Kit for detecting four enteroviruses and detection method thereof
A kit and detection probe technology, applied in the field of virus detection kits, can solve problems such as the inability to simultaneously detect multiple types of Coxsackieviruses
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Embodiment 1
[0082] [Example 1] A kit for simultaneously detecting four types of Coxsackieviruses
[0083] The kit includes RT-PCR reaction solution, nucleic acid composition for simultaneous detection of four types of Coxsackie virus, enzyme mixed solution, positive control and negative control. Wherein, the RT-PCR reaction solution includes 250mM Tris-base, 0.25% TritonX-100, 25mmol / L MgCl 2 .
[0084] Nucleic acid compositions include:
[0085] Coxsackievirus type A9 amplification primers whose sequences are shown in SEQ ID No.1 and SEQ ID No.2;
[0086] Coxsackievirus A10 type amplification primer pair whose sequences are shown in SEQ ID No.4 and SEQ ID No.5;
[0087] Coxsackievirus type B2 amplification primer pair whose sequences are shown in SEQ ID No.7 and SEQ ID No.8;
[0088] Coxsackievirus type B5 amplification primer pair whose sequences are shown in SEQ ID No.10 and SEQ ID No.11;
[0089] The coxsackie virus A9 type detection probe whose sequence is shown in SEQ ID No.3, ...
Embodiment 2
[0093] [Example 2] Determination of the sensitivity of the test kit for the simultaneous detection of four types of Coxsackievirus in Example 1
[0094] (1) Coxsackievirus A9 type positive standard, Coxsackievirus A10 type positive standard, Coxsackievirus B2 type positive standard, Coxsackievirus B5 positive standard in the kit of Example 1 Equal amounts were mixed and made into a mixed solution to obtain a positive test article (the concentration of each virus positive standard substance was 10 6 copies / mL).
[0095] (2) Perform a 10-fold serial dilution of the positive sample to be tested (i.e. 10copies / mL~10 5 copies / mL), using the kit of Example 1 to perform multiple fluorescent quantitative PCR detection on the positive test items under each gradient, the system of multiple fluorescent quantitative PCR reactions is shown in Table 1. Wherein the volume ratio of the hot start Taq enzyme, MMLV reverse transcriptase, and RNase inhibitor in the enzyme mixture is 4:1:3. All...
Embodiment 3
[0101] (1) Coxsackievirus A9 type positive standard, Coxsackievirus A10 type positive standard, Coxsackievirus B2 type positive standard, Coxsackievirus B5 type positive standard ( The concentration of each virus positive standard was 10 6copies / mL) were mixed and made into a mixed solution to obtain a positive test article; normal saline that did not contain the above-mentioned four-type Coxsackievirus positive control substance was a negative test article.
[0102] (2) The experiment is divided into an experimental group, a control group 1 and a control group 2, and the kit of the experimental group is the kit of Example 1. The kit components of control group 1 and control group 2 are commercial kits in use, and their components include: dNTPs, PCR reaction solution, amplification primers and probes for four viruses, enzyme mixture, positive standard , Negative standard. The kits of control groups 1 and 2 are approximately the same as those of Example 1.
[0103] (3) The ...
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