Composition and method for detecting group A streptococcus,
A composition, streptococcal technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem of unsatisfactory sensitivity
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Embodiment 1
[0075] Example 1 is designed to detect primers and probes for GAS
[0076] According to the spe B gene sequence of GAS, a total of 10 upstream primers, downstream primers and probe combinations were designed. Table 1 below takes three groups as examples to show the specific sequences and detection results of each primer and probe.
[0077] Table 1 Example of primer probe sequence based on GAS-based spe B gene sequence design
[0078]
Embodiment 2
[0079] Embodiment 2 detects GAS in the sample
[0080] 2.1 Extraction of DNA from GAS
[0081] Use the magnetic bead method nucleic acid extraction or purification reagent produced by Shengxiang Biotechnology Co., Ltd. (Xiangchang Machinery Equipment 20150021), and follow the manufacturer's instructions to extract the nucleic acid of GAS in the sample.
[0082] 2.2 Prepare PCR reaction reagents
[0083] 2.2.1 Experimental materials
[0084] Internal standard template: a recombinant of a 132 base pair artificially synthesized DNA sequence inserted into the pMD18T vector, wherein the sequence of 132 base pairs is shown in SEQ ID NO:4:
[0085] Internal standard upstream primer: as shown in SEQ ID NO:5;
[0086] Internal standard downstream primer: as shown in SEQ ID NO:6;
[0087] Internal standard probe: 5'HEX-CGCGCTTCTGGAAAGGCTTCTCAGT-BHQ1-3';
[0088] Positive control: calibrate the plasmid with known concentration, the concentration is 5.00E+05copies / ml;
[0089] Negat...
Embodiment 3
[0101] Embodiment 3 compares the detection effect of different combinations
[0102] Using combinations 1 to 3 in Example 1, 5 cases of GAS clinically positive samples were detected according to the method in Example 2, wherein the fluorescent quantitative PCR instrument was Hongshi SLAN-96, and the results were respectively: Figure 1 ~ Figure 3 The mean Ct values are summarized in Table 3, as indicated.
[0103] Table 3 Summary of test results of combinations 1 to 3
[0104]
[0105] Such as Figure 1~3 As shown in Table 3, the Ct value of combination 1 is higher, indicating that its amplification efficiency and sensitivity are higher.
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