovirus (HCMV), herpes Simplex Virus (HSV), mycobacterium Tuberculosis (TB), JC polyoma virus (JCV) and Toxoplasma Gondii (TG) can be detected simultaneously from the nucleic acid sample, and compared with the existing laboratory PCR technology aiming at single pathogen detection, the detection method is simpler, more convenient and faster, saves cost and has good repeatability, and can rapidly and effectively judge intracranial space occupying lesions (BML) of a Central Nervous System (CNS) of an HIV infected person.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.
Claims (10)
1. The PCR detection reagent is characterized by comprising one or more of an EBV DNA amplification reagent, an HCMV DNA amplification reagent, an HSV DNA amplification reagent, a TB DNA amplification reagent, a JCV DNA amplification reagent and a TG DNA amplification reagent;
the EBV DNA amplification reagent comprises an EBV forward primer and an EBV reverse primer; the sequence of the EBV forward primer is: CAGCTTTGACGATGGAGTAG; the sequence of the EBV reverse primer is: CAYCTCCDTCATCTCCGTCAT;
the HCMV DNA amplification reagent comprises a forward primer of HCMV and a reverse primer of HCMV; the sequence of the HCMV forward primer is: CTTCTTATCACCATCAGCG; the sequence of the HCMV reverse primer is: GACGCTGGTGGGGGTCGGC;
the HSV DNA amplification reagent comprises a forward primer and a reverse primer of HSV; the sequence of HSV forward primer is: GGYCTGYCCCATCCGAACG; the sequence of the HSV reverse primer is: CAGGTACGTRCCSGCGGTCT;
the TB DNA amplification reagent comprises a TB forward primer and a TB reverse primer; the sequence of the TB forward primer is: CTCCGTACCCGGAGCGCCAA; the sequences of the TB reverse primer were: GAATCGGTCTGGACGTCAAGG;
the JCV DNA amplification reagent comprises a JCV forward primer and a JCV reverse primer; the sequence of the JCV forward primer is: GCTCATACCTAGGGAGCC; the JCV reverse primer sequences were: AGGGGTTTCACTATAACTG;
the TG DNA amplification reagent comprises a forward primer and a TG reverse primer of TG; the sequence of the TG forward primer is: GAGCACGCAACCCTGGACC; the sequence of the TG reverse primer is: CACCTCAGTCTCTTGCCTGC.
2. A PCR detection reagent according to claim 1, wherein,
the EBV DNA amplification reagent also comprises an EBV fluorescent probe, and the sequence of the EBV fluorescent probe is as follows: FAM-CGGCAGCCCCTTCCACCATAGG-BHQ1; and/or the number of the groups of groups,
the HCMV DNA amplification reagent also comprises a HCMV fluorescent probe, and the sequence of the HCMV fluorescent probe is as follows: FAM-CCACGCTGACAACCCACTCTT-BHQ1; and/or the number of the groups of groups,
the HSV DNA amplification reagent also comprises an HSV fluorescent probe, and the sequence of the HSV fluorescent probe is as follows: AGCGCCGTCAGCGAGGAT; and/or the number of the groups of groups,
the TB DNA amplification reagent also comprises a TB fluorescent probe, and the sequence of the TB fluorescent probe is as follows: FAM-TCTCCTTCGCTAAGCTGCGC-BHQ1; and/or the number of the groups of groups,
the JCV DNA amplification reagent also comprises JCV fluorescent probes, and the sequences of the JCV fluorescent probes are as follows: FAM-CAGCCAGAGGGAGCCCT-BHQ1; and/or the number of the groups of groups,
the TG DNA amplification reagent also comprises a TG fluorescent probe, and the sequence of the TG fluorescent probe is FAM-TAGCGGAATGAGCAATTGTGC-BHQ1.
3. Use of a PCR detection reagent according to any one of claims 1-2, characterized in that the use comprises any one of the following:
detecting pathogens causing infection of the central nervous system of AIDS;
preparing a kit for detecting pathogens causing infection of the central nervous system of AIDS;
detecting whether a sample to be detected contains pathogens causing infection of the central nervous system of AIDS;
preparing a kit for detecting whether a sample to be tested contains pathogens causing infection of the central nervous system of AIDS.
4. A PCR detection kit for aids central nervous system infection, comprising six amplification reagents of the PCR detection reagent according to any one of claims 1-2.
5. The PCR detection kit of claim 4, wherein the EBV forward primer has an initial concentration of 50 μΜ and a final concentration of 300nM in a 25 μl reaction system; the original concentration of the EBV reverse primer was 50. Mu.M, and the final concentration in the 25. Mu.L reaction system was 300nM; the original concentration of the EBV fluorescent probe was 50. Mu.M, and the final concentration in the 25. Mu.L reaction system was 150nM.
6. The PCR detection kit according to claim 4, wherein the EBV DNA amplification reagent, HCMV DNA amplification reagent, HSV DNA amplification reagent, TB DNA amplification reagent, JCV DNA amplification reagent and TG DNA amplification reagent further comprise Taq DNA polymerase, magnesium chloride solution, potassium chloride solution, dNTPs, tris-HCl buffer solution, respectively, and the pH of the Tris-HCl buffer solution is 8.0.
7. The PCR detection kit of claim 6, wherein,
the original concentration of the Taq DNA polymerase is 5U/. Mu.L, and the final concentration in a 25. Mu.L reaction system is 1U/. Mu.L;
the initial concentration of the magnesium chloride solution was 1M, and the final concentration in the 25. Mu.L reaction system was 1mM;
the original concentration of the potassium chloride solution was 1M, and the final concentration in the 25. Mu.L reaction system was 30mM;
the original concentration of dNTPs was 10mM, and the final concentration in the 25. Mu.L reaction system was 0.4mM;
the original concentration of the Tris-HCl buffer (pH 8.0) was 200mM, and the final concentration in the 25. Mu.L reaction system was 20mM.
8. The PCR detection kit of any one of claims 4 to 7, wherein the kit is more than 6 wells, each well containing an amplification reagent.
9. A PCR method for detecting pathogens of aids central nervous system infection, characterized in that the PCR detection kit of claim 8 is used for detection, comprising the steps of:
s1: extracting DNA of a sample to be detected;
s2: preparing an amplification system to complete a PCR amplification reaction;
s3: and collecting FAM detection channel fluorescence signals to obtain detection results.
10. The method according to claim 9, wherein in step S2, the conditions for the PCR amplification reaction are: predenaturation temperature: 95 ℃; pre-denaturation time: 5min; fluorescence detection was then performed at 95℃for 15s and at 60℃for 35s, with 50 cycles alternating.
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