CN113186304A

CN113186304A - Fluorescence isothermal amplification primer, probe, kit and detection method for orientia tsutsutsugamushi nucleic acid

Info

Publication number: CN113186304A
Application number: CN202110642084.9A
Authority: CN
Inventors: 邬强; 乔斌; 陈倩
Original assignee: Hainan Medical College
Current assignee: Hainan Medical College
Priority date: 2021-06-09
Filing date: 2021-06-09
Publication date: 2021-07-30

Abstract

A fluorescence isothermal amplification primer of Orientia tsutsugamushi disease nucleic acid, wherein the primer screen is selected from an Ot1 sequence, the primer comprises an upstream primer and a downstream primer, the upstream primer has a sequence characteristic of SEQ ID NO.1, and the downstream primer has a sequence characteristic of SEQ ID NO. 2; a fluorescent isothermal amplification probe of Orientia tsutsugamushi disease nucleic acid, wherein the probe screen is selected from an Ot1 sequence, and the probe (Ot-P) has a sequence characteristic of SEQ ID NO. 3; a fluorescence isothermal amplification kit for Orientia tsutsugamushi nucleic acid, said kit comprising primers and probes; the detection method applying the fluorescence isothermal amplification kit for Orientia tsutsugamushi nucleic acid comprises the following steps: s1: processing the template; s2: the template, lyophilized enzyme powder, primers, probe, ultra pure water (UP water, resistivity UP to 18 M.OMEGA.. multidot.cm) and activator were treated and reacted at 35-41 ℃ for 20-40 minutes. The primer and the probe have strong specificity and high sensitivity; the invention has short detection time, does not need large expensive instruments and temperature change systems, can complete nucleic acid amplification at 37 ℃ for 30 minutes, and is easy for on-site rapid nucleic acid detection and popularization.

Description

Fluorescence isothermal amplification primer, probe, kit and detection method for orientia tsutsutsugamushi nucleic acid

Technical Field

The invention relates to the field of molecular biological detection of orientia tsutsugamushi, in particular to a primer, a probe, a kit and a detection method for fluorescence isothermal amplification of orientia tsutsutsugamushi nucleic acid.

Background

Tsutsugamushi disease, also known as jungle typhus, is a zoonotic infectious disease transmitted by the bite of the larvae of tsutsugamushi disease oriental (Ot). The patient with tsutsugamushi mainly shows fever, rash, eschar or ulcer, headache, muscle soreness, lymph node swelling, etc. clinically. Research reports that about 100 million people are infected with tsutsugamushi disease every year around the world, and nearly 10 million people are under the threat of being infected with tsutsutsugamushi disease. In 2006 + 2016, the number of the attacks and the incidence rate of tsutsugamushi disease in China are increased year by year, and most of the attacks occur in southern areas of China. Tsutsutsugamushi disease is similar to other febrile diseases in symptoms and signs, is often misdiagnosed, delays the course of the disease, and finally causes other complications such as acute respiratory distress syndrome and renal failure, thereby endangering the life safety of patients.

The current laboratory diagnosis method of tsutsugamushi disease mainly comprises pathogen isolation culture, serum immunological detection and molecular biological detection. The pathogen isolation culture is the gold standard for clinical diagnosis of tsutsugamushi disease. Mainly injects the blood of the patient with tsutsugamushi into the mouse through the abdominal cavity, and the disease is generally developed 7 to 9 days later. The pathogen isolation culture detection is accurate, but the detection needs long time, needs professional operators to carry out intraperitoneal injection and a large number of mice and standard breeding environments; the serological examination method mainly tests antibodies in blood of patients suffering from tsutsugamushi disease, and has a disadvantage of delayed detection. While the appearance of antibodies has a "blank window period"; the molecular biology examination mainly includes Polymerase Chain Reaction (PCR) and its extension-related detection method. Although the PCR detection is sensitive, the detection cost is high, special equipment is required, and professional technicians are required, so that the wide practical application is limited. It is of great significance to establish a rapid, sensitive and field-applicable detection method for tsutsugamushi disease.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a fluorescence isothermal amplification primer, a probe and a kit for the orientia tsutsugamushi nucleic acid, which have short detection time, do not need large and expensive instruments and temperature change systems, can complete nucleic acid amplification at 37 ℃ for 30 minutes, and are easy for on-site rapid nucleic acid detection and popularization.

The purpose of the invention is realized by the following technical scheme:

a fluorescence isothermal amplification primer of Orientia tsutsugamushi nucleic acid, wherein the primer screen is selected from an Ot1 sequence, the primer comprises an upstream primer and a downstream primer, the upstream primer has a sequence characteristic of SEQ ID NO.1, and the downstream primer has a sequence characteristic of SEQ ID NO. 2:

SEQ ID NO.1(7-38)：5′-AGATATATAGTGATATAAAGCCATTCGCTGAT-3′

SEQ ID NO.2(135-103)：5′-CTTCCAATAGATCGTTTAATTCTTGCATTTTAT-3′。

the fluorescent isothermal amplification probe for the orientia tsutsugamushi nucleic acid is characterized in that the probe sieve is selected from an Ot1 sequence, the probe has the sequence characteristics of SEQ ID NO.3, and the sequence of SEQ ID NO.3 is as follows:

SEQ ID NO.3：5′-AGCTGGTATTGATGTTCCTGATACTAGTT/iFAMdT/G/idSp/C/

iBHQ1dT/AATAGTGCATCTG-C3spacer-3′

the 30 th base T of the SEQ ID NO.3 sequence is modified by a fluorescent dye, the 34 th base T of the SEQ ID NO.3 sequence is modified by a fluorescence quenching group, the 32 th base of the SEQ ID NO.3 sequence is a nucleic acid analogue, and the 3' last base of the SEQ ID NO.3 sequence is modified by a blocking extension group.

Further, the fluorescent dye is one of FAM, FITC, CY3, CY5, CY5.5, ROX, HEX, SYBR, JOE, VIC or TAMRA; the fluorescence quenching group is one of BHQ1, BHQ2 or BHQ3, the nucleic acid analogue is tetrahydrofuran, and the blocking extension group is C3spacer or other blocking groups such as phosphate group.

A fluorescence isothermal amplification kit for Orientia tsutsugamushi nucleic acid, said kit comprising primers and probes;

the primer screen is selected from an Ot1 sequence, the primer comprises 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 sequence characteristics of SEQ ID NO. 2:

SEQ ID NO.1(7-38)：5′-AGATATATAGTGATATAAAGCCATTCGCTGAT-3′

SEQ ID NO.2(135-103)：5′-CTTCCAATAGATCGTTTAATTCTTGCATTTTAT-3′

the probe screen is selected from an Ot1 sequence, the probe has the sequence characteristics of SEQ ID NO.3, and the sequence of SEQ ID NO.3 is as follows:

SEQ ID NO.3：5′-AGCTGGTATTGATGTTCCTGATACTAGTT/iFAMdT/G/idSp/C/

iBHQ1dT/AATAGTGCATCTG-C3spacer-3′

Further, the kit also comprises freeze-dried enzyme powder and an activator.

Further, the freeze-dried enzyme powder comprises recombinase, polymerase, nuclease, single-stranded DNA binding protein, dNTP and salt ions.

Further, the volume parts of the primer and the probe are respectively as follows:

1.1-3.5 parts by volume of a primer;

0.9-1.5 volume parts of probe.

Further, the volume fraction of the primer is 2.8 volume fractions, and the volume fraction of the probe is 1.2 volume fractions.

Further, the concentration of the upstream primer and the concentration of the downstream primer in the primers are both 10 mu mol/L; the concentration of the probe was 5. mu. mol/L.

The detection method applying the fluorescence isothermal amplification kit for Orientia tsutsugamushi nucleic acid comprises the following steps:

s1: processing the template;

s2: treating the template, the freeze-dried enzyme powder, the primer, the probe, the ultrapure water and the activator, and reacting for 20-40 minutes at 35-41 ℃.

Further, the volume parts of the primers and the probes in the step S2 are respectively:

1.1-3.5 parts by volume of a primer;

0.9-1.5 volume parts of probe.

The invention has the beneficial effects that:

in recent years, isothermal nucleic acid amplification technology has been rapidly developed and is known as a nucleic acid detection technology capable of replacing PCR. Enzymatic recombinant isothermal Amplification (ERA) developed by Suzhou Xianda Gene technology, Inc. simulates in vivo DNA replication based on Recombinase, single-stranded binding protein and DNA polymerase, amplifies a DNA template without a temperature-changing system, and can amplify at 37-43 ℃. The detection technology has low requirements on experimental hardware, short amplification time and simple sample treatment, and is very suitable for the aspect of rapid pathogen detection.

The present invention provides specific primers and probes for orientia tsutsugamushi, and a detection kit capable of specifically, rapidly and sensitively detecting orientia tsutsugamushi. The primer and the probe have strong specificity and high sensitivity; short detection time, no need of large expensive instrument and temperature varying system, capability of completing nucleic acid amplification at 37 deg.c for 30min, and easy in-situ fast nucleic acid detection and popularization.

Drawings

FIG. 1 shows the results of detection of fluorescence nucleic acid of Oriental tsutsugamushi by Recombinase Polymerase Amplification (RPA) with different primer concentrations: 1.2 and 3 represent addition primersThe samples (10. mu.M/L) were 1.1, 2.1 and 2.8. mu.L in volume, and template plasmid (1.0X 10)⁵copies/. mu.L); 4. 5 and 6 represent primer volumes of 1.1, 2.1 and 2.8. mu.L added, but the template added was deionized water, which is a negative control.

FIG. 2 shows the results of detection of Orientia tsutsugamushi based on Recombinase Polymerase Amplification (RPA) fluorescent nucleic acid detection with different probe concentrations: 1.2 and 3 represent the addition of 1.5, 1.2 and 0.9. mu.L probe (5. mu.M/L) and the loading of template plasmid (1.0X 10)⁵copies/. mu.L); 4. 5 and 6 represent the amount of probe added as 1.5, 1.2 and 0.9. mu.L, but the template added was deionized water, which is a negative control.

FIG. 3 shows the result of sensitivity test 10 of recombinant enzyme polymerase amplification (RPA) based fluorescent nucleic acid detection method of Orientia tsutsutsugamushi⁶、10⁵、10⁴、10³、10²10, 1 and negatives represent concentrations of 1.0X 10⁶copies/μL、1.0×10⁵copies/μL、1.0×10⁴copies/μL、1.0×10³copies/μL、1.0×10²copies/μL、1.0×10¹copies/. mu.L, 1.0 copies/. mu.L and negative control (deionized water).

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It is to be noted that the features in the following embodiments and examples may be combined with each other without conflict.

It should be noted that the drawings provided in the following embodiments are only for illustrating the basic idea of the present invention, and the components related to the present invention are only shown in the drawings rather than drawn according to the number, shape and size of the components in actual implementation, and the type, quantity and proportion of the components in actual implementation may be changed freely, and the layout of the components may be more complicated.

The embodiments referred to below are conventional unless otherwise indicated.

The experimental procedures relating to molecular biology in the examples described below were carried out according to the protocols of the molecular cloning guidelines of Sambrook et al, or with reference to the kit instructions.

Materials, reagents and the like according to the following embodiments are commercially available unless otherwise specified.

The first embodiment is as follows:

SEQ ID NO.1(7-38)：5′-AGATATATAGTGATATAAAGCCATTCGCTGAT-3′

SEQ ID NO.2(135-103)：5′-CTTCCAATAGATCGTTTAATTCTTGCATTTTAT-3′

the length of the primer is more than 30 bp.

SEQ ID NO.3：5′-AGCTGGTATTGATGTTCCTGATACTAGTT/iFAMdT/G/idSp/C/

iBHQ1dT/AATAGTGCATCTG-C3spacer-3′

the 30 th base T of the SEQ ID NO.3 sequence is modified by a fluorescent dye, the 34 th base T of the SEQ ID NO.3 sequence is modified by a fluorescence quenching group, the 32 th base of the SEQ ID NO.3 sequence is a nucleic acid analogue, and the 3' last base of the SEQ ID NO.3 sequence is modified by an extension blocking group to prevent the extension of DNA polymerase.

In the sequence of SEQ ID NO.3, iFAMdT represents a fluorescein dye FAM (6-carboxyfluoroscein) -modified thymidine nucleotide; idSp represents a tetrahydrofuran (tetrahydrofuran) nucleic acid analog; iBHQ1dT represents thymine deoxynucleotide modified by fluorescence quenching group BHQ 1(black hole quencher 1); c3spacer represents a polymerase extension blocking group.

The probe comprises a5 'terminal sequence, a fluorescent dye, idSp, a fluorescent dye corresponding quenching group, a 3' terminal sequence and an extension blocking group.

The fluorescent dye is one of FAM, FITC, CY3, CY5, CY5.5, ROX, HEX, SYBR, JOE, VIC or TAMRA; the fluorescence quenching group is BHQ1 or BHQ2 or BHQ3, the nucleic acid analogue is tetrahydrofuran (THF or idSp), and the extension blocking group is C3spacer or other blocking groups such as phosphate group.

The probe is 47 bases in length.

FAM for carboxyfluorescein, FITC for fluorescein isothiocyanate, BHQ1 for Black Hole Quencher 1(Black Hole Quencher 1), and C3spacer for 3-CH₂。

The length of the probe is 47bp, wherein the 5 'end is 31bp, the middle (32 th position) is idSp, and the 3' end is 15 bp.

The sequences of the primers and probes are shown in Table 1.

Table 1 screening of the primer and Probe sequences

Aiming at the specific conserved region of Oriental tsutsugamushi disease as an amplification target region, a specific amplification primer and an RPA fluorescent probe are designed, and the primer and the probe are synthesized by Jinzhi Biotechnology Limited, Suzhou.

The primer and probe for fluorescence isothermal amplification of Oriental tsutsugamushi disease are designed based on conserved sequence of outer membrane protein Sta56 of Oriental tsutsutsugamushi disease, the conserved sequence of Oriental tsutsugamushi protein Sta56 gene has Ot1 sequence, Ot1 sequence (SEQ ID NO.4) as follows, wherein black bold is primer sequence, underlined part is probe sequence:

the primer aims at common conserved genes of the orientia tsutsutsugamushi, specifically the outer membrane protein Sta56 with strong conservation as a kit amplification target, and is constructed into a vector puc57 to prepare a positive plasmid of the orientia tsutsutsugamushi, which is synthesized by Nanjing Jinwei Zhi Biotechnology Limited.

SEQ ID NO.1(7-38)：5′-AGATATATAGTGATATAAAGCCATTCGCTGAT-3′

SEQ ID NO.2(135-103)：5′-CTTCCAATAGATCGTTTAATTCTTGCATTTTAT-3′

SEQ ID NO.3：5′-AGCTGGTATTGATGTTCCTGATACTAGTT/iFAMdT/G/idSp/C/

iBHQ1dT/AATAGTGCATCTG-C3spacer-3′

The kit also comprises freeze-dried enzyme powder and an activator.

The freeze-dried enzyme powder comprises recombinase, polymerase, nuclease, single-stranded DNA binding protein, dNTP and salt ions.

Example two:

the detection method of the tsutsugamushi disease oriental nucleic acid fluorescence isothermal amplification kit applied to the embodiment I comprises the following steps:

s1: processing the template: the plasmid was diluted to 100. mu.M/L with TE buffer to 1.0X 10 according to the plasmid copy number calculation formula⁶copies/μL。

The plasmid is positive plasmid of Orientia tsutsugamushi, and is obtained by: obtaining a Sta56 gene conserved region of Oriental tsutsugamushi from NCBI website, selecting a part of Sta56 region as a target amplification segment, constructing the target amplification segment in a vector puc57-Amp, and preparing a positive plasmid quality control product of Oriental tsugamushi, wherein the plasmid is synthesized by Suzhou Jinwei Zhi Biotech limited.

Considering the biological safety of the laboratory, the nucleic acid extraction of pathogenic microorganisms does not carry out relevant experiments.

S2: treating the template, the dissolving agent, the freeze-dried enzyme powder, the primer, the probe, the ultrapure water and the activating agent, and reacting for 20-40 minutes at 35-41 ℃.

In the step S2, the volume parts of the template, the dissolving agent, the freeze-dried enzyme powder, the primer, the probe, the ultrapure water and the activator are respectively as follows:

the concentrations of the upstream primer and the downstream primer in the primers are both 10 mu mol/L; the concentration of the probe was 5. mu. mol/L.

In this example, a 50. mu.L reaction system was used, specifically comprising

0.2mL of the reaction tube was charged with: the sample loading of the template is 2 mu L; firstly, 20 mu L of gene fluorescent nucleic acid amplification freeze-dried enzyme powder is obtained; the concentrations of the upstream primer and the downstream primer are both 10 mu mol/L, and the sample loading amount is 2.8 mu L; the concentration of the fluorescent probe is 5 mu mol/L, and the sample loading amount is 1.2 mu L; 22 microliter of ultrapure water; then 2 mul of activator is added to the reaction tube cover; also, 20. mu.L of a dissolving agent was added to the reaction tube.

Then, the mixture is subjected to sequential separation, fully mixed, centrifuged again, and subjected to constant temperature amplification reaction for 30min at 37 ℃ in a metal bath.

Specifically, the mixture was centrifuged at 2000rpm/min for 15s, and thoroughly mixed by shaking.

The collected signal is detected by a GS8 isothermal fluorescence amplification apparatus from Xizhou Xianda Gene technology, Inc., or other PCR apparatus or other isothermal fluorescence amplification apparatus capable of collecting fluorescence signals.

In this example, the specific operation is described in the fluorescent nucleic acid amplification kit of Xianda Gene technology, Inc., Suzhou.

EXAMPLE III

s1: processing the template: the plasmid was diluted to 100. mu.M/L with TE buffer to 1.0X 10 according to the plasmid copy number calculation formula⁵copies/. mu.L, negative control in the experiment was ultrapure water.

S2: the template, the dissolving agent, the lyophilized enzyme powder (the lyophilized enzyme powder is in the reaction tube), the primer, the probe, the ultrapure water and the activator are treated and reacted at 35-41 ℃ for 20-40 minutes.

In this example, the RPA fluorescent detection system was used to amplify specific sequences of orientia tsutsugamushi, and the volume of each reagent added is shown in table 2:

TABLE 2 reagent table of EXAMPLE III

After sufficient shaking and brief centrifugation at 2000rpm for 15s, the reaction tube was opened, 2. mu.L of activator was added to the reaction tube cap, and the reaction tube was briefly centrifuged at 2000rpm for 15s, followed by sufficient shaking and brief centrifugation at 2000rpm for 15 s.

The reaction tube was placed in a fluorescence isothermal amplification apparatus GS8 (Suzhou Xianda Gene science and technology Co., Ltd.) under amplification conditions of 37 ℃ for 30 minutes. The requirements of positive control and negative control during amplification need to be met in the same experiment, otherwise, the experiment is invalid.

And (4) result judgment standard: the fluorescence detection instrument obviously detects FAM fluorescence amplification signals within 30 minutes, has obvious S-shaped amplification trend and judges the FAM fluorescence amplification signals to be positive; the fluorescence detection instrument has no increase of FAM fluorescence signals and no amplification trend within 30 minutes, is basically a relatively flat straight line, and is judged to be negative.

And (4) judging a result:

negative control: the FAM channel has no amplification curve and is basically a smooth straight line;

positive control: FAM channels have distinct sigmoidal amplification curves.

Example four:

the detection method of tsutsugamushi disease eastern cubic nucleic acid fluorescence isothermal amplification kit of the first example was applied, and this example employs 3 sets of experiments:

the detection method comprises the following steps:

s1: processing the template: the plasmid was diluted to 100. mu.M/L with TE buffer, as calculated by plasmid copy number, to 10⁵copies/. mu.L, negative control in the experiment was ultrapure water.

In this example, the RPA fluorescent detection system was used to amplify specific sequences of orientia tsutsugamushi, and the volume of each reagent added is shown in table 3:

table 3 reagent table of example four

In 3 experiments, the primers, i.e., the mixture of Ot-F1 and Ot-R1 (10. mu.M/L), were added in an amount of 2.8. mu.L, 2.1. mu.L, and 1.1. mu.L, respectively, and the remainder, which was less than the final reaction volume (i.e., volume sum), was filled with ultrapure water.

As shown in FIG. 1, the amount of the primer mixture added was 2.8. mu.L, which was more effective in amplification.

EXAMPLE five

the detection method comprises the following steps:

In this example, the RPA fluorescent detection system was used to amplify specific sequences of orientia tsutsugamushi, and the volume of each reagent added is shown in table 4:

table 4 reagent table for example five

The volume of the probe (5. mu.M/L) in the 3 experiments was 0.9. mu.L, 1.2. mu.L and 1.5. mu.L, respectively, and the remainder was not filled with the final reaction volume (i.e., volume sum) in ultrapure water.

As shown in FIG. 2, the amplification effect was improved when the amount of the probe added was 1.2. mu.L.

EXAMPLE six

Using the detection method of fluorescence isothermal amplification kit of Oriental tsutsugamushi disease nucleic acid of the first example, this example uses several sets of experiments (for diluted concentrations of plasmid):

the detection method comprises the following steps:

s1: processing the template: the plasmid was diluted to 100. mu.M/L with TE buffer to 1.0X 10 according to the plasmid copy number calculation formula⁶copies/. mu.L, diluted with TE buffer at 10-fold gradient and used as template for amplification detection at 1.0X 10⁶copies/μL、1.0×10⁵copies/μL、1.0×10⁴copies/μL、1.0×10³copies/μL、1.0×10²copies/μL、1.0×10¹copies/. mu.L and 1 copies/. mu.L. The negative control in the experiment was ultrapure water.

In this example, the RPA fluorescent detection system was used to amplify specific sequences of orientia tsutsugamushi, and the volume of each reagent added is shown in table 5:

table 5 reagent table of example six

The results are shown in FIG. 3, and the lowest detection limit of the kit can reach 10 copies/. mu.L.

The experiment verifies that the lowest detection limit of the detection method adopts the concentration of 1.0 multiplied by 10⁶copies/μL、1.0×10⁵copies/μL、1.0×10⁴copies/μL、1.0×10³copies/μL、1.0×10²copies/μL、1.0×10¹copies/. mu.L and 1.0 copies/. mu.L positive plasmids were used as reference samples for detection sensitivity, and ultrapure water without plasmid was used as a negative control. As shown in FIG. 3, the detection limit of the detection method can reach 10 copies/. mu.L.

10 in fig. 3⁶、10⁵、10⁴、10³、10²10, 1 respectively represent concentrations of 1.0X 10⁶copies/μL、1.0×10⁵copies/μL、1.0×10⁴copies/μL、1.0×10³copies/μL、1.0×10²copies/μL、1.0×10¹copies/. mu.L, 1.0 copy/. mu.L positive plasmid amplification curve, negative represents negative quality control sample, abscissa represents reaction time (min), and ordinate represents fluorescence value. The result shows that the detection limit of the detection method reaches 10 copies/mu L.

The invention provides a fluorescence isothermal detection kit (ERA detection system) for nucleic acid of orientia tsutsugamushi, which provides a specific primer and a probe for orientia tsutsugamushi, and a detection kit capable of specifically, rapidly and sensitively detecting orientia tsutsugamushi.

The invention relates to a Recombinase Polymerase Amplification (RPA) based fluorescence nucleic acid detection method for Orientia tsutsugamushi, in particular to a nucleic acid fluorescence detection primer and a probe, wherein the detection target is outer membrane protein Sta56 of Karp strain of Orientia tsutsugamushi in Hainan province, and the detection target relates to a gene conserved sequence Ot1, amplification primers Ot-F1, Ot-R1 and an amplification probe Ot-P. The primer and the probe have strong specificity and high sensitivity; the detection technology can be used for auxiliary diagnosis of tsutsugamushi disease infection, has short detection time, does not need large and expensive instruments and temperature change systems, can complete nucleic acid amplification at 37 ℃ for 30 minutes, and is easy for on-site rapid nucleic acid detection and popularization.

The invention is an isothermal amplification system for fluorescence detection of nucleic acid of orientia tsutsugamushi, which greatly shortens the detection time of orientia tsutsugamushi, the reaction condition is 37 ℃, 30min, the whole detection process can be completed within 1 hour, and the time is greatly shortened compared with the several-hour amplification of real-time fluorescence quantitative PCR.

The invention relates to an isothermal amplification system for detecting orientia tsutsugamushi nucleic acid by fluorescence, which can complete an experiment only by keeping the temperature at 37 ℃ relative to a 50-95 ℃ temperature-changing system of fluorescence quantitative PCR and about 60 ℃ of other isothermal nucleic acid expansions, and the temperature is easy to obtain without a temperature-changing system of an expensive instrument.

The isothermal amplification system for detecting the orientia tsutsugamushi nucleic acid by fluorescence is simple to operate and also has small-sized portable amplification equipment; the enzyme and other products required by amplification can be freeze-dried and stored, and only enzyme amplification buffer solution, a template, a primer, a probe, water and a magnesium ion activator need to be added during amplification.

The invention is an isothermal amplification system for detecting tsutsugamushi oriental nucleic acid by fluorescence, and has high sensitivity and good specificity. Can be used for field site or bedside detection, and has great application market potential.

The above-mentioned embodiments only express the specific embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Sequence listing

<110> Hainan college of medicine

<120> primer, probe and kit for fluorescence isothermal amplification of orientia tsutsugamushi nucleic acid

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agatatatag tgatataaag ccattcgctg at 32

<210> 2

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cttccaatag atcgtttaat tcttgcattt tat 33

<210> 3

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

agctggtatt gatgttcctg atactagttt gctaatagtg catctg 46

<210> 4

<211> 634

<212> DNA

<213> Orientia tsutsugamushi

<400> 4

ttactcagat atatagtgat ataaagccat tcgctgatat agctggtatt gatgttcctg 60

atactagttt gcctaatagt gcatctgtcg aacagataca gaataaaatg caagaattaa 120

acgatctatt ggaagagctc agagaatctt ttgatgggta tcttggtggt aatgcttttg 180

ctaatcagat acagttgaat tttgtcatgc cgcagcaagc acagcagcag gggcaagggc 240

agcaacagca agctcaagct acagcgcaag aagcagtagc agcagcagct gttaggcttt 300

taaatggcaa tgatcagatt gcgcagttat ataaagatct tgttaaattg cagcgtcatg 360

caggaattaa gaaagcgatg gaaaaattag ctgcccaaca agaagaagat gcaaagaatc 420

aaggtgaagg tgactgcaag cagcaacaag gaacatctga aaaatctaaa aaaggaaaag 480

acaaagaggc agagtttgat ctgagtatga ttgtcggcca agttaaactc tatgctgacg 540

taatgataac tgaatcagtc tcaatatatg ctggtgttgg tgcagggtta gcttatactt 600

ctggaaaaat agataataag gatattaaag ggca 634

Claims

1. A fluorescence isothermal amplification primer for Orientia tsutsugamushi disease nucleic acid is characterized in that: the primer screen is selected from an Ot1 sequence, the primer comprises 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 sequence characteristics of SEQ ID NO. 2:

SEQ ID NO.1(7-38)：5′-AGATATATAGTGATATAAAGCCATTCGCTGAT-3′

SEQ ID NO.2(135-103)：5′-CTTCCAATAGATCGTTTAATTCTTGCATTTTAT-3′。

2. a fluorescence isothermal amplification probe for Orientia tsutsugamushi disease nucleic acid is characterized in that: the probe screen is selected from an Ot1 sequence, the probe has the sequence characteristics of SEQ ID NO.3, and the sequence of SEQ ID NO.3 is as follows:

SEQ ID NO.3：5′-AGCTGGTATTGATGTTCCTGATACTAGTT/iFAMdT/G/idSp/C/

iBHQ1dT/AATAGTGCATCTG-C3spacer-3′

3. The fluorescent isothermal amplification probe of orientia tsutsugamushi nucleic acid according to claim 2, characterized in that: the fluorescent dye is one of FAM, FITC, CY3, CY5, CY5.5, ROX, HEX, SYBR, JOE, VIC or TAMRA; the fluorescence quenching group is one of BHQ1, BHQ2 or BHQ3, the nucleic acid analogue is tetrahydrofuran, and the blocking extension group is C3spacer or other blocking groups such as phosphate group.

4. A fluorescence isothermal amplification kit for Orientia tsutsugamushi nucleic acid is characterized in that: the kit comprises a primer and a probe;

SEQ ID NO.1(7-38)：5′-AGATATATAGTGATATAAAGCCATTCGCTGAT-3′

SEQ ID NO.2(135-103)：5′-CTTCCAATAGATCGTTTAATTCTTGCATTTTAT-3′

SEQ ID NO.3：5′-AGCTGGTATTGATGTTCCTGATACTAGTT/iFAMdT/G/idSp/C/

iBHQ1dT/AATAGTGCATCTG-C3spacer-3′

5. The fluorescent isothermal amplification kit of orientia tsutsugamushi nucleic acid according to claim 4, characterized in that: the kit also comprises freeze-dried enzyme powder and an activator.

6. The fluorescent isothermal amplification kit of orientia tsutsugamushi nucleic acid according to claim 5, characterized in that: the freeze-dried enzyme powder comprises recombinase, polymerase, nuclease, single-stranded DNA binding protein, dNTP and salt ions.

7. The detection method using the nucleic acid fluorescence isothermal amplification kit of Orientia tsutusgamushi as described in any one of claims 4-5, wherein said kit comprises: the method comprises the following steps:

s1: processing the template;

8. The detection method using a fluorescent isothermal amplification kit of orientia tsutsugamushi nucleic acid as claimed in claim 7, wherein: the volume parts of the primers and the probes in the step S2 are respectively as follows:

1.1-3.5 parts by volume of a primer;

0.9-1.5 volume parts of probe.

9. The detection method using a fluorescent isothermal amplification kit of orientia tsutsugamushi nucleic acid as claimed in claim 8, wherein: the volume parts of the primers are 2.8 volume parts, and the volume parts of the probes are 1.2 volume parts.

10. The detection method using a fluorescent isothermal amplification kit of orientia tsutsugamushi nucleic acid as claimed in claim 8, wherein: the concentrations of the upstream primer and the downstream primer in the primers are both 10 mu mol/L; the concentration of the probe was 5. mu. mol/L.