Human enterovirus four-color fluorescence RT-PCR detection kit and detection method

Abstract

The invention discloses a human enterovirus four-color fluorescence RT-PCR detection kit and a detection method; the detection kit comprises multiple fluorescence RT-PCR reaction mother liquor, AMV reverse transcriptase liquid, Taq DNA polymerase liquid, a positive control, and a negative control; the detection kit is characterized by further comprising an internal control, a multiple 10*PCR reaction buffer, bovine serum albumin with a concentration of 20 mg / ml, various forward primers and reverse primers with a concentration of 100 muM, and various specific probes with a concentration of 50 muM, wherein sequences of the forward primers, reverse primers, specific probes and the internal control are as shown in SEQIDNO. 1, NO. 2, NO.3, NO.4, NO.5, NO.6, NO.7. NO.8, NO.9, NO.10., NO.11., NO.12., and NO.13. The detection kit of the invention has the advantages of high detection sensitivity, good specificity, accuracy, high speed, and no false positivity.

Description

technical field

[0001] The invention relates to a fluorescent PCR kit in the field of biotechnology, in particular to a four-color fluorescent RT-PCR detection kit containing an internal control for general detection of human enteroviruses and type detection of enteroviruses EV71 and CoxA16 . Background technique

[0002] Enteroviruses include 71 serotypes including poliovirus, Coxsackievirus, enterocytopathic human orphan virus (ECHO) and new enteroviruses. The hand, foot and mouth disease caused by enterovirus is a kind of acute infectious disease. The main clinical features are fever and skin rashes on the hands, feet, and oral cavity. Maculopapular rash and herpes appear on the hands, feet and buttocks, which mostly occur in preschool children, especially in the age group under 3 years old. This type of disease is distributed all over the world and occurs throughout the year in the tropics and subtropics.

[0003] The main causes of hand, foot and mouth disease are ty...

Examples

specific Embodiment 1

[0051] Enterovirus four-color fluorescent RT-PCR kit

[0052] 1. RT-PCR kit composition

[0053] This kit includes multiple fluorescent RT-PCR reaction master solution, positive control substance, negative control substance, internal control, AMV reverse transcriptase, Taq polymerase. The multiple fluorescent RT-PCR reaction master solution contains multiple 10× fluorescent RT-PCR reaction buffer, EV universal primers, EV universal probes, EV71 primers, EV71 probes, CoxA16 primers, CoxA16 probes, internal Control primers, internal control probes, bovine serum albumin; multiplex 10× fluorescent RT-PCR reaction buffer contains tris hydrochloride, potassium chloride, glycerin, four kinds of mononucleotide monomers, and magnesium chloride.

[0054] The multiple fluorescent RT-PCR reaction master solution is prepared as follows (the corresponding substance concentrations are shown in brackets):

[0055] Multiplex 10×PCR reaction buffer 5ul

[0056] Bovine serum albumin (20mg / ml)...

specific Embodiment 2

[0088] Four-color fluorescent RT-PCR detection method for human enterovirus, positive control and negative control should be set up for each detection

[0089] 1. Sample processing:

[0090] Collect throat swabs, herpes fluid, cerebrospinal fluid or human enterovirus isolation culture from suspected patients, and quickly put them into a commercial sampling tube (Youkang Jiye, MT0301-1) containing 1-2ml of virus preservation solution as the sample to be tested use;

[0091] 2. Extraction of viral nucleic acid RNA:

[0092] Strictly follow the commercial nucleic acid RNA extraction kit QIAGEN Viral RNA Mini Extraction Kit (QIAGEN, CAT: 52904) to extract viral RNA from clinical specimens or virus isolation cultures; add 10ul internal control (concentration: 10 4 copies / ml) to participate in the extraction of nucleic acid samples simultaneously;

[0093] 3. RT-PCR reaction system preparation

[0094] Take 10ul extracted sample nucleic acid, 10ul positive control substance and ...

specific Embodiment 3

[0105] Detection Kit Sensitivity and Specificity Analysis

[0106] 1. Experimental material virus strain

[0107] Target strain group: Enterovirus EV71 strain, Coxsackie virus group A type 16 strain, Coxsackie virus group A type 1, Coxsackie virus group A type 11, Coxsackie virus group A type 13 , Coxsackie virus group B type 3, enterovirus EV68 type, echovirus type 5, echovirus type 13; non-target strain group: rotavirus, norovirus type I and II, type A H1N1 Influenza virus and influenza B virus, the above-mentioned strains were purchased from the American Culture Collection and Management Center, or isolated and preserved by the Ningbo Center for Disease Control and Prevention.

[0108] 2. Test results

[0109] Enterovirus four-color fluorescent RT-PCR kit was used to detect the above strains, and the target strain group included Enterovirus EV71 strain, Coxsackievirus A group 16 strain, Coxsackievirus A group 1 Coxsackievirus A group 11, Coxsackievirus A group 13, Coxsac...

technical field

[0001] The invention relates to a fluorescent PCR kit in the field of biotechnology, in particular to a four-color fluorescent RT-PCR detection kit containing an internal control for general detection of human enteroviruses and type detection of enteroviruses EV71 and CoxA16 . Background technique

[0002] Enteroviruses include 71 serotypes including poliovirus, Coxsackievirus, enterocytopathic human orphan virus (ECHO) and new enteroviruses. The hand, foot and mouth disease caused by enterovirus is a kind of acute infectious disease. The main clinical features are fever and skin rashes on the hands, feet, and oral cavity. Maculopapular rash and herpes appear on the hands, feet and buttocks, which mostly occur in preschool children, especially in the age group under 3 years old. This type of disease is distributed all over the world and occurs throughout the year in the tropics and subtropics.

[0003] The main causes of hand, foot and mouth disease are ty...

Examples

specific Embodiment 1

[0051] Enterovirus four-color fluorescent RT-PCR kit

[0052] 1. RT-PCR kit composition

[0053] This kit includes multiple fluorescent RT-PCR reaction master solution, positive control substance, negative control substance, internal control, AMV reverse transcriptase, Taq polymerase. The multiple fluorescent RT-PCR reaction master solution contains multiple 10× fluorescent RT-PCR reaction buffer, EV universal primers, EV universal probes, EV71 primers, EV71 probes, CoxA16 primers, CoxA16 probes, internal Control primers, internal control probes, bovine serum albumin; multiplex 10× fluorescent RT-PCR reaction buffer contains tris hydrochloride, potassium chloride, glycerin, four kinds of mononucleotide monomers, and magnesium chloride.

[0054] The multiple fluorescent RT-PCR reaction master solution is prepared as follows (the corresponding substance concentrations are shown in brackets):

[0055] Multiplex 10×PCR reaction buffer 5ul

[0056] Bovine serum albumin (20mg / ml)...

specific Embodiment 2

[0088] Four-color fluorescent RT-PCR detection method for human enterovirus, positive control and negative control should be set up for each detection

[0089] 1. Sample processing:

[0090] Collect throat swabs, herpes fluid, cerebrospinal fluid or human enterovirus isolation culture from suspected patients, and quickly put them into a commercial sampling tube (Youkang Jiye, MT0301-1) containing 1-2ml of virus preservation solution as the sample to be tested use;

[0091] 2. Extraction of viral nucleic acid RNA:

[0092] Strictly follow the commercial nucleic acid RNA extraction kit QIAGEN Viral RNA Mini Extraction Kit (QIAGEN, CAT: 52904) to extract viral RNA from clinical specimens or virus isolation cultures; add 10ul internal control (concentration: 10 4 copies / ml) to participate in the extraction of nucleic acid samples simultaneously;

[0093] 3. RT-PCR reaction system preparation

[0094] Take 10ul extracted sample nucleic acid, 10ul positive control substance and ...

specific Embodiment 3

[0105] Detection Kit Sensitivity and Specificity Analysis

[0106] 1. Experimental material virus strain

[0107] Target strain group: Enterovirus EV71 strain, Coxsackie virus group A type 16 strain, Coxsackie virus group A type 1, Coxsackie virus group A type 11, Coxsackie virus group A type 13 , Coxsackie virus group B type 3, enterovirus EV68 type, echovirus type 5, echovirus type 13; non-target strain group: rotavirus, norovirus type I and II, type A H1N1 Influenza virus and influenza B virus, the above-mentioned strains were purchased from the American Culture Collection and Management Center, or isolated and preserved by the Ningbo Center for Disease Control and Prevention.

[0108] 2. Test results

[0109] Enterovirus four-color fluorescent RT-PCR kit was used to detect the above strains, and the target strain group included Enterovirus EV71 strain, Coxsackievirus A group 16 strain, Coxsackievirus A group 1 Coxsackievirus A group 11, Coxsackievirus A group 13, Coxsac...

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