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Orientia tsutsugamushi nucleic acid fluorescence isothermal amplification primer, probe, kit and detection method

A technique for isothermal amplification of Orientia tsutsugamushi, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve the problem of high detection cost

Pending Publication Date: 2021-07-30
HAINAN MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although PCR detection is sensitive, its high detection cost, the need for specific equipment, professional technicians and other defects limit its wide practical application

Method used

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  • Orientia tsutsugamushi nucleic acid fluorescence isothermal amplification primer, probe, kit and detection method
  • Orientia tsutsugamushi nucleic acid fluorescence isothermal amplification primer, probe, kit and detection method
  • Orientia tsutsugamushi nucleic acid fluorescence isothermal amplification primer, probe, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] A fluorescent isothermal amplification primer for Orientia tsutsugamushi nucleic acid, the primer is screened from the Ot1 sequence, the primer includes an upstream primer and a downstream primer, the upstream primer has the sequence characteristics of SEQ ID NO.1, and the downstream primer has the SEQ ID NO.2 sequence features:

[0050] SEQ ID NO.1 (7-38): 5'-AGATATATAGTGATATAAAGCCATTCGCTGAT-3'

[0051] SEQ ID NO.2(135-103): 5'-CTTCCAATAGATCGTTTAATTCTTGCATTTTAT-3'

[0052] The length of the primer is more than 30bp.

[0053] A fluorescent isothermal amplification probe for Orientia tsutsugamushi nucleic acid, the probe is screened from the Ot1 sequence, the probe has the sequence characteristics of SEQ ID NO.3, and the SEQ ID NO.3 sequence is:

[0054] SEQ ID NO. 3: 5'-AGCTGGTATTGATGTTCCTGATACTAGTT / iFAMdT / G / idSp / C /

[0055] iBHQ1dT / AATAGTGCATCTG-C3spacer-3′

[0056] The 30th base T of the SEQ ID NO.3 sequence is modified by a fluorescent dye, the 34th base T of the...

Embodiment 3

[0097] The detection method of a kind of Orientia tsutsugamushi nucleic acid fluorescence isothermal amplification kit of application embodiment 1 comprises the following steps:

[0098] S1: Processing template: Dilute the plasmid to 100 μM / L with TE buffer and dilute to 1.0×10 according to the formula for calculating the number of plasmid copies. 5 copies / μL, the negative control in the experiment is ultrapure water.

[0099] S2: Treat the template, dissolving agent, lyophilized enzyme powder (the lyophilized enzyme powder itself is in the reaction tube), primers, probes, ultrapure water and activator and react at 35-41° C. for 20-40 minutes.

[0100] In this example, the RPA fluorescence detection system was used to amplify the specific sequence of Orientia tsutsugamushi, and the volume of each reagent added is shown in Table 2:

[0101] The reagent table of table 2 embodiment three

[0102]

[0103] After sufficient shaking and brief centrifugation at 2000rpm / min for 1...

Embodiment 4

[0110] The detection method of a kind of Orientia tsutsugamushi nucleic acid fluorescence isothermal amplification kit of application embodiment one, the present embodiment adopts 3 groups of experiments:

[0111] The detection method comprises the following steps:

[0112] S1: Processing template: Dilute the plasmid to 100 μM / L with TE buffer and dilute to 10 according to the formula for calculating the number of plasmid copies. 5 copies / μL, the negative control in the experiment is ultrapure water.

[0113] S2: Treat the template, dissolving agent, lyophilized enzyme powder (the lyophilized enzyme powder itself is in the reaction tube), primers, probes, ultrapure water and activator and react at 35-41° C. for 20-40 minutes.

[0114] In this example, the RPA fluorescence detection system was used to amplify the specific sequence of Orientia tsutsugamushi, and the volume of each reagent added is shown in Table 3:

[0115] The reagent table of table 3 embodiment four

[0116...

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Abstract

The invention provides an orientia tsutsugamushi nucleic acid fluorescence isothermal amplification primer. The primer is screened from an Ot1 sequence, the primer comprises an upstream primer and a downstream primer, the upstream primer has the sequence characteristic of SEQ ID NO.1, and the downstream primer has the sequence characteristic of SEQ ID NO.2. The invention provides an orientia tsutsugamushi nucleic acid fluorescence isothermal amplification probe, the probe is screened from an Ot1 sequence, and the probe (Ot-P) has the sequence characteristic of SEQ ID NO.3. The invention discloses an orientia tsutsugamushi nucleic acid fluorescence isothermal amplification kit. The kit comprises the primer and the probe. The invention provides a detection method using the orientia tsutsugamushi nucleic acid fluorescence isothermal amplification kit. The detection method comprises the following steps: S1, treating a template; s2, treating the template, the freeze-dried enzyme powder, the primer, the probe, ultrapure water (Ultrapure water, UP water, the resistivity of which reaches 18 M omega.cm) and an activating agent, and reacting at the temperature of 35-41 DEG C for 20-40 minutes. The primer and the probe are high in specificity and sensitivity; the detection time is short, large expensive instruments and variable temperature systems are not needed, nucleic acid amplification can be completed at 37 DEG C within 30 minutes, and on-site rapid nucleic acid detection and popularization are easy.

Description

technical field [0001] The invention relates to the field of molecular biology detection of Orientia tsutsugamushi, in particular to a fluorescent isothermal amplification primer, probe, kit and detection method of Orientia tsutsugamushi nucleic acid. Background technique [0002] Scrub typhus, also known as scrub typhus, is a zoonotic infectious disease transmitted by Orientia tsutsugamushi (Ot) through the bites of chigger larvae. The main clinical manifestations of scrub typhus patients are fever, rash, eschar or ulcer, headache, muscle aches, and swollen lymph nodes. Studies have reported that about 1 million people in the world are infected with scrub typhus every year, and nearly 1 billion people are at risk of being infected with scrub typhus. From 2006 to 2016, the number and incidence of scrub typhus in my country increased year by year, mostly in southern China. Scrub typhus is similar to other febrile diseases in terms of symptoms and signs. It is often misdiagn...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6888C12Q1/6844
Inventor 邬强乔斌陈倩
Owner HAINAN MEDICAL COLLEGE
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