Kit for detecting haemophilus influenzae through loop-mediated isothermal amplification and using method
A Haemophilus influenzae, ring-mediated isotherm technology, applied in the field of biomedicine, can solve the problems of high cost, long detection time and complicated operation of Haemophilus influenzae, and achieve short time-consuming, high specificity and simple operation Effect
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Embodiment 1
[0029] Example 1: Primer Design
[0030] The basic principle of primer design in the LAMP reaction system is that the Tm value of the external primer set is controlled at 55–63°C, and the base length is 15–25. The Tm value of about 20 bases at the 5' end of the internal primer set is higher than that of other primer sets, controlled at 60–68°C, and the Tm value of about 20 bases at the 3’ end of the internal primer set is at 55–63°C. The transcription site is between the 5' end and the 3' end of the internal primer set, and the 5' sequence and 3' sequence of the transcription region should be fine-tuned according to the secondary structure of the primer itself, so that the secondary structure of the primer is minimized, and the base length of the entire internal primer set is between Between 50–70. The Tm value of the loop primer set is 60–66°C, and the base length is 15–25 bases. The GC content of all primers is controlled at 30%-65%, and it is necessary to avoid the format...
Embodiment 2
[0038] Example 2: Loop-mediated isothermal amplification detection kit for detection of Haemophilus influenzae detection of clinical samples
[0039] The following is a further description of the present invention in conjunction with specific examples, but it should not be regarded as a limitation of the present invention.
[0040] 1) Sample source
[0041] Selected from the Clinical Microbiology Department of the Laboratory Department of Shanghai Dongfang Hospital, two cases of sputum samples were positive and negative for Haemophilus influenzae identified by routine culture, and the samples were stored in PBS buffer. The positive quality control standard product is the pMD19-T plasmid carrying the specific DNA fragment of Haemophilus influenzae (sequence shown in SEQ ID NO.6), and the negative quality control product is PBS buffer.
[0042] 2) Use the QIAamp DNA Mini Kit Extraction Kit to extract DNA from the above samples according to the kit instructions.
[0043] 3) LAMP ...
Embodiment 3
[0047] Example 3 Sensitivity Analysis of Loop-mediated Isothermal Amplification Detection Kit for Haemophilus Influenzae
[0048] 1) Sample processing: Haemophilus influenzae (standard strain ATCC 9007) was extracted using the QIAamp DNA Mini Kit, and the sample DNA was extracted according to the kit instructions.
[0049] 2) Use a spectrophotometer to measure the DNA concentration, calculate the copy number of the DNA template by the molecular weight and dilute the DNA template to 10 5 , 10 4 , 10 3 , 10 2 , 10 and 1 copies / 5 μL.
[0050] 3) Take 200 μL centrifuge tubes and mark them as 10 5 , 10 4 , 10 3 , 10 2 , 10 and 1 copy number and negative control, add 5 μL template to each centrifuge tube, and add other reaction mixtures, the reaction mixtures are shown in Table 1.
[0051] 4) LAMP reaction: place the reaction tube in a real-time fluorescence detector and set the reaction temperature to 63°C for 45 minutes. After the reaction, according to the real-time fluor...
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