CN116042902A

CN116042902A - Real-time fluorescent nucleic acid isothermal amplification detection kit for simultaneously detecting six candida species and special primer and probe thereof

Info

Publication number: CN116042902A
Application number: CN202310045595.1A
Authority: CN
Inventors: 居金良; 崔振玲; 钱卫国; 沈晓宁
Original assignee: Shanghai Rendu Biotechnology Co ltd
Current assignee: Shanghai Rendu Biotechnology Co ltd
Priority date: 2023-01-30
Filing date: 2023-01-30
Publication date: 2023-05-02

Abstract

The invention discloses a real-time fluorescent nucleic acid isothermal amplification detection kit for simultaneously detecting six candida and a special primer and a probe thereof, and further provides a real-time fluorescent nucleic acid isothermal amplification detection kit for simultaneously detecting six candida and trichomonas vaginalis and a special primer and a probe thereof, belonging to the technical field of biomedical detection. The kit provided by the invention comprises nucleic acid extract, detection solution and SAT enzyme solution for six candida species or further for trichomonas vaginalis, primers and probes which are more suitable for detection of the microorganisms are optimally designed, and each component is added step by step in the detection process to perform step reaction, so that real-time and rapid detection of the six candida species or the candida vaginalis combined with the trichomonas vaginalis can be realized, the sensitivity is high, the specificity is good, the amplified product RNA is easy to degrade, no environmental pollution is caused, and no sample cross contamination is caused in the detection process.

Description

Real-time fluorescent nucleic acid isothermal amplification detection kit for simultaneously detecting six candida species and special primer and probe thereof

Technical Field

The invention belongs to the technical field of biomedical detection, in particular to a real-time fluorescent nucleic acid isothermal amplification detection kit for simultaneously detecting six candida and a special primer and a probe thereof, and also relates to a real-time fluorescent nucleic acid isothermal amplification detection kit for simultaneously detecting six candida and trichomonas vaginalis and a special primer and a probe thereof, in particular to a primer, a probe and a related kit used in real-time fluorescent nucleic acid isothermal amplification detection of candida and trichomonas vaginalis by combining a specific target capturing technology and a real-time fluorescent nucleic acid isothermal amplification detection technology (Simultaneous Amplification and Test, SAT).

Background

Vaginitis (Vaginitis), a group of conditions that lead to vulvovaginal symptoms such as itching, burning, irritation and abnormal fluid flow. Clinically, there are common: bacterial vaginosis (accounting for 22-50% of symptomatic women), candidal vaginitis (17-39%), trichomonas vaginitis (4-35%), senile vaginitis and juvenile vaginitis.

Candida vaginitis is caused by fungal infections of the genus Candida (also known as Candida albicans, VVC), of which Candida albicans (Candida albicans) is the most common pathogen, accounting for about 85% to 95%. Among infections caused by non-Candida albicans, candida glabra (Candida glabra) and Candida tropicalis (Candida tropicalis) are the most common, and in addition Candida krusei (Candida krusei), candida parapsilosis (Candida parapsilosis), candida dublinii (canada dubliniemis) and the like, occur mainly in immunocompromised women. In particular, in recent years, with the widespread use of antibiotics and the like, candidal vaginitis has been changed in the pathogenic bacterial spectrum, and cases of candidal vaginitis caused by non-candida albicans have been increasing, and have been resistant to the conventional therapies, with the highest proportion of candida glabrata and candida krusei resistance. Therefore, the method has important epidemiological and clinical therapeutic significance for the targeted detection of candida, in particular candida glabrata, candida krusei and the like in the candida.

Trichomonas vaginitis is caused by infection of trichomonas vaginalis (Trichomonas vaginalis, TV), which is a parasite but invisible to the naked eye, and is a broad pear-shaped or oval trichomonas with a length of 10-30 μm and a width of 10-20 μm, and 4 flagella with equal length to the trichomonas body on the head, which can be clearly seen under a microscope. TV is estimated to be 1.8 billion women worldwide each year infected throughout the world. Therefore, the targeted detection of trichomonas vaginalis also of great epidemiological and clinical therapeutic significance.

At present, the method for detecting pathogenic candida and trichomonas vaginalis mainly molecular biology method and mainly comprises DNA probe hybridization technology and PCR technology. For example, patent document CN101638688A (hereinafter referred to as document 1) discloses a specific probe for identifying pathogenic candida including candida albicans, candida glabrata, candida krusei, candida parapsilosis, candida dublinii and candida tropicalis, which is amplified by PCR using a pair of universal primers for candida and hybridized with DNA probes for each candida respectively, and the presence or absence of each candida is determined based on the hybridization result. As another example, patent document CN110551840a (hereinafter, referred to as document 2) discloses a nucleic acid reagent for detecting invasive fungi, which comprises a tube a, a tube B and a tube C, wherein the tube a contains a pair of universal primers for four candida species (candida albicans, candida glabrata, candida parapsilosis, candida tropicalis) and fluorescent-labeled probes for the four candida species, respectively, capable of detecting the above four candida species simultaneously. Also disclosed in patent document CN109971883a (hereinafter, referred to as document 3) is a primer probe combination for candida species detection, which comprises PCR amplification primers and detection probes for candida albicans, candida glabrata, candida parapsilosis, candida tropicalis, and candida krusei, respectively, capable of detecting one or more infections of the above five candidas at a time. However, the above-mentioned document 1 uses the principle of hybridization of DNA probes, and is capable of detecting and distinguishing various candida species, but the sensitivity is generally poor, and the time required for the detection is long, usually about 6 hours, and the efficiency is low. Moreover, the methods disclosed in the above-mentioned documents 1 to 3 are all based on the basic principle of PCR, which requires temperature rise and fall and circulation during the PCR reaction, and thus the required detection time is also long (generally 2 to 3 hours are required), and the efficiency is low; in addition, the reaction product of PCR is DNA, which is not easy to degrade and can easily cause sample cross contamination and experimental environment pollution. Furthermore, the above-mentioned documents 2 to 3 can detect only four or five kinds of candida at the same time, but cannot detect six kinds of candida at the same time, and further cannot detect six kinds of candida and trichomonas vaginalis at the same time.

While there have been methods in the art for simultaneous detection of candida and trichomonas vaginalis, for example, patent document US20210332421A1 (hereinafter, document 4) discloses a method and composition for multiplex detection of candida species (including candida glabrata, candida albicans, candida tropicalis, candida dublinii, candida parapsilosis, candida krusei) and trichomonas vaginalis, which includes primers and probes for detection of candida glabrata, universal primers and probes for detection of four candida albicans, candida tropicalis, candida dublinii and candida parapsilosis, primers and probes for detection of candida criptii, and primers and probes for detection of trichomonas vaginalis, these candida glabrata and trichomonas vaginalis can be detected simultaneously. However, the method disclosed in this document 4 is still based on the basic principle of PCR, and has the drawbacks of low efficiency and easiness in causing cross-contamination of samples and contamination of experimental environments.

Disclosure of Invention

In view of one or more problems of the prior art, one aspect of the present invention provides a real-time fluorescent nucleic acid isothermal amplification detection kit for simultaneously detecting six candida species, designated as a first kit, comprising:

(T1) nucleic acid extract: comprising a solid support comprising a first specific capture probe for capturing detection sequences of candida albicans, candida tropicalis, candida parapsilosis and/or candida dublinii (collectively referred to as Cspp), a second specific capture probe for capturing detection sequences of candida glabrata, and a third specific capture probe for capturing detection sequences of candida krusei;

(T2) detection solution: the kit comprises a first primer, a second primer, a first target detection probe, a third primer, a fourth primer, a second target detection probe, a fifth primer, a sixth primer and a third target detection probe; wherein the first primer, the second primer and the first target detection probe cooperate to detect a target sequence of Cspp; the third primer, the fourth primer and the second target detection probe are matched and used for detecting a target sequence of candida glabrata; the fifth primer, the sixth primer and the third target detection probe are matched and used for detecting a target sequence of candida krusei; the two ends of the nucleotide sequences of the first target detection probe, the second target detection probe and the third target detection probe are respectively provided with a fluorescence report group and a quenching group, and the fluorescence report groups carried by the first target detection probe, the second target detection probe and the third target detection probe are different;

(T3) SAT enzyme solution: comprising at least one RNA polymerase and an M-MLV reverse transcriptase;

wherein:

the first specific capture probe comprises a nucleotide sequence shown as SEQ ID NO. 17, the second specific capture probe comprises a nucleotide sequence shown as SEQ ID NO. 18, and the third specific capture probe comprises a nucleotide sequence shown as SEQ ID NO. 19;

the first primer comprises a nucleotide sequence shown as SEQ ID NO. 1, the second primer comprises a nucleotide sequence shown as SEQ ID NO. 9, and the first target detection probe comprises a nucleotide sequence shown as SEQ ID NO. 21;

the third primer comprises a nucleotide sequence shown as SEQ ID NO. 3, the fourth primer comprises a nucleotide sequence shown as SEQ ID NO. 11, and the second target detection probe comprises a nucleotide sequence shown as SEQ ID NO. 23;

the fifth primer comprises a nucleotide sequence shown as SEQ ID NO. 5, the sixth primer comprises a nucleotide sequence shown as SEQ ID NO. 13, and the third target detection probe comprises a nucleotide sequence shown as SEQ ID NO. 25.

In another aspect, the present invention provides a kit for real-time isothermal amplification of fluorescent nucleic acids for simultaneously detecting six candida species and trichomonas vaginalis, named as a second kit, which further comprises, based on the first kit, the following components:

(a) Fourth specific capture probe: which is present in the nucleic acid extract for capturing the detection sequence of trichomonas vaginalis and the fourth specific capture probe comprises the nucleotide sequence shown as SEQ ID No. 20;

(b) Seventh primer, eighth primer, and fourth target detection probe: the third primer and the fourth primer are matched in the detection solution and used for detecting the target sequence of trichomonas vaginalis, wherein the seventh primer comprises a nucleotide sequence shown as SEQ ID NO. 7, the eighth primer comprises a nucleotide sequence shown as SEQ ID NO. 15, the fourth target detection probe comprises a nucleotide sequence shown as SEQ ID NO. 27, fluorescent reporter groups and quenching groups are respectively carried at two ends of the nucleotide sequence of the fourth target detection probe, and the carried fluorescent reporter groups are different from the fluorescent reporter groups carried by the first target detection probe, the second target detection probe and the third target detection probe.

In some embodiments, the kit further comprises:

(M1) washing solution: it contains NaCl and SDS; preferably 5-50mM HEPES, 50-500mM NaCl, 0.5-1.5% SDS, 1-10mM EDTA; and/or

(M2) mineral oil; and/or

(M3) positive control: a system comprising Cspp, candida glabrata, candida krusei nucleic acid, or further comprising trichomonas vaginalis nucleic acid; and/or

(M4) negative control: a system that does not contain Cspp, candida glabrata, candida krusei nucleic acid, or further does not contain trichomonas vaginalis nucleic acid.

In some embodiments, the components of the nucleic acid extraction solution include: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfate), 1-50. Mu.M of first specific capture probe, 1-50. Mu.M of second specific capture probe, 1-50. Mu.M of third specific capture probe, 50-500mg/L magnetic beads; optionally, the components of the nucleic acid extraction solution further comprise a fourth specific capture probe in the range of 1-50. Mu.M.

In some embodiments, the components of the test solution include: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl ₂ 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of a first primer, 250-750pmol/mL of a second primer, 250-750pmol/mL of a third primer, 250-750pmol/mL of a fourth primer, 250-750pmol/mL of a fifth primer, 250-750pmol/mL of a sixth primer, 250-750pmol/mL of a first target detection probe, 250-750pmol/mL of a second target detection probe, 250-750pmol/mL of a third target detection probe; optionally, the components of the detection solution further comprise 250-750pmol/mL of a seventh primer, 250-750pmol/mL of an eighth primer, and 250-750pmol/mL of a fourth target detection probe.

In some embodiments, the components of the SAT enzyme solution include: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10mM HEPES pH7.5, 10-100mM of N-acetyl-L-cysteine, 0.04-0.4mM of zinc acetate, 10-100mM of trehalose, 40-200mM of Tris-HCl pH 8.0, 40-200mM of KCl, 0.01-0.5mM of EDTA, 0.1-1% (v/v) of Triton X-100 and 20-50% (v/v) of glycerol.

In yet another aspect, the present invention provides a primer and probe combination for real-time isothermal amplification detection of fluorescent nucleic acids for simultaneous detection of six candida species, designated as a first combination, comprising:

(i) Primers and probes for detecting candida albicans, candida tropicalis, candida parapsilosis and/or candida dublinii, comprising: a first specific capture probe comprising the nucleotide sequence shown as SEQ ID NO. 17, a first primer comprising the nucleotide sequence shown as SEQ ID NO. 1, a second primer comprising the nucleotide sequence shown as SEQ ID NO. 9, and a first target detection probe comprising the nucleotide sequence shown as SEQ ID NO. 21;

(ii) Primers and probes for detecting candida glabrata comprising: a second specific capture probe comprising the nucleotide sequence shown as SEQ ID NO. 18, a third primer comprising the nucleotide sequence shown as SEQ ID NO. 3, a fourth primer comprising the nucleotide sequence shown as SEQ ID NO. 11, and a second target detection probe comprising the nucleotide sequence shown as SEQ ID NO. 23;

(iii) Primers and probes for detecting candida krusei comprising: a third specific capture probe comprising the nucleotide sequence shown as SEQ ID NO. 19, a fifth primer comprising the nucleotide sequence shown as SEQ ID NO. 5, a sixth primer comprising the nucleotide sequence shown as SEQ ID NO. 13 and a third target detection probe comprising the nucleotide sequence shown as SEQ ID NO. 25;

the two ends of the nucleotide sequences of the first target detection probe, the second target detection probe and the third target detection probe respectively carry a fluorescence report group and a quenching group, and the fluorescence report groups carried by the first target detection probe, the second target detection probe and the third target detection probe are different.

In still another aspect, the present invention provides a primer and probe combination for real-time isothermal amplification detection of fluorescent nucleic acids for simultaneously detecting six candida species and trichomonas vaginalis, named a second combination, further comprising, based on the first combination:

(iv) Primers and probes for detecting trichomonas vaginalis comprising: a third specific capture probe comprising the nucleotide sequence shown as SEQ ID NO. 20, a seventh primer comprising the nucleotide sequence shown as SEQ ID NO. 7, an eighth primer comprising the nucleotide sequence shown as SEQ ID NO. 15 and a fourth target detection probe comprising the nucleotide sequence shown as SEQ ID NO. 27; the nucleotide sequence of the fourth target detection probe carries a fluorescent reporter group and a quenching group at two ends respectively, and the carried fluorescent reporter group is different from the fluorescent reporter groups carried by the first, second and third target detection probes.

In yet another aspect, the present invention provides a non-disease diagnosis method for simultaneously detecting six kinds of candida, named as a first method, comprising the steps of:

1) Adding a nucleic acid extracting solution into a sample to be detected for nucleic acid extraction to obtain an analysis detection sample;

2) Adding detection liquid into the analysis detection sample to perform a first-step reaction to obtain a first-step reaction liquid;

3) Adding SAT enzyme solution into the first-step reaction solution to perform a second-step reaction, and performing real-time fluorescence detection to obtain a dt value of the real-time fluorescence detection;

4) And 3) judging the result according to the dt value of the real-time fluorescence detection obtained in the step 3):

if the channel corresponding to the first target detection probe has an S-shaped amplification curve and dt is less than or equal to 35, the sample to be detected contains Cspp nucleic acid, namely one or more of candida albicans, candida tropicalis, candida parapsilosis and candida dublinii nucleic acid; if the S-shaped amplification curve does not appear or the S-shaped amplification curve appears, but dt is more than 35, the sample to be detected does not contain Cspnucleic acid;

if the channel corresponding to the second target detection probe has an S-shaped amplification curve and dt is less than or equal to 35, the sample to be detected contains candida glabrata nucleic acid; if the S-shaped amplification curve does not appear or the S-shaped amplification curve appears, but dt is more than 35, the sample to be detected does not contain candida glabrata nucleic acid;

If the channel corresponding to the third target detection probe has an S-shaped amplification curve and dt is less than or equal to 35, the sample to be detected contains candida krusei nucleic acid; if the S-shaped amplification curve does not appear or the S-shaped amplification curve appears, but dt is more than 35, the sample to be detected does not contain candida krusei nucleic acid.

In yet another aspect, the present invention provides a method for diagnosing a non-disease in which six kinds of candida and trichomonas vaginalis are simultaneously detected, which further includes the following criteria based on the first method described above: if the channel corresponding to the fourth target detection probe has an S-shaped amplification curve and dt is less than or equal to 35, the sample to be detected contains trichomonas vaginalis nucleic acid; if the S-shaped amplification curve does not appear or the S-shaped amplification curve appears, but dt is more than 35, the sample to be tested does not contain trichomonas vaginalis nucleic acid.

In some embodiments, the conditions of the first step reaction in step 2) are 40 ℃ to 45 ℃ for 3 to 15 minutes.

In some embodiments, the SAT enzyme solution in step 3) is preheated prior to use at a temperature of 41-43 ℃.

In some embodiments, the conditions of the second reaction step in step 3) are 41 ℃ to 43 ℃ for 30 to 50 minutes.

In some embodiments, the sample to be tested comprises a medical sample and a non-medical sample of sources including sputum, blood products, dairy products.

The special primer and the probe for simultaneously detecting the real-time fluorescent nucleic acid isothermal amplification detection of six candida are obtained based on the optimized design of candida Ribonuclease P RNA (Ribonuclease P RNA, RNAseP RNA), and the special primer and the probe for simultaneously detecting the real-time fluorescent nucleic acid isothermal amplification detection of six candida and trichomonas vaginalis are obtained based on the optimized design of candida Ribonuclease P RNA (Ribonuclease P RNA, RNAseP RNA) and 16S rRNA of trichomonas vaginalis, and the real-time fluorescent nucleic acid isothermal amplification detection kit provided based on the primer and the probe comprises nucleic acid extracting solution, detection solution, SAT enzyme solution and the like. Wherein the nucleic acid extract may comprise a first specific capture probe commonly used for binding to candida albicans, candida tropicalis, candida parapsilosis and candida dublinii (collectively referred to as Cspp) detection sequences, a second specific capture probe for binding to candida glabrata detection sequences, a third specific capture probe for binding to candida krusei detection sequences, or further comprises a fourth specific capture probe for binding to trichomonas vaginalis detection sequences. The detection solution comprises a primer and a probe for specifically binding to a Cspp target sequence, a primer and a probe for specifically binding to a candida glabrata target sequence, a primer and a probe for specifically binding to a candida krusei target sequence, or a primer and a probe for specifically binding to a trichomonas vaginalis target sequence, and the like. The SAT enzyme solution contains RNA polymerase, reverse transcriptase, etc. required in the reaction process. Based on SAT isothermal amplification detection technology, all primers and probes are included in the same detection liquid in the use process, the system reaction is simpler, six kinds of candida or simultaneous detection of six kinds of candida and trichomonas vaginalis can be realized, and candida glabrata and candida krusei with higher drug resistance can be distinguished. The invention can be used for detecting candida and trichomonas vaginalis in the field of medical reproduction, detecting candida in wounds and skin, detecting non-medical samples such as sputum, blood products, dairy products and the like, and is suitable for being popularized and used in a large range.

Compared with the existing detection method, the method has the following advantages:

(1) The primer and the probe are obtained by optimizing design according to the RNAseP RNA of candida or further according to the 16S rRNA of trichomonas vaginalis, and six candida species can be detected simultaneously or six candida species and trichomonas vaginalis can be detected simultaneously in the detection process by combining the upstream primer, the downstream primer and the probe into one detection liquid, and candida glabrata and candida krusei with high drug resistance can be distinguished.

(2) According to the invention, higher sensitivity detection can be realized by adding different components step by step in the detection process. The results of the examples show that the detection limits of the kit and the method provided by the invention for candida albicans, candida tropicalis, candida parapsilosis, candida dublinii, candida glabrata, candida krusei and trichomonas vaginalis are 100 copies/reaction, which are obviously lower than the detection limits (1000 copies/reaction) for the above six candidas or the combination of the six candidas and trichomonas vaginalis by the method disclosed in the document 4. Therefore, the kit and the method provided by the invention can meet the requirement of higher sensitivity.

(3) The invention adopts the real-time fluorescent nucleic acid isothermal amplification detection technology to detect candida or the combination of candida and trichomonas vaginalis, avoids the operations of heating, centrifuging, in-vitro reverse transcription and the like for extracting RNA in the documents 1-4, has simpler experimental steps and reaction systems, is easy to realize automation, reduces errors caused by personnel operation, and reduces the risk of infection of operators.

(4) The invention carries out the amplification and detection of nucleic acid in the same closed system at the same time, and the whole process has no temperature rise and circulation, so compared with the detection method disclosed in the above documents 1-4, the time required for detection is greatly shortened, the detection efficiency can be improved, and the design and production cost of the PCR instrument used is reduced.

(5) The amplified product of the invention is RNA which is easy to degrade in nature, and compared with the PCR amplified DNA in the above documents 1-4, the amplified product has easy control of pollution and small cross influence, and can not cause environmental pollution.

Drawings

FIG. 1 is an amplification curve of primer and probe combinations of group 1 (A), group 2 (B), group 3 (C) and group 4 (D) in example 1 for Candida albicans in Cspp, respectively;

FIG. 2 is an amplification curve of primer and probe combinations of group 1 (A), group 2 (B), group 3 (C) and group 4 (D), respectively, for Candida tropicalis in Cspp in example 1;

FIG. 3 is an amplification curve of primer and probe combinations of group 1 (A), group 2 (B), group 3 (C) and group 4 (D), respectively, for C.parapsilosis in Cspp, in example 1;

FIG. 4 is an amplification curve of primer and probe combinations of group 1 (A), group 2 (B), group 3 (C) and group 4 (D), respectively, for Dublin in Cspp, in example 1;

FIG. 5 is an amplification curve of the primer and probe combinations of group 1 (A), group 2 (B), group 3 (C) and group 4 (D), respectively, for C.glabrata in example 1;

FIG. 6 is an amplification curve of the primer and probe combinations of group 1 (A), group 2 (B), group 3 (C) and group 4 (D), respectively, for C.krypton in example 1;

FIG. 7 is an amplification curve of primer and probe combinations of group 5 (A), group 6 (B), group 7 (C) and group 8 (D), respectively, for Trichomonas vaginalis in example 2;

FIG. 8 is an amplification curve of Cspp culture-1 (A), cspp culture-2 (B), candida glabrata culture (C), candida krusei culture (D), and Trichomonas vaginalis culture (E) using the kit provided in example 5 in example 6.

Detailed Description

Aiming at the defects of candida and trichomonas vaginalis detection methods in the prior art, the invention simultaneously detects six candida species and trichomonas vaginalis species by using a nucleic acid constant-temperature synchronous amplification detection method, and also provides a nucleic acid constant-temperature simultaneous amplification detection kit for combined detection of six candida species and trichomonas vaginalis species, a special primer, a special probe and a detection method thereof.

The present invention will be described in detail with reference to specific embodiments and drawings.

Hereinafter, only certain exemplary embodiments are briefly described. As will be recognized by those of skill in the pertinent art, the described embodiments may be modified in various different ways without departing from the spirit or scope of the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

The methods used in the examples below are conventional methods unless otherwise specified, and specific steps can be found in: molecular cloning guidelines (Molecular Cloning: A Laboratory Manual) Sambrook, j., russell, david w., molecular Cloning: A Laboratory Manual,3rd edition,2001,NY,Cold Spring Harbor).

The various biomaterials described in the examples were obtained by merely providing a means of experimental acquisition for the purposes of specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples.

All primers, capture probes, fluorescent probes and in vitro transcribed RNA products mentioned in the present invention are synthesized using the prior art.

Example 1: special primer and probe design for detecting six candida by real-time fluorescent nucleic acid isothermal amplification

The inventors have determined primer designs and amplified probes for simultaneously performing amplification of nucleic acid species in real time according to the principle that primers are highly conserved among Candida albicans, candida parapsilosis and Candida parapsilosis (these four species are collectively called Cspp) and specific to Candida glabrata and Candida parapsilosis respectively and have great difference with other species according to the published Genbank RNA nucleic acid sequence of the Candida albicans RNAseP, wherein the Genbank accession number of the Candida albicans RNAseP RNA nucleic acid sequence is XR_002086389.1, the Genbank accession number of the Candida parapsilosis RNAseP RNA nucleic acid sequence is DQ660439.1, the Genbank accession number of the Candida parapsilosis RNAseP RNA nucleic acid sequence is DQ660436.1, the Genbank accession number of the Candida parapsilosis RNAseP RNA nucleic acid sequence is DQ660438.1, the Genbank accession number of the Candida parapsilosis RNAseP RNA nucleic acid sequence is GQ251341.1, and the Genbank accession number of the Candida parapsilosis RNA nucleic acid sequence is DQ 660435.1.

In this example, a total of sets of primers and probes were designed, wherein 4 sets of primers and probes were selected (set 1, set 2, set 3 and set 4; primer probe combinations for Cspp in each set could reach a minimum detection limit of 10 copies/reaction when candida albicans, candida tropicalis, candida parapsilosis and candida dublinii were detected separately, primer probe combinations for candida glabrata in each set could reach a minimum detection limit of 10 copies/reaction when candida glabrata was detected separately, and primer probe combinations for candida krusei in each set could reach a minimum detection limit of 10 copies/reaction when candida krusei were detected separately), and real-time fluorescent isothermal amplification of nucleic acids was performed on candida positive controls (detailed below) and negative controls (systems without the six candida target nucleic acid sequences or without the six candida glabrata, such as deionized water or sample stock (commercially available)) respectively (specific detection methods can be seen in example 3 below), and the primers for six candida glabrata can be selected from the sets of primers that can be used for detecting both candida glabrata and can be distinguished between good sensitivity.

Group 1:

first specific capture probe for Cspp:

TGGGAAATTCGGTGGTACGCTCCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:17)；

First primer for Cspp: ATGGGCGGCGTTACAAGAA (SEQ ID NO: 1);

second primer for Cspp:

AATTTAATACGACTCACTATAGGGAGACATATTGCACTAAACAGC(SEQ ID NO:9)；

first target detection probe for Cspp: CGCCUUGGAUGGUUGGCUGGCG (SEQ ID NO: 21);

wherein the 5 'end and the 3' end of the first target detection probe are respectively marked with a FAM fluorescence report group and a fluorescence quenching group (such as DABCYL, BHQ-1 or BHQ-2, etc.), and the following steps are the same;

a second specific capture probe for candida glabrata:

TTTTCCTCTTCACCTTTCTGCTTGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA A(SEQ ID NO:18)；

third primer for candida glabrata: GGGGAACCCGGCCGGTAAGATTA (SEQ ID NO: 3);

fourth primer for candida glabrata:

AATTTAATACGACTCACTATAGGGAGATAGACAGGCCCATACGTCTCT(SEQ IDNO:11)；

second target detection probe for candida glabrata: CGACGUGCUGAAAUCUGUCGUCG (SEQ ID NO: 23);

wherein the 5 'end and the 3' end of the second target detection probe are respectively marked with a ROX fluorescence report group and a fluorescence quenching group (such as DABCYL, BHQ-1, BHQ-2, etc.); and the 3' -end labeled quenching group may be the same as or different from the primary target detection probe, as follows.

Third specific capture probe against candida krusei:

GGGGGGCGATGCGGAGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:19)；

fifth primer for candida krusei: CTCCACTGGGAGTGTTCT (SEQ ID NO: 5);

sixth primer for candida krusei:

AATTTAATACGACTCACTATAGGGAGAAGGATCCACGCATTGCACTCAA(SEQ IDNO:13)；

third target detection probe for candida glabrata: CGCCGUGCUUGUACAACUUUGGCG (SEQ ID NO: 25);

Wherein the 5 'end and the 3' end of the third target detection probe are respectively marked with a CY5 fluorescence report group and a fluorescence quenching group (such as DABCYL, BHQ-1 or BHQ-2, etc.); and the 3' end-labeled quenching group may be the same as or different from the primary target detection probe or the secondary target detection probe, as follows.

Group 2:

a first specific capture probe for Cspp (SEQ ID NO: 17);

first primer-1 for Cspp: GCGCATGGGCGGCGTTACAA (SEQ ID NO: 2);

second primer-1 for Cspp:

AATTTAATACGACTCACTATAGGGAGAGTTCGCATATTGCACTAAA(SEQ ID NO:10)；

first target detection probe-1 for Cspp: CGCACUAUGGGAAUGGCGUGCG (SEQ ID NO: 22);

wherein the 5 'end and the 3' end of the first target detection probe-1 are respectively marked with a FAM fluorescence report group and a fluorescence quenching group (such as DABCYL, BHQ-1 or BHQ-2, etc.);

a second specific capture probe for Candida glabrata (SEQ ID NO: 18);

third primer-1 for candida glabrata: ACCCGGCCGGTAAGATTAAGT (SEQ ID NO: 4);

fourth primer-1 for candida glabrata:

AATTTAATACGACTCACTATAGGGAGATACGTCTCTGCCCTACCCTTATC(SEQ IDNO:12)；

second target detection probe-1 for candida glabrata: CGAGCUAUCUGCUGAAAUCAGCUCG (SEQ ID NO: 24);

wherein the 5 'end and the 3' end of the second target detection probe-1 are respectively marked with a ROX fluorescence report group and a fluorescence quenching group (such as DABCYL, BHQ-1, BHQ-2, etc.); and the 3' -end labeled quenching group may be the same as or different from the primary target detection probe-1, as follows.

A third specific capture probe for C.krusei (SEQ ID NO: 19);

fifth primer-1 for candida krusei: TTCCCACGAATCCCGTCTCT (SEQ ID NO: 6);

sixth primer-1 for candida krusei:

AATTTAATACGACTCACTATAGGGAGATCAAGATGACAAGGATCCACG(SEQ IDNO:14)；

third target detection probe-1 for candida glabrata: CACCUUGCUUGUACAACUAGGUG (SEQ ID NO: 26);

wherein the 5 'end and the 3' end of the third target detection probe-1 are respectively marked with a CY5 fluorescence report group and a fluorescence quenching group (such as DABCYL, BHQ-1 or BHQ-2, etc.); and the 3' -end labeled quenching group may be the same as or different from the first target detection probe-1 or the second target detection probe-1, as follows.

Group 3:

a first specific capture probe for Cspp (SEQ ID NO: 17);

a first primer for CspP (SEQ ID NO: 1);

a second primer-1 (SEQ ID NO: 10) directed against Cspp;

a first target detection probe for Cspp (SEQ ID NO: 21);

a second specific capture probe for Candida glabrata (SEQ ID NO: 18);

a third primer (SEQ ID NO: 3) directed against Candida glabrata;

a fourth primer-1 (SEQ ID NO: 12) directed against Candida glabrata;

a second target detection probe (SEQ ID NO: 23) for Candida glabrata;

a third specific capture probe for C.krusei (SEQ ID NO: 19);

A fifth primer (SEQ ID NO: 5) directed against C.krusei;

sixth primer-1 for C.krusei (SEQ ID NO: 14);

a third target detection probe for Candida glabrata (SEQ ID NO: 25).

Group 4:

a first specific capture probe for Cspp (SEQ ID NO: 17);

a first primer-1 (SEQ ID NO: 2) directed against Cspp;

a second primer for CspP (SEQ ID NO: 9);

a first target detection probe for Cspp (SEQ ID NO: 21);

a second specific capture probe for Candida glabrata (SEQ ID NO: 18);

third primer-1 (SEQ ID NO: 4) directed against Candida glabrata;

a fourth primer (SEQ ID NO: 11) directed against Candida glabrata;

a second target detection probe (SEQ ID NO: 23) for Candida glabrata;

a third specific capture probe for C.krusei (SEQ ID NO: 19);

fifth primer-1 (SEQ ID NO: 6) for Candida krusei;

a sixth primer directed against C.krusei (SEQ ID NO: 13);

a third target detection probe for Candida glabrata (SEQ ID NO: 25).

The positive control in this example was prepared by the following steps:

(1) Synthesizing candida albicans, candida tropicalis, candida parapsilosis, candida dublinii, candida glabrata and candida krusei RNAseP RNA fragments (corresponding to XR_002086389.1, DQ660439.1, DQ660436.1, DQ660438.1, GQ251341.1 and DQ660435.1 respectively) respectively by a chemical synthesis method, and constructing the fragments on a common plasmid vector containing a T7 promoter sequence;

(2) RNA fragments were transcribed using a commercial T7 promoter external transcription kit (sigma), and after purification, RNA copy numbers were calculated by UV, and Candida albicans, candida tropicalis, candida parapsilosis, candida dubliniensis, candida glabrata, and Candida krusei in vitro transcribed RNA were used as positive controls, respectively.

Each of the four primer sets and the probe sets (set 1, set 2, set 3 and set 4) was used to detect the presence of Candida (Cspp (herePositive control gradient concentration samples of candida albicans, candida tropicalis, candida parapsilosis, candida dublinii), candida glabrata and candida krusei (wherein the gradient concentrations of the in vitro transcribed RNAs of Cspp, candida glabrata and candida krusei are each 10) ⁵ cobies/reaction, 10 ⁴ cobies/reaction, 10 ³ cobies/reaction, 10 ² The copies/reaction) and the negative control were subjected to real-time fluorescent nucleic acid isothermal amplification detection (triple detection). Specific detection methods are described in example 4 below. For convenience of illustration, the amplification curves for the

above groups

1, 2, 3 and 4 are shown separately in FIGS. 1-6, where 1, 2, 3, 4 represent RNA concentrations of 10, respectively ⁵ cobies/reaction, 10 ⁴ cobies/reaction, 10 ³ cobies/reaction, 10 ² cobies/reactions. Wherein the A-D panels of FIG. 1 are the amplification curves of the primer and probe combinations in group 1, group 2, group 3 and group 4, respectively, for Candida albicans in Cspp, and it can be seen that the primer and probe combinations in group 1 and group 2 can reach a minimum limit of 10 for detecting Candida albicans ² cobies/reaction, while the primers and probes in groups 3 and 4 detected candida albicans at a minimum limit of only 10 ³ cobies/reactions; the A-D panels of FIG. 2 are amplification curves for Candida tropicalis in the primer and probe pairs Cspp for group 1, group 2, group 3 and group 4, respectively, and it can be seen that the primer and probe in group 1 and group 3 have a minimum limit of up to 10 for detection of Candida tropicalis ² The probes/reactions were performed, while the primers and probes in groups 2 and 4 detected candida tropicalis at a minimum of only 10 ³ cobies/reactions; the amplification curves of primers and probes in groups 1, 2, 3 and 4 for Candida parapsilosis, respectively, are shown in panels A-D of FIG. 3, where the primer and probe in group 1 detect Candida parapsilosis at a minimum of 10 ² The probes/reactions were performed while the primers and probes in groups 2, 3 and 4 detected Candida parapsilosis at a minimum of only 10 ³ cobies/reactions; the amplification curves of primers and probes in groups 1, 2, 3 and 4 for Dublin candida, respectively, are shown in panels A-D of FIG. 4, where the primers and probes in groups 1 and 4 can be found to be up to 10 at the minimum for detection of Dublin candida ² COPIES/reaction, while primers and probes in groups 2 and 3 detect the best of Dublin candidaThe lower limit is only 10 ³ cobies/reactions; the amplification curves of primers and probes for Candida glabrata in groups 1, 2, 3 and 4, respectively, are shown in panels A-D of FIG. 5, and it can be seen that the primer and probe in groups 1 and 4 detect Candida glabrata at a minimum of 10 ² The primers and probes in groups 2 and 3 detected candida glabrata at a minimum of 10 per copes/reaction ³ cobies/reactions; the amplification curves of the primers and probes in groups 1, 2, 3 and 4 against C.krusei are shown in panels A-D of FIG. 6, respectively, and it can be seen that the detection sensitivity of the primers and probes in groups 1, 2 and 4 for C.krusei can reach 10 ² The primers and probes in group 3 detected C.krusei at a minimum of 10 per copes/reaction ³ cobies/reactions.

Combining the above sensitivity test results, it can be seen that when multiplex tests are performed on primer probe combinations having good sensitivity test effects for each candida species, the test sensitivity may be reduced due to mutual interference between different primer probes, but the effect of different combinations on the final sensitivity test effect is different, wherein the primer and probe combinations of group 1 have higher sensitivity in the triple test than the primer and probe combinations of group 2-group 4 for three candida species, and in another test it is also verified that the primer and probe combinations of group 1 have the same test sensitivity (all can reach 10) when three-fold tests are performed on a gradient concentration sample containing positive controls of candida species (Cspp (here, candida albicans, candida tropicalis, candida parapsilosis, and candida dublinii in vitro transcribed RNAs are mixed at a concentration of 1:1:1:1), candida glabrata and candida krusei) ² cobies/reactions), the present invention determines that the primers and probes of group 1 are useful for simultaneous real-time fluorescent nucleic acid isothermal amplification detection of the above six candida species, and at the same time, can distinguish candida smooth from candida krusei.

In this example, the inventors also synthesized primers and probes for Cspp (Candida albicans, candida tropicalis, candida parapsilosis, candida dubliniensis), primers and probes for Candida glabrata, and Kluyveromyces, respectively, as disclosed in the above-mentioned document 4The primer and probe of candida are used for carrying out triple detection on the sample with the gradient concentration containing the positive control of each candida by utilizing a PCR method, and the result shows that the primer probe combination disclosed in the document 4 can only detect 10 aiming at Cspp, candida glabrata and candida krusei ³ The probes/reaction is significantly higher than the minimum detection limit of the primer and probe combination of the set 1 determined by the invention on the six candida species, and compared with the PCR method of the document 4, which generally takes 2-3 hours, the invention can be completed within 1 hour (even 40 minutes), the detection efficiency is significantly improved, and the amplified product of the invention is RNA, which is easy to degrade and basically does not cause cross contamination of samples or environmental pollution of a laboratory.

Example 2: special primer and probe design for detecting six candida and trichomonas vaginalis by real-time fluorescent nucleic acid isothermal amplification

2.1 design and screening of real-time fluorescent nucleic acid isothermal amplification primers and probes for separately detecting trichomonas vaginalis

The inventor determines a detection sequence from a section of sequence which is highly conserved and has larger difference with other similar pathogens according to the trichomonas vaginalis 16S RNA (Genbank accession number of trichomonas vaginalis 16S RNA gene sequence is U17510.1) published by Genbank database, and designs a primer and a probe according to a primer and probe design principle so as to carry out real-time fluorescent nucleic acid isothermal amplification detection on trichomonas vaginalis.

This example designed multiple sets of primers and probes for trichomonas vaginalis, wherein the following 4 sets of primers and probes (16S-1, 16S-2, 16S-3 and 16S-4, as shown in Table 1 below) were selected for each of a series of concentration samples (concentrations of 10 each) containing a trichomonas vaginalis positive control (detailed below) ⁵ cobies/reaction, 10 ⁴ cobies/reaction, 10 ³ cobies/reaction, 10 ² Real-time fluorescent isothermal amplification detection of nucleic acids (specific detection methods can be seen in example 4 below) by performing a real-time fluorescent isothermal amplification detection of nucleic acids (specific detection methods can be seen in example 4 below) on a system without trichomonas vaginalis nucleic acid sequence, such as deionized water or sample preservation solution (commercially available), with 1 copies/reaction and a negative control (system without trichomonas vaginalis nucleic acid sequence), from which trichomonas vaginalis can be selected for high sensitivity detection Is a primer and probe combination of (a). The results are shown in Table 2 below, where "+" indicates detected and "-" indicates undetected.

The positive control for trichomonas vaginalis in this example was prepared by the following steps:

(1) Synthesizing trichomonas vaginalis 16S rRNA fragments (corresponding to U17510.1) respectively by a chemical synthesis method, and constructing the fragments on a common plasmid vector containing a T7 promoter sequence;

(2) RNA fragments were transcribed using a commercial T7 promoter external transcription kit (sigma), and after purification, RNA copy numbers were calculated by UV and trichomonas vaginalis in vitro transcribed RNA was used as a positive control.

Table 1: 4 sets of primer and probe information for real-time fluorescent nucleic acid isothermal amplification detection of trichomonas vaginalis

Table 2: the results of detection of 4 sets of primers and probes shown in Table 1 against different concentrations of Trichomonas vaginalis, respectively

As can be seen from the sensitivity results of the individual detection of trichomonas vaginalis positive standard by the primer probes of each group shown in Table 2, the 4 groups of primer and probe combinations (16S-1 to 16S-4) can detect the positive standard with the concentration of 10 copies/reaction when trichomonas vaginalis is detected individually, and therefore, the 16S-1 to 16S-4 primer and probe combinations can be used for detecting trichomonas vaginalis individually with high sensitivity.

2.2 determination of primer and Probe combinations for real-time fluorescent nucleic acid isothermal amplification detection of six candida and trichomonas vaginalis

The primer probe combinations (16S-1 to 16S-4) determined in the above step 2.1, which can be used alone for high-sensitivity detection of trichomonas vaginalis, were combined with the primers and probes of the group 1, which can be used alone for high-sensitivity detection of six candida species, determined in example 1, respectively, to obtain 4 groups in totalThe novel primers and probes (set 5, set 6, set 7 and set 8, specific combinations are shown in Table 3 below) were used to obtain gradient concentration samples of positive controls for Candida (Cspp, candida glabrata and Trichomonas vaginalis, where the concentration of in vitro transcribed RNA of Cspp, candida glabrata, candida parapsilosis and Trichomonas dubli was 1:1:1) and Trichomonas vaginalis, respectively, using these 4 sets of novel primers and probes (Cspp, candida glabrata and Trichomonas vaginalis, respectively, at a gradient concentration of 10 ⁵ cobies/reaction, 10 ⁴ cobies/reaction, 10 ³ cobies/reaction, 10 ² The copies/reaction) and the negative control were subjected to real-time fluorescent nucleic acid isothermal amplification detection (quadruple detection). Specific detection methods are described in example 6 below. The detection results are shown in Table 4 below, wherein "+" indicates detected and "-" indicates undetected.

From the results set forth in Table 4, it can be seen that only the primer and probe combinations of group 5 reached a minimum of 10 for six candida and trichomonas vaginalis in the quadruple assay ² cobies/reactions; while the primer and probe combinations of set 6 detected 10 in the quadruple detection against trichomonas vaginalis ² The copies/reaction was only detectable to 10 for both candida glabrata and candida krusei ³ cobies/reactions; the primer and probe combinations of group 7 were only detectable to 10 for trichomonas vaginalis ³ The copies/reaction, primer and probe combinations of group 8 were only detectable to 10 for both trichomonas vaginalis and candida krusei ³ cobies/reactions. FIG. 7, panels A-D, schematically shows amplification curves for primer and probe combinations of sets 5-8 for Trichomonas vaginalis, where 1, 2, 3, 4 represent RNA concentrations of 10, respectively ⁵ cobies/reaction, 10 ⁴ cobies/reaction, 10 ³ cobies/reaction, 10 ² cobies/reactions.

Table 3: different primer and probe combinations for detecting six candida and trichomonas vaginalis by real-time fluorescent nucleic acid isothermal amplification

Primer probe combination numbering	Combination mode of primer probe
		Group
5	16S-1+group 1
		Group 6	16S-2+ group 1
Group 7	16S-3+ group 1
		Group 8	16S-4+ group 1

Table 4: the results of sensitivity detection of different primer and probe combinations shown in Table 3 and the method disclosed in document 4

From the above results, it was confirmed that even though the primer probe combinations 16S-1 to 16S-4 each had a good sensitivity test result (each of which could reach 10 copies/reaction) when the trichomonas vaginalis was individually tested, the primer probe combinations were recombined with the primer probe combination (group 1) for simultaneously testing six candida determined in example 1 to simultaneously perform four-fold tests on six candida and trichomonas vaginalis, and the sensitivity results of the four-fold tests could be affected. Specifically, when the primer probes of 16S-2 to 16S-4 are further combined with the primer probe of group 1, all of them will be guidedThe detection limit of the novel primer probe combination (i.e. group 6 to group 8) on one or more of candida and trichomonas vaginalis cannot reach 10 at the minimum ² The probes/reactions, however, can still be performed with higher sensitivity (up to 10 at the lowest limit) only when the 16S-1 primer probe is further combined with the group 1 primer probe (i.e., group 5) ² cobies/response) were tested simultaneously for six candida species and trichomonas vaginalis. Thus, the present invention identifies the primer and probe combinations of set 5 as optimal combinations for simultaneous detection of six candida and trichomonas vaginalis and suitable for use in real-time fluorescent nucleic acid isothermal amplification detection methods.

In this example, the inventors further synthesized primers and probes for trichomonas vaginalis according to the disclosure of the above document 4, and performed quadruple detection on the above positive control gradient concentration samples containing candida and trichomonas vaginalis by PCR method, and the results are shown in the above table 4. It can be seen that the primer probe combination disclosed in this document 4 is only detectable to 10 for Cspp, candida glabrata, candida krusei and trichomonas vaginalis ³ The copies/response was significantly higher than the minimum detection of the above six candida and trichomonas vaginalis by the primer and probe combinations of set 5 as determined by the present invention.

Example 3: real-time fluorescent nucleic acid isothermal amplification detection kit for simultaneously detecting six candida

The kit for detection of Cspp, candida glabrata and candida krusei provided in this example is a kit based on the principle of isothermal amplification detection of RNA nucleic acids (also referred to herein as a first kit), and specifically comprises the following components:

(T1) nucleic acid extract: for extracting and purifying candida nucleic acids in a sample, which may comprise a solid support (e.g., magnetic beads) comprising a first specific capture probe (SEQ ID NO: 17), a second specific capture probe (SEQ ID NO: 18), a third specific capture probe (SEQ ID NO: 19), wherein the first specific capture probe is used to capture a Cspp detection sequence; the second specific capture probe is used for capturing candida glabrata detection sequences; a third specific capture probe is used to capture candida krusei detection sequences. Specifically, the nucleic acid extraction solution may contain: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfate), 1-50. Mu.M of first specific capture probe, 1-50. Mu.M of second specific capture probe, 1-50. Mu.M of third specific capture probe, 50-500mg/L magnetic beads;

(T2) detection solution: it comprises a first primer (SEQ ID NO: 1), a second primer (SEQ ID NO: 9), a first target detection probe (SEQ ID NO: 21), a third primer (SEQ ID NO: 3), a fourth primer (SEQ ID NO: 11), a second target detection probe (SEQ ID NO: 23), a fifth primer (SEQ ID NO: 5), a sixth primer (SEQ ID NO: 13), a third target detection probe (SEQ ID NO: 25); wherein the first primer is complexed with the second primer and the first target detection probe for detecting a target sequence of Cspp; the third primer is matched with the fourth primer and the second target detection probe and is used for detecting a target sequence of candida glabrata; the fifth primer is matched with the sixth primer and the third target detection probe and is used for detecting a target sequence of candida krusei. Specifically, the detection liquid may contain: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl ₂ 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of a first primer, 250-750pmol/mL of a second primer, 250-750pmol/mL of a third primer, 250-750pmol/mL of a fourth primer, 250-750pmol/mL of a fifth primer, 250-750pmol/mL of a sixth primer, 250-750pmol/mL of a first target detection probe, 250-750pmol/mL of a second target detection probe, 250-750pmol/mL of a third target detection probe; and

(T3) SAT enzyme solution: it may comprise at least one RNA polymerase and an M-MLV reverse transcriptase; specifically, the SAT enzyme solution comprises: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10mM HEPES pH7.5, 10-100mM N-acetyl-L-cysteine, 0.04-0.4mM zinc acetate, 10-100mM trehalose, 40-200mM Tris-HCl pH 8.0, 40-200mM KCl, 0.01-0.5mM EDTA, 0.1-1% (v/v) Triton X-100, and 20-50% (v/v) glychol (glycerol).

For convenience of detection, the kit provided in this embodiment may further comprise the following components:

(M1) washing solution: the magnetic bead washing agent is used for washing magnetic beads in water phase, and the formula of the magnetic bead washing agent can be HEPES 5-50mM, naCl 50-500mM, SDS 0.5-1.5% and EDTA 1-10mM;

(M2) mineral oil: mineral oil for washing the magnetic bead organic phase;

(M3) a positive control; dilutions of in vitro transcribed RNAs that can be Cspp, candida glabrata, and candida krusei (prepared in example 1);

(M4) negative control: can be a system that does not contain the above-mentioned six candida nucleic acid sequences or the above-mentioned six candida species, such as physiological saline and sample preservation fluid (which contains a high concentration of detergent and physiological saline).

Example 4: real-time fluorescent nucleic acid isothermal amplification method for simultaneously detecting six candida

The method of this example is based on the RNA isothermal amplification detection principle to detect candida albicans, candida tropicalis, candida parapsilosis, candida dublinii (the above four candida are collectively referred to as Cspp), candida glabrata and candida krusei simultaneously, and it uses the kit provided in the above example 3 to detect whether the sample contains the above six candida nucleic acids, and the specific operation steps are:

4.1, sample preparation

2mL of the microorganism culture was mixed with 2mL of a sample-keeping solution (Shanghai Reed Biotechnology Co., ltd.) at a ratio of 1:1 to obtain a sample to be measured.

4.2 RNA extraction

(1) Adding 100-800 mu L of nucleic acid extracting solution and 400 mu L of sample to be detected into a sample treatment tube, uniformly mixing, preserving heat at 60 ℃ for 10 minutes, and standing at room temperature for 10 minutes;

(2) The sample treatment tube is placed on a magnetic bead separation device and kept stand for 3-5 minutes. After the magnetic beads are adsorbed on the tube wall, the sample treatment tube is kept on the magnetic bead separation device, waste liquid is sucked and discarded, and the magnetic beads are reserved. Adding 1mL of washing liquid, shaking uniformly, standing for 3-5 minutes, adding 800 mu L of washing liquid and 200 mu L of mineral oil, shaking uniformly, standing for 3-5 minutes, sucking waste liquid, and retaining magnetic beads;

(3) The sample processing tube is removed from the magnetic bead separation device and the tube is filled with a magnetic bead-nucleic acid complex for use (the beads should be clearly visible in this step).

4.3 detection of SAT nucleic acid amplification

(1) Adding 40 mu L of detection solution into the sample treatment tube (the magnetic bead-nucleic acid compound in the tube) treated in the step 4.2, and oscillating and resuspending the magnetic beads;

(2) Taking 40 mu L of detection liquid which is uniformly mixed by shaking to a clean micro-reaction tube, adding 50 mu L of mineral oil into the micro-reaction tube, preheating the micro-reaction tube for 5-10min under the reaction condition of 42 ℃, and adding 13 mu L of SAT enzyme liquid preheated to 42 ℃ into the micro-reaction tube. The micro reaction tube is quickly transferred to a constant temperature fluorescence detection instrument for reaction for 40 minutes at 42 ℃, and fluorescent signals are collected once per minute. The fluorescein channel selects FAM, ROX and CY5 channels (namely, the 5' end of a first target detection probe in the detection liquid is marked with a FAM fluorescence report group, the 5' end of a second target detection probe is marked with a ROX fluorescence report group, and the 5' end of a third target detection probe is marked with a CY5 fluorescence report group). Negative controls were tested synchronously as above.

4.4, result determination

And automatically reading the dt value by software according to a curve obtained by the SAT amplification result, and judging the result.

The result judgment criteria were:

if the FAM channel dt is less than or equal to 35, the sample is Csppositive, namely the sample contains Cspnucleic acid;

FAM channel dt >35, the sample is Cspnegative, i.e., the sample does not contain Cspnucleic acid;

the ROX channel dt is less than or equal to 35, the sample is candida glabrata positive, namely the sample contains candida glabrata nucleic acid;

the ROX channel dt is more than 35, the sample is candida glabrata negative, namely the sample does not contain candida glabrata nucleic acid;

if the CY5 channel dt is less than or equal to 35, the sample is candida krusei positive, namely the sample contains candida krusei nucleic acid;

and if the CY5 channel dt >35, the sample is candida krusei negative, i.e. the sample does not contain candida krusei nucleic acid.

19 vaginal cultures of common microorganisms or pathogens (wherein sample 1 was candida Albicans (ATCC)18804 Culture, sample 2 was candida tropicalis (ATCC 750) culture, sample 3 was candida parapsilosis (ATCC 22019) culture, sample 4 was candida dublinii (MYA-646) culture, sample 5 was candida glabrata (ATCC 2001) culture, sample 6 was candida krusei (ATCC 32196) culture, sample 7 was candida albicans, candida tropicalis, candida parapsilosis, candida dublinii, a mixed culture of candida glabrata and candida krusei, sample 8 was candida quaternary also (ATCC 14242) culture, sample 9 was candida lactis (ATCC 4135) culture, sample 10 was candida staphylococci (ATCC 34449) culture, sample 11 was lactobacillus crispatus (ATCC 33820) culture, sample 12 was lactobacillus gasseri (ATCC 33323) culture, sample 13 was candida vaginalis (ATCC 14018) culture, sample 14 was candida (ATCC 25922) culture, sample 15 was candida genitalia (ATCC 33530) culture, sample 16 was candida glabrata (ATCC) was candida (ATCC 5718) and sample c (ATCC 571) was a plurality of staphylococcus aureus (ATCC 19) and sample c (ATCC 19) was amplified at a constant temperature of equal to that was equal to one of the sample concentration of staphylococcus (ATCC 19) was equal to that was amplified (ATCC 1) was a sample of staphylococcus (ATCC 19) and (ATCC 1) was a sample was amplified (c) was cultured, and a sample was a sample of c1 was a sample was amplified sample was a sample of a sample was prepared ⁶ CFU/mL or 1X 10 ⁵ cell/mL, and positive controls (Candida albicans, candida glabrata, and Candida krusei exocrine prepared in example 1 were assayed simultaneously for external transcribed RNA (4X 10) ⁵ cobies/mL) mixture) and negative control. The results are shown in the following table 5, and it can be seen that 4 samples (samples 1-4) in 19 common vaginal microbial or pathogen culture samples are positive for FAM channel, 1 sample (sample 5) is positive for ROX channel, 1 sample (sample 6) is positive for CY5 channel, 1 sample (sample 7) is positive for FAM, ROX, CY three channels, and 12 samples (samples 8-19) is negative, which is consistent with the actual condition of the samples, and the positive and negative control detection results are normal, so that the method is effective, has good detection specificity, can be used for simultaneously detecting Cspp, candida glabrata and candida krusei, and can distinguish candida glabrata from candida krusei.

Table 5: detection results of 19 common vaginal microorganisms or pathogen cultures

With reference to this example, other medical samples (e.g., wounds, skin, etc.) or non-medical samples (sputum, blood products, dairy products, etc.) can also be tested in the same way (particularly steps 4.1-4.4).

Example 5: real-time fluorescent nucleic acid isothermal amplification detection kit for simultaneously detecting six candida and trichomonas vaginalis

The kit for detection of Cspp, candida glabrata, candida krusei and trichomonas vaginalis provided in this example is a kit (also referred to herein as a second kit) based on the principle of isothermal amplification detection of RNA nucleic acids, and specifically comprises the following components:

(T1) nucleic acid extract: for extracting and purifying candida and trichomonas vaginalis nucleic acids in a sample, which may comprise a solid support (e.g., magnetic beads) comprising a first specific capture probe (SEQ ID NO: 17), a second specific capture probe (SEQ ID NO: 18), a third specific capture probe (SEQ ID NO: 19), and a fourth specific capture probe (SEQ ID NO: 20), wherein the first specific capture probe is used to capture Cspp detection sequences; the second specific capture probe is used for capturing candida glabrata detection sequences; the third specific capture probe is used for capturing candida krusei detection sequences; the fourth specific capture probe was used to capture trichomonas vaginalis detection sequences. Specifically, the nucleic acid extraction solution may contain: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfate), 1-50. Mu.M of first specific capture probe, 1-50. Mu.M of second specific capture probe, 1-50. Mu.M of third specific capture probe, 1-50. Mu.M of fourth specific capture probe, 50-500mg/L magnetic beads;

(T2) detection solution: comprising a first primer (SEQ ID NO: 1), a second primer (SEQ ID NO: 9), a first target detection probe (SEQ ID NO. 21), third primer (SEQ ID NO. 3), fourth primer (SEQ ID NO. 11), second target detection probe (SEQ ID NO. 23), fifth primer (SEQ ID NO. 5), sixth primer (SEQ ID NO. 13), third target detection probe (SEQ ID NO. 25), seventh primer (SEQ ID NO. 7), eighth primer (SEQ ID NO. 15), fourth target detection probe (SEQ ID NO. 27); wherein the first primer is complexed with the second primer and the first target detection probe for detecting a target sequence of Cspp; the third primer is matched with the fourth primer and the second target detection probe and is used for detecting a target sequence of candida glabrata; the fifth primer is matched with the sixth primer and the third target detection probe and is used for detecting a target sequence of candida krusei; the seventh primer is matched with the eighth primer and the fourth target detection probe and is used for detecting a target sequence of trichomonas vaginalis. Specifically, the detection liquid may contain: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl ₂ 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of a first primer, 250-750pmol/mL of a second primer, 250-750pmol/mL of a third primer, 250-750pmol/mL of a fourth primer, 250-750pmol/mL of a fifth primer, 250-750pmol/mL of a sixth primer, 250-750pmol/mL of a seventh primer, 250-750pmol/mL of an eighth primer, 250-750pmol/mL of a first target detection probe, 250-750pmol/mL of a second target detection probe, 250-750pmol/mL of a third target detection probe, 250-750pmol/mL of a fourth target detection probe; and

(M2) mineral oil: mineral oil for washing the magnetic bead organic phase;

(M3) a positive control; dilutions of in vitro transcribed RNAs that can be Cspp, candida glabrata, candida krusei, and trichomonas vaginalis (prepared in example 1);

(M4) negative control: can be a system that does not contain the above-mentioned six candida and trichomonas vaginalis nucleic acid sequences or the above-mentioned six candida and trichomonas vaginalis, such as physiological saline and sample preservation solutions (which contain high concentrations of detergent and physiological saline).

Example 6: real-time fluorescent nucleic acid isothermal amplification method for simultaneously detecting six candida species and trichomonas vaginalis and sensitivity test

The method of this example is based on the principle of RNA isothermal amplification detection to detect simultaneously Candida albicans, candida tropicalis, candida parapsilosis, candida dublinii (the above four candida are collectively referred to as Cspp), candida glabrata, candida krusei and Trichomonas vaginalis, and uses the kit provided in example 5 above to detect whether a sample (e.g., a microbial culture) contains the above six candida and Trichomonas vaginalis nucleic acids, and the specific procedure is described in steps 4.1 to 4.4 of example 4. The only differences are that:

(1) In step 4.3, the fluorescein channel is selected from FAM, ROX, CY and HEX channels (i.e., the 5 'end of the first target detection probe in the detection solution is labeled with a FAM fluorescent reporter group, the 5' end of the second target detection probe is labeled with a ROX fluorescent reporter group, the 5 'end of the third target detection probe is labeled with a CY5 fluorescent reporter group, and the 5' end of the fourth target detection probe is labeled with a HEX fluorescent reporter group).

(2) In step 4.4, the result criterion is:

the CY5 channel dt >35, the sample is candida krusei negative, i.e. the sample does not contain candida krusei nucleic acid;

if the HEX channel dt is less than or equal to 35, the sample is positive to trichomonas vaginalis, namely the sample contains trichomonas vaginalis nucleic acid;

HEX channel dt >35, the sample is trichomonas vaginalis negative, i.e., trichomonas vaginalis nucleic acid is not contained in the sample.

The Cspp culture-1 (which is a mixture of Candida albicans (ATCC 18804) culture, candida tropicalis (ATCC 750) culture, candida parapsilosis (ATCC 22 019) culture, and Candida dubliniensis (MYA-646) culture at a concentration ratio of 1:1:1:1), candida glabrata (ATCC 2001) culture, candida krusei (ATCC 32196) culture, and Trichomonas vaginalis (ATCC PRA-98) culture were mixed by the above-mentioned method to obtain a gradient concentration mixture (in which the gradient concentrations of Cspp culture-1, candida glabrata culture, candida krusei culture were 10, respectively) ⁴ CFU/mL、10 ³ CFU/mL、10 ² CFU/mL、10 ¹ CFU/mL、10 ⁰ CFU/mL; the gradient concentrations of trichomonas vaginalis cultures were 1, 0.1, 0.01, 0.001, 0.0001Cells/mL, respectively, and four-fold assays were performed on the gradient concentration mixtures (gradient concentrations as above) of Cspp culture-2 (candida albicans (ATCC 18804), candida glabrata (ATCC 2001), candida krusei (ATCC 32196), and trichomonas vaginalis (ATCC PRA-98) cultures, respectively.

The results are shown in FIG. 8, and the amplification curves for Cspp, candida glabrata, candida krusei, and Trichomonas vaginalis cultures are shown separately, as shown in panels A-E in FIG. 8, and are shown schematically for quadruple detection of Cspp culture-1, cspp culture-2, candida glabrata culture, candida krusei culture, and Trichomonas vaginalis culture, respectively, using the kit provided in example 5. It can be seen that the lower detection limit for Cspp cultures (Cspp culture-1 or Cspp culture-2), candida glabrata cultures, and Candida krusei cultures can all be up to 10CFU/mL, and the lower detection limit for trichomonas vaginalis cultures can be up to 0.001Cells/mL.

Example 7: real-time fluorescent nucleic acid isothermal amplification specificity test for simultaneously detecting six candida species and trichomonas vaginalis

The method of this example is based on the principle of RNA isothermal amplification detection to detect simultaneously Candida albicans, candida tropicalis, candida parapsilosis, candida dublinii (the above four candidiasis are collectively referred to as Cspp), candida glabrata, candida krusei and Trichomonas vaginalis, and it uses the kit provided in example 5 to detect whether the sample contains the above six Candida and Trichomonas vaginalis nucleic acids, and the specific procedure is described in example 6.

For 20 vaginal regular microorganisms or pathogen cultures (where sample 1 is candida albicans (ATCC 18804) culture, sample 2 is candida tropicalis (ATCC 750) culture, sample 3 is candida parapsilosis (ATCC 22019) culture, sample 4 is candida dublinii (MYA-646) culture, sample 5 is candida glabrata (ATCC 2001) culture, sample 6 is candida krusei (ATCC 32196) culture, sample 7 is trichomonas vaginalis (ATCC PRA-98) culture, sample 8 is candida albicans, candida tropicalis, candida glabrata, candida parapsilosis, candida dublinii, candida krusei and trichomonas vaginalis mixed culture, sample 9 is candida parapsilosis (ATCC 14242) culture, sample 10 is candida lactis (ATCC 4135) culture, sample 11 is candida vinis (ATCC 34449) culture, sample 12 is lactobacillus crispatus (ATCC 33820) culture, sample 13 is candida garginis candida glabra (ATCC 33323) culture, sample 14 is candida vaginalis (ATCC 37) culture, sample 35) is candida vaginalis (ATCC 35) culture, sample 35 is sample 35) is 15, and sample 35 is 15 is neisseria (ATCC 37) is a sample 35) is prepared by the above method, respectively Culture, sample 19 was Chlamydia trachomatis (ATCC VR-571B) culture, sample 20 was Staphylococcus aureus (ATCC 12600) culture, and detection of isothermal amplification of fluorescent nucleic acid of Candida was performed at a concentration of 1X 10 ⁶ CFU/mL or 1X 10 ⁵ Cells/mL, and positive controls (candida albicans, candida glabrata, candida krusei prepared in example 1, and trichomonas vaginalis in vitro transcribed RNA prepared in example 2 (4×10 ⁵ cobies/mL) and a negative control. As shown in table 6 below, it can be seen that the 20 common vaginal microorganism or pathogen cultures samples were positive for FAM channel in 4 samples (samples 1-4), positive for ROX channel in 1 sample (sample 5), positive for CY5 channel in 1 sample (sample 6), positive for HEX channel in 1 sample (sample 7), positive for FAM, ROX, CY and HEX four channels in 1 sample (sample 8), negative in 12 samples (samples 9-20), which is identical to the actual condition of the samples, and the negative and positive control test results were normal, proving that the method was effective, and had good detection specificity, and can be used for simultaneous detection of Cspp, smoothness, candida krusei and trichomonas vaginalis.

Table 6: detection results of 20 samples of common vaginal microorganisms or pathogen cultures

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With reference to this embodiment, other medical samples (e.g., wounds, skin, etc.) or non-medical samples (sputum, blood products, dairy products, etc.) may also be tested in the same manner.

Example 8: detection of clinical vaginal swab samples

This example uses the kit provided in example 5 above to detect whether 1194 clinical vaginal swab samples (in 2mL of saline) contain Cspp, candida glabrata, candida krusei, and trichomonas vaginalis nucleic acid, and the procedure is as described in example 6 above.

As shown in table 7 below, 215 positive samples were detected in total from 1194 clinical vaginal swab samples tested, and the ratios of Cspp positive, candida glabrata positive, candida krusei positive, and trichomonas vaginalis positive samples were 76.74%, 17.21%, 3.26%, and 5.12%, respectively, which substantially match the ratios of candida vaginalis positive samples and trichomonas vaginalis positive samples reported in the prior art documents, demonstrating that the method and kit are effective and can be used for clinical sample testing.

Table 7: detection results of 1194 cases of clinical vaginal swab samples

Target(s)	Clinical vaginal sample (1194 cases) duty cycle	Positive sample (1194 cases) duty cycle
			Cspp positive	13.9％(165/1194)	76.74％(165/215)
Candida glabrata positivity	3.09％(37/1194)	17.21％(37/215)
			Candida krusei positive	0.51％(7/1194)	3.26％(7/215)
Trichomonas vaginalis positive	0.92％(11/1194)	5.12％(11/215)
			Cspp&Trichomonas vaginalis mixed positive	0.08％(1/1194)	0.46％(1/215)
Cspp&Smooth mix positives	0.16％(2/1194)	0.93％(2/215)
			Smooth and crunchy mix positive	0.16％(2/1194)	0.93％(2/215)
Cspp, smooth and crunchy mix positives	0.08％(1/1194)	0.46％(1/215)

Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or that equivalents may be substituted for part of the technical features thereof. Any equivalent substitutions, modifications and the like made within the spirit and principles of the present invention should fall within the present disclosure.

Claims

1. A real-time fluorescent nucleic acid isothermal amplification detection kit for simultaneously detecting six candida species, named a first kit, comprising:

wherein:

2. A real-time fluorescent nucleic acid isothermal amplification detection kit for simultaneously detecting six candida species and trichomonas vaginalis, designated as a second kit, wherein the second kit further comprises, on the basis of the first kit of claim 1:

3. The kit of claim 1 or 2, wherein the kit further comprises:

(M2) mineral oil; and/or

4. The kit according to any one of claim 1 to 3,

the nucleic acid extraction solution comprises the following components: 250-800mM HEPES, 4-10% LLS (lithium dodecyl sulfonate), 1-50 μΜ of the first specific capture probe, 1-50 μΜ of the second specific capture probe, 1-50 μΜ of the third specific capture probe, 50-500mg/L of the magnetic beads; optionally, the components of the nucleic acid extraction solution further comprise a fourth specific capture probe of 1-50 μΜ;

the components of the detection liquid comprise: 10-50mM Tris, 5-40mM KCl, 10-40mM MgCl ₂ 1-20mM NTP, 0.1-10mM dNTPs, 1-10% PVP40, 250-750pmol/mL of a first primer, 250-750pmol/mL of a second primer, 250-750pmol/mL of a third primer, 250-750pmol/mL of a fourth primer, 250-750pmol/mL of a fifth primer, 250-750pmol/mL of a sixth primer, 250-750pmol/mL of a first target detection probe, 250-750pmol/mL of a second target detection probe, 250-750pmol/mL of a third target detection probe; optionally, the components of the detection solution further comprise 250-750pmol/mL of a seventh primer, 250-750pmol/mL of an eighth primer, and 250-750pmol/mL of a fourth target detection probe;

the SAT enzyme solution comprises the following components: 16000-160000U/mL of M-MLV reverse transcriptase, 8000-80000U/mL of RNA polymerase, 2-10mM HEPES pH7.5, 10-100mM of N-acetyl-L-cysteine, 0.04-0.4mM of zinc acetate, 10-100mM of trehalose, 40-200mM of Tris-HCl pH 8.0, 40-200mM of KCl, 0.01-0.5mM of EDTA, 0.1-1% (v/v) of Triton X-100 and 20-50% (v/v) of glycerol.

5. A primer and probe combination for real-time fluorescent nucleic acid isothermal amplification detection for simultaneous detection of six candida species, designated as a first combination, comprising:

6. A primer and probe combination for simultaneous detection of real-time fluorescent nucleic acid isothermal amplification of six candida and trichomonas vaginalis, designated as a second combination, further comprising, on the basis of the first combination of claim 5:

7. A non-disease diagnostic method for simultaneously detecting six kinds of candida, named a first method, characterized in that the first method comprises the steps of:

8. A non-disease diagnostic method for simultaneous detection of six kinds of candida and trichomonas vaginalis, designated as a second method, characterized in that the second method further comprises the following decision criteria on the basis of the first method of claim 7: if the channel corresponding to the fourth target detection probe has an S-shaped amplification curve and dt is less than or equal to 35, the sample to be detected contains trichomonas vaginalis nucleic acid; if the S-shaped amplification curve does not appear or the S-shaped amplification curve appears, but dt is more than 35, the sample to be tested does not contain trichomonas vaginalis nucleic acid.

9. The method according to claim 7 or 8, wherein the conditions of the first reaction in step 2) are 40 ℃ to 45 ℃ for 3 to 15min; and/or

Preheating the SAT enzyme solution in the step 3) before use, wherein the preheating temperature is 41-43 ℃; and/or

The condition of the second step reaction in the step 3) is 41-43 ℃ for 30-50min.

10. The method according to any one of claims 7-9, wherein the sample to be tested comprises a medical sample and a non-medical sample of sources including sputum, blood products, dairy products.