CN112680541B

CN112680541B - LNA-Taqman-multiplex fluorescence PCR technology and application thereof in rapid detection of candida

Info

Publication number: CN112680541B
Application number: CN202110076817.7A
Authority: CN
Inventors: 殷耀宏; 王校; 乔毅
Original assignee: Zhongxun Youjian Biotechnology Jiangsu Co ltd
Current assignee: Zhongxun Youjian Biotechnology Jiangsu Co ltd
Priority date: 2021-01-20
Filing date: 2021-01-20
Publication date: 2021-10-12
Anticipated expiration: 2041-01-20
Also published as: CN112680541A

Abstract

The invention relates to the technical field of biology, in particular to a multiplex fluorescence PCR based on LNA-Taqman and application thereof in candida detection. The system of the specific amplification primer and the LNA-Taqman probe can be used for rapidly amplifying and detecting common candida in female genital tracts, and compared with the traditional detection means, the detection efficiency, the sensitivity, the specificity and the multiple detection rate are improved.

Description

LNA-Taqman-multiplex fluorescence PCR technology and application thereof in rapid detection of candida

Technical Field

The invention relates to the field of biotechnology, in particular to a technology based on LNA-Taqman-multiplex fluorescence PCR and application thereof in rapid detection of candida in female genital secretion.

Background

Vaginal yeast infections, also known as candidal vaginitis and vaginal thrush, are caused by overgrowth of candida. These yeasts are usually present in small amounts in the vagina. The most common symptoms are vaginal itching, other symptoms include burning pain during urination, thick white band, and symptoms often worsen in the woman immediately before delivery. About 75% of women are infected with at least one vaginal yeast at some point in life, while nearly half of women are infected with at least two. Approximately 5% of people infect more than three within a year. This is the second most common cause of vaginal inflammation following bacterial vaginosis.

Although Candida albicans is the most common yeast associated with vaginal thrush, infections with other types of yeast produce similar symptoms. A study of 370 confirmed vaginal yeast infected patients identified the following types of infections: candida albicans (85.7%), non-candida albicans (13.2%), others (1.1%). Non-albicans is often found in complex cases of vaginal thrush, where first line treatment is ineffective. These conditions are more likely to occur in immunocompromised persons.

At present, according to the national industry standard' SN/T2801-; as indicated in the infectious candidiasis quarantine technical Specification, "candidal vaginitis and guide thrush are usually diagnosed in one of three ways: vaginal wet microscopy, microbial culture and antigen detection. These conventional identification methods usually require professional personnel and long detection time, are easily affected by human factors, cause low accuracy and the like, and are not favorable for early treatment of diseases.

The TaqMan-multiplex fluorescence PCR technology is a molecular biological diagnosis technology based on a nucleic acid amplification technology. The technology has the advantages of short time consumption, strong specificity, high sensitivity, real-time detection and the like, and provides more reliable help in clinical application requiring early diagnosis, timely treatment and the like. At present, the traditional fluorescent PCR detection method is mainly adopted for the products for detecting candida, which have been taken to the registration certificate of CFDA medical instruments in China, and the method still has a space for improving the detection sensitivity and the detection specificity.

In order to further improve the efficiency and accuracy of detection, the art still searches for a detection method capable of detecting candida in the female genital tract more quickly and accurately.

In view of this, the present invention is proposed.

Disclosure of Invention

One of the purposes of the application is to find a detection method for the candida in the female genital tract, wherein the detection efficiency and the detection accuracy are obviously improved;

the other purpose of the application is to find a reagent/kit for detecting the candida in the female genital tract, wherein the detection efficiency and the detection accuracy are both obviously improved.

In order to realize the aim, the invention provides an LNA-Taqman-multiplex fluorescence PCR technology, which combines the Locked Nucleic Acid (LNA) technology, the Taqman probe detection technology, the multiplex PCR technology and other technologies to establish a nucleic acid amplification reagent for the candida in the female genital tract; the reagent can be used for amplifying five common candida in the genital tract in a reaction tube, so as to finish multiple real-time fluorescence detection of candida albicans, candida glabrata, candida tropicalis, candida parapsilosis and candida krusei and realize specific differential detection of the candida in the female genital tract.

Specifically, the invention firstly provides a PCR amplification primer and an LNA-Taqman probe set, wherein the PCR amplification primer and the LNA-Taqman probe set comprise: primer/LNA-Taqman probe for SEQ ID No.1 of Candida albicans, primer/LNA-Taqman probe for SEQ ID No.2 of Candida glabrata, primer/LNA-Taqman probe for SEQ ID No.3 of Candida tropicalis, primer/LNA-Taqman probe for SEQ ID No.4 of Candida parapsilosis, primer/LNA-Taqman probe for SEQ ID No.5 of Candida krusei.

Further, the PCR amplification primers and the LNA-Taqman probe sequences are as follows:

primer/Probe name	Primer sequence (5'-3')
		Candida albicans primer Cal-F	CTGATTTGCTTAATTGCACC
Candida albicans primer Cal-P +	CAACCAATTTTTT+AT+C+AACTTGTC
		Candida glabrata Cgla-F	TTCTGCAGTGGGGGGAGGGA
Candida glabrata Cgla-P +	TTCCAAAGGA+GGT+GTTTTATCACA
		Candida tropicalis Ctro-F	AAGGATCATTACTGATTTGCTTAATT
Candida tropicalis Ctro-P +	ATTTCTTTG+GT+GG+CGGGAGC
		Candida parapsilosis Cpa-F	TAGGTGAACCTGCGGAAGGATC
Candida parapsilosis Cpa-P +	CTTAACTG+C+A+TTTTTTCTTACAC
		Candida krusei Ckru-F	AGGATCATTACTGTGATTTAGTA
Candida krusei Ckru-P +	AAAACAACAA+CA+CCTAAAATGTGG
		Reverse general CTY-R	TATCGCATTTCGCTGCGCTCTTC

The invention also provides a composition, which is characterized by comprising the PCR amplification primer and the LNA-Taqman probe set; preferably, the composition is an amplification composition.

The invention also provides a rapid candida detection reagent/kit, which is characterized by comprising the PCR amplification primer and the LNA-Taqman probe group;

further, the nucleic acid amplification reagent may contain a DNA polymerase, a buffer, and the like;

further, the nucleic acid amplification reagent comprises water and the following components at final concentrations: the nucleic acid amplification reagent comprises water and the following components in final concentration:

DNA polymerase	0.3U～0.4U
		KCl	50mmol/L
MgCl₂	0.4mmol/L～0.8mmol/L
		Tris-Hcl(pH＝8.0)	20mmol/L
Tween 20	0.1％
		dNTPs	0.4mmol/L～1.6mmol/L

Preferably, the nucleic acid amplification reagent comprises water and the following components at final concentrations:

DNA polymerase	0.4U
		KCl	50mmol/L
MgCl₂	0.5mmol/L
		Tris-Hcl(pH＝8.0)	20mmol/L
Tween 20	0.1％
		dNTPs	1mmol/L

Furthermore, in the nucleic acid amplification reagent of the present invention, the concentration of each component is a working concentration, and the concentration of each component may be 2 to 100 times the working concentration during storage and transportation. The components can be independently present or mixed, for example, the nucleic acid reagent comprises four independently present reagents of enzyme, buffer solution, PCR detection primer and LNA-Taqman probe.

Furthermore, a positive control and a negative control can be included in the reagent or the kit;

preferably, the positive control is a nucleic acid DNA equal ratio mixture of Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei.

In some specific embodiments, the kit comprises 10. mu.L of the nucleic acid amplification reagent, 0.3mmol/L of Candida albicans PCR amplification primer and 0.3. mu.L of LNA-Taqman probe, respectively, and water to make up for 25. mu.L.

The invention also provides a rapid detection method of candida in female genital secretion, which is based on the PCR amplification primer and the LNA-Taqman probe set or utilizes the reagent/kit for detection.

In some embodiments, after extracting nucleic acid from the sample, the sample is amplified on a fluorescence quantitative PCR instrument based on the nucleic acid amplification reagent and the PCR amplification primer/LNA-Taqman probe, and whether Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei are contained is judged according to an S-shaped curve of a real-time fluorescence amplification curve.

Further, the amplification system comprises: 5 mul of sample nucleic acid, 10 mul of the nucleic acid amplification reagent, 0.3mmol/L of Candida albicans PCR amplification primer and LNA-Taqman probe, 0.3 mul of each, and 25 mul of water.

The amplification conditions may be:

it can be understood that in the rapid detection method of candida, the detected candida can be from the environment in or outside the body; preferably, the external environment includes candida species in various external environments or habitats, such as candida albicans, candida glabrata, candida tropicalis, candida parapsilosis, and candida krusei; the organism can be originated from various tissues in the body, preferably, secretion of female genital tract, and the like, such as candida albicans, candida glabrata, candida tropicalis, candida parapsilosis and candida krusei.

The invention also provides application of the PCR amplification primer and the LNA-Taqman probe set in rapid detection of candida or preparation of a rapid detection kit of the candida.

Preferably, the candida species mentioned above are candida species in female genital secretions.

More preferably, the female genital tract secretions are from a vaginal swab.

Compared with the prior art, the invention has at least the following advantages:

(1) the novel LNA-Taqman-multiplex fluorescence PCR technology provided by the invention is applied to rapid detection of candida for the first time, and is prepared into a corresponding rapid detection kit.

(2) According to the invention, a set of detection system suitable for common candida is obtained through sequence and combined screening optimization of primers/probes, five kinds of common candida in female reproductive tract are detected based on the system, and a fluorescent quantitative PCR is combined, so that a multiple amplification result can be distinguished in real time, a complex traditional detection means is avoided, and the detection efficiency is improved.

(3) The detection sensitivity of the LNA-Taqman multiplex fluorescence PCR amplification primer and the LNA-Taqman probe qualitative test adopted by the invention can reach 10 copy/mu L (DNA concentration), and the lower detection limit is 10 copies per reaction system.

(4) The primer/probe system can realize the multiplex detection of candida albicans, candida glabrata, candida tropicalis, candida parapsilosis and candida krusei, and the detection result is highly specific.

(5) The invention has simple operation and is suitable for industrial popularization and use.

Drawings

FIG. 1 shows the results of the CAL PCR amplification primers and LNA-Taqman probe amplification of 1000 copy/. mu.l Candida albicans nucleic acid genomic DNA;

FIG. 2 shows the results of PCR amplification primers of Cgla group and LNA-Taqman probe amplifying 1000 copy/. mu.l Candida glabrata nucleic acid genome DNA;

FIG. 3 shows the results of PCR amplification primers of Ctro group and LNA-Taqman probe amplifying 1000 copy/. mu.l Candida tropicalis nucleic acid genome DNA;

FIG. 4 shows the results of amplification of 1000 copy/. mu.l Candida parapsilosis nucleic acid genomic DNA with Cpa group PCR amplification primers and LNA-Taqman probe;

FIG. 5 shows the results of PCR amplification primers in Ckru group and LNA-Taqman probe amplifying 1000 copy/. mu.l Candida krusei nucleic acid genomic DNA;

FIG. 6 shows the results of the CAL 2PCR amplification primer set and LNA-Taqman probe set after amplifying 1000 copy/. mu.l Candida albicans nucleic acid genomic DNA;

FIG. 7 shows the results of amplification of 1000 copy/. mu.l Candida glabrata genomic DNA with Cgla2 set of PCR amplification primers and LNA-Taqman probe;

FIG. 8 shows the results of PCR amplification primers of Ctro2 set and LNA-Taqman probe amplifying 1000 copy/. mu.l Candida tropicalis nucleic acid genome DNA;

FIG. 9 shows the results of PCR amplification primers of Cpa2 set and LNA-Taqman probe amplifying 1000 copy/. mu.l Candida parapsilosis nucleic acid genomic DNA;

FIG. 10 shows the results of PCR amplification primers of Ckru2 set and LNA-Taqman probe amplifying 1000 copy/. mu.l of Candida krusei nucleic acid genomic DNA;

FIG. 11 shows the results of the CAL3 set of PCR amplification primers and LNA-Taqman probe amplifying 1000 copy/. mu.l Candida albicans nucleic acid genomic DNA;

FIG. 12 shows the results of amplification of 1000 copy/. mu.l Candida glabrata genomic DNA with Cgla3 set of PCR amplification primers and LNA-Taqman probe;

FIG. 13 shows the results of PCR amplification primers of Ctro3 set and LNA-Taqman probe amplifying 1000 copy/. mu.l Candida tropicalis nucleic acid genome DNA;

FIG. 14 shows the results of amplification of 1000 copy/. mu.l Candida parapsilosis nucleic acid genomic DNA using the Cpa3 set of PCR amplification primers and LNA-Taqman probe;

FIG. 15 shows the results of PCR amplification primers of Ckru3 set and LNA-Taqman probe amplifying 1000 copy/. mu.l of Candida krusei nucleic acid genomic DNA;

FIG. 16 shows the amplification results of the Cal-P + group PCR amplification primers and LNA-Taqman probe;

FIG. 17 shows the amplification results of the Cal-P + 2PCR amplification primers and LNA-Taqman probe;

FIG. 18 shows the amplification results of Cgla-P + group PCR amplification primers and LNA-Taqman probe;

FIG. 19 shows the amplification results of Cgla-P + 2PCR amplification primers and LNA-Taqman probe;

FIG. 20 shows the amplification results of the PCR amplification primers and LNA-Taqman probe in the Ctro-P + group;

FIG. 21 shows the amplification results of Ctro-P + 2PCR amplification primers and LNA-Taqman probe;

FIG. 22 shows the amplification results of the Cpa-P + group PCR amplification primers and the LNA-Taqman probe;

FIG. 23 shows the amplification results of the Cpa-P + 2PCR amplification primers and the LNA-Taqman probe;

FIG. 24 shows the amplification results of the PCR amplification primers and LNA-Taqman probe in the Ckru-P + group;

FIG. 25 shows the amplification results of the Ckru-P + 2PCR amplification primers and LNA-Taqman probe;

FIG. 26 shows the results of PCR amplification primers and LNA-Taqman probe determined in example 1 after amplifying a mixture of Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei genomic DNA at 1000 copy/. mu.l, 100 copy/. mu.l and 10 copy/. mu.l.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

Definition of

Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.

The terms "about" and "substantially" in the present invention denote an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.

EXAMPLE 1 design and screening of primer/Probe System

1. Preparation of nucleic acid amplification reagents

The DNA polymerase includes: 1 μ L of 5U/. mu.L DNA polymerase;

the buffer solution comprises: 2 μ L of 1M KCl; 0.5 μ L of 0.4M MgCl 2; 1 μ L of 10% tween 20; 4 μ L of 25mM dNTPs; 1.5 μ L of 0.2M Tris-HCl (pH 8.0).

2. Design of PCR amplification primer and Taqman probe

According to the invention, firstly, the sequence segments of the determined intraspecific and interspecific conservation sequence are screened according to the standard sequences of Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei recorded on GenBank, and are specifically shown as SEQ ID NO. 1-5; and then, manually designing a PCR amplification primer and a Taqman probe sequence according to the determined sequence section, and comparing and analyzing. All primer sequences were synthesized by the firm Biotech, China.

Specifically, the method comprises the following steps: designing PCR amplification primers and Taqman probes for detecting Candida albicans ITS1-5.8S-ITS2 gene aiming at a target sequence SEQ ID NO. 1; designing PCR amplification primers and Taqman probes for detecting Candida glabrata ITS1-5.8S-ITS2 genes aiming at a target sequence SEQ ID NO. 2; design and detection of candida tropicalis aiming at target sequence SEQ ID NO.3

PCR amplification primers and Taqman probes of ITS1-5.8S-ITS2 gene; designing PCR amplification primers and Taqman probes for detecting Candida parapsilosis ITS1-5.8S-ITS2 genes aiming at a target sequence SEQ ID NO. 4; designing PCR amplification primers and Taqman probes for detecting Candida krusei ITS1-5.8S-ITS2 genes aiming at a target sequence SEQ ID NO. 5; the nucleotide sequences of the primer sets preliminarily designed are shown in Table 1.

TABLE 1 PCR amplification primer and Taqman probe for detecting female genital secretion candida

In table 1, numbers 1, 2 and 11 constitute a primer set, abbreviated as Cal set, numbers 3, 4 and 11 constitute a primer set, abbreviated as Cgla set, numbers 5, 6 and 11 constitute a primer set, abbreviated as Ctro set, numbers 7, 8 and 11 constitute a primer set, abbreviated as Cpa set, numbers 9, 10 and 11 constitute a primer set, abbreviated as Ckru set. In table 1, numbers 12, 13, and 11 constitute a primer set, abbreviated as Cal2, numbers 14, 15, and 11 constitute a primer set, abbreviated as Cgla2, numbers 16, 17, and 11 constitute a primer set, abbreviated as Ctro2, numbers 18, 19, and 11 constitute a primer set, abbreviated as Cpa2, numbers 20, 21, and 11 constitute a primer set, abbreviated as Ckru2, numbers 22, 23, and 11 constitute a primer set, abbreviated as Cal3, numbers 24, 25, and 11 constitute a primer set, abbreviated as Cgla3, numbers 26, 27, and 11 constitute a primer set, abbreviated as Ctro3, numbers 28, 29, and 11 constitute a primer set, abbreviated as Cpa3, numbers 30, 31, and 11 constitute a primer set, and abbreviated as Ckru 3.

3. PCR amplification primer and screening of Taqman probe

The PCR amplification primers and LNA-Taqman probes corresponding to each other were screened using genomic DNAs of Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei, respectively, at a nucleic acid concentration of 1000copy/μ l under the reaction conditions:

as shown in fig. 1, fig. 6 and fig. 11, when PCR amplification detection is performed on the Cal group, the Cal2 group and the Cal3 group of candida albicans, the peak time of the Cal group is fastest, that is, the Cal group detects candida albicans most quickly and has the most excellent performance. As shown in fig. 2, 7 and 12, PCR amplification tests were performed on the Cgla group, Cgla2 group and Cgla3 of candida glabrata, and the peak time of the Cgla group was the fastest. As shown in FIG. 3, FIG. 8 and FIG. 13, when PCR amplification detection is performed on Ctro group, Ctro2 group and Ctro3 group of Candida tropicalis, the Ctro group has the fastest peak emergence time. As shown in fig. 4, 9 and 14, PCR amplification tests were performed on Cpa group, Cpa2 group and Cpa3 group of candida parapsilosis, and Cpa group showed the fastest peak time. As shown in fig. 5, 10 and 15, PCR amplification tests were performed on the Ckru group, Ckru2 group and Ckru3 group of candida krusei, and the Ckru group showed the fastest peak time.

Therefore, Cal group, Cgla group, Ctro group, Cpa group and Ckru group were selected as candidates for the detection of Candida as a female genital secretion.

4. Design and screening of LNA-Taqman probe

In this embodiment, based on the Taqman probes in the Cal group, the Cgla group, the Ctro group, the Cpa group, and the Ckru group, various LNA modifications are performed on the selected probes to make the selected probes become LNA-Taqman probes, with the purpose of improving the conservation and specificity of the Taqman probes. And comparing the effects of the designed LNA-Taqman probes, and comparing and analyzing the designed LNA-Taqman probes with Taqman probes which are not modified by LNA. For reasons of space, only the partial sequence is listed as an example, such as: the nucleotide sequences of the part of LNA-Taqman probes preliminarily designed at the previous stage are shown in Table 2.

TABLE 2 LNA-Taqman probes for detection of Candida female genital secretion

(Note: N stands for 4 kinds of DNA nucleotides A, T, C, G, + N stands for locked nucleic acid modification)

In the above table 2, the PCR amplification primers for candida albicans, No. 32 and the above screening, constitute a detection group; 33 and the screened candida albicans PCR amplification primers form a detection group; 34 and the screened candida glabrata PCR amplification primers form a detection group; 35 and the screened candida glabrata PCR amplification primers form a detection group; 36 and the PCR amplification primers of the screened candida tropicalis form a detection group; 37 and the PCR amplification primers of the screened candida tropicalis form a detection group; 38 and the PCR amplification primers of the screened candida parapsilosis form a detection group; 39 and the PCR amplification primers of the screened candida parapsilosis form a detection group; 40 and the PCR amplification primers of the screened candida krusei form a detection group; 41 and the PCR amplification primers of the screened candida krusei constitute a detection group. The PCR amplification detection comparison results are as follows:

from the results, the Taqman probe modified by LNA obviously further improves the peak time, the detection results of the P + group are obviously superior to those of the P +2 group, and finally the primer/probe combination disclosed by the invention is established as follows.

Example 2 Performance verification of PCR amplification primers and LNA-Taqman probes

1. Specificity verification

By utilizing the Cal group primer probe combination, the reaction system and the detection method established in the embodiment 1, LNA-Taqman multiple fluorescence PCR detection is carried out on 1 strain of Candida albicans genome DNA and 4 other Candida species; by utilizing the Cgla group primer probe combination, the reaction system and the detection method determined in the embodiment 1, LNA-Taqman multiple fluorescence PCR detection is carried out on 1 strain Candida glabrata genome DNA and 4 other Candida species; by utilizing the Ctro group primer probe combination, the reaction system and the detection method obtained in the embodiment 1, LNA-Taqman multiplex fluorescence PCR detection is carried out on 1 strain of Candida tropicalis genome DNA and 4 other Candida species; by utilizing the Cpa group primer probe combination, the reaction system and the detection method obtained in the embodiment 1, LNA-Taqman multiplex fluorescence PCR detection is carried out on 1 Nematoda endive genome DNA and 4 other Candida species; by using the Ckru group primer probe combination, the reaction system and the detection method obtained in the embodiment 1, LNA-Taqman multiplex fluorescence PCR detection is carried out on 1 strain of Candida krusei genomic DNA and 4 other Candida species; the detection results are shown in table 3, no cross problem exists, and the specificity is obvious.

TABLE 3 test strains and LNA-Taqman multiplex fluorescence PCR amplification test results

Note: CGMCC is China general microbiological culture Collection center.

2. Sensitivity verification

Respectively inoculating Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei to a YBD liquid culture medium, normally culturing at 37 ℃ for 16h, taking 1mL of the cultured bacterial liquid, and extracting the genome DNA of the Candida according to a conventional method. The concentration and purity of Candida genome DNA were determined using a NanoDrop 2000C ultramicro spectrophotometer, and the copy number was calculated. Mixing and diluting genome nucleic acid DNA templates of candida albicans, candida glabrata, candida tropicalis, candida parapsilosis and candida krusei to copy number final concentrations respectively as follows: a: 100 copy/. mu.l, b:10 copy/. mu.l, c:1 copy/. mu.l. And finally, the DNA template is determined by using the LNA-Taqman multiplex fluorescence PCR amplification primer and the LNA-Taqman probe detection method.

When the copy number of the genomic nucleic acid DNA containing the five candida is 10 copy/microliter, the positive reaction result of detecting the candida albicans has the interpretation time less than 29 Cycles; the positive reaction result of the candida glabrata detection has the interpretation time less than 31 Cycles; the positive reaction result of the candida tropicalis is detected, and the interpretation time is less than 27.9 Cycles; the positive reaction result of the candida parapsilosis detection has interpretation time less than 34 Cycles; the positive reaction result of the candida krusei is detected, and the interpretation time is less than 27.6 Cycles; the results are shown in FIG. 26.

Therefore, the detection sensitivity of the LNA-Taqman multiplex fluorescence PCR amplification primer and the LNA-Taqman probe qualitative test adopted by the invention can reach 10 copy/mu L (DNA concentration), namely the lower detection limit is 10 copies per reaction system, and the method has obvious advantages.

In conclusion, the PCR amplification primers and the LNA-Taman probe for detecting Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei provided by the invention have high sensitivity and strong specificity, and can be used for rapidly and accurately detecting the existence of Candida in female genital secretion.

Example 3 clinical test and comparison of different test methods

1. Collection of clinical specimens and preparation of nucleic acid genomic DNA of clinical specimens

Genital secretion samples (i.e. vaginal swabs) were collected from 993 women at 16 weeks gestation, provided by the Beijing women hospital. DNA extraction of clinical specimens: extraction of genomic DNA from clinical specimens was carried out using a magnetic bead method universal genomic DNA extraction kit (catalog No. DP705) from Tiangen corporation. The operation is carried out with particular reference to the step H in the description. And (3) determining the concentration and purity of the candida genomic DNA by using a NanoDrop 2000C ultramicro spectrophotometer after extraction of the clinical sample genomic DNA, and storing the candida genomic DNA in a refrigerator at the temperature of-20 ℃ for later use.

2. Detection and identification of clinical samples by LNA-TaqMan multiplex fluorescence PCR

Preparing and amplifying conditions of the LNA-Taqman multiplex fluorescence PCR reaction system: according to the system preparation concentration in the example 1, a reaction system for detecting candida in female genital secretion is prepared, and the clinical samples are subjected to multiplex amplification detection according to the reaction conditions in the example 1, and the detection results are shown in table 3.

3. Control method-detection and identification of clinical samples by phenotypic biochemical method

Strain isolation culture: inoculating the specimen into a fungus plate and a blood plate, culturing in an incubator (28-35 ℃) for 24-48h, picking out a suspicious colony smear, observing under a mirror after gram staining to suspected yeast-like fungus, and separating and purifying by using the blood plate or the fungus plate.

Identification of candida: after single candida species are separated, a color development culture medium (Haibozbiol, China) of the kema candida species according to an enzyme substrate method is adopted, and different candida species are distinguished through color development after the culture is carried out for 48 hours at 25-28 ℃, such as: candida albicans develops blue-green, Candida tropicalis develops blue-grey or iron-blue, Candida glabrata develops purple, Candida krusei develops pink, other Candida albicans develops white, and the bacteria are inhibited. The results are shown in Table 4.

4. Control method-clinical sample assay identification based on ITS sequencing analysis

Extraction of genomic DNA from clinical specimens was carried out using a magnetic bead method universal genomic DNA extraction kit (catalog No. DP705) from Tiangen corporation. And then amplifying the clinical sample by using ITS universal PCR amplification primers, completing the sequence determination of PCR amplification products by Shanghai biological engineering technology service, and completing the analysis of sequence results by a BLAST tool of an NCBI website according to sequencing results. The results are shown in Table 4.

Wherein the ITS general PCR amplification primer sequence is as follows:

forward PCR amplification primers: ITS3(5'-GCATATCAATAAGCGGAGGA-3')

Reverse PCR amplification primers: ITS4(5'-GCATCGATGAAGAACGCAGC-3')

TABLE 4.3 comparison of the results of the identification of the detection methods

5. Statistical analysis of clinical sample test results

The test results of the clinical samples were statistically analyzed based on chi-square test statistics of statistical analysis software "SPSS 22.0", and the differences of P <0.005 were statistically significant.

After 993 secretion samples of the genital tract of women with 16 weeks of gestation were analyzed by the ITS sequencing method, LNA-TaqMan fluorescent multiplex PCR method and the traditional phenotypic biochemical method, Candida colonization was found in 228 clinical samples, as follows (see Table 3): 1) 183 cases of Candida albicans, 30 cases of Candida glabrata, 2 cases of Candida tropicalis, 3 cases of Candida parapsilosis, and 5 cases of Candida krusei; 2) complex infection: candida albicans + Candida glabrata in case 3, Candida albicans + Candida tropicalis in case 1, and Candida albicans + Candida parapsilosis in case 1.

The ITS sequence determination result is used as a gold standard, the accuracy of 993 clinical samples is identified by statistical analysis of other 2 methods, the LNA-TaqMan multiplex fluorescence PCR method is 98.89%, the phenotype biochemical method is 90.13%, and the accuracy difference of the 2 detection methods has no statistical significance (Chi)²0.38, P > 0.05); the identification accuracy of 183 Candida albicans colonization samples by 2 methods is as follows: the LNA-TaqMan multiplex fluorescence PCR method is 93.99%, the phenotypic biochemical method is 62.30%, the difference between the LNA-TaqMan multiplex fluorescence PCR method and the phenotypic biochemical method has statistical significance (x 2 is 53.81, P is less than 0.01; the identification accuracy of various Candida species except Candida albicans: the LNA-TaqMan multiplex fluorescence PCR method is 100%, the phenotypic biochemical method is 35.56%, and the difference has statistical significance compared with 2 detection methods (x 2 is)²42.79, P <0.0 (see table 5).

TABLE 5.3 comparison of the accuracy of the detection methods

Comparing the results of LNA-TaqMan multiplex fluorescence PCR and conventional phenotypic biochemical methods, the positive match rate was 12.59% (125 cases), the negative match rate was 77.04% (765 cases), and the mismatch rate was 10.37% (103 cases) (see Table 6).

TABLE 6 comparison of the coincidence of LNA-TaqMan multiplex fluorescent PCR method with phenotypic Biochemical method

The results of the 103 inconsistent identifications were compared to those obtained by the ITS sequence assay as a gold standard (see tables 7 and 8): 78 samples negative to candida infection detected by a phenotypic biochemical method actually comprise 60 samples infected by candida albicans and 8 samples infected by non-candida albicans; the LNA-TaqMan multiplex fluorescence PCR method is used for detecting 11 samples negative to candida infection, and actually comprises 11 samples infected by candida albicans; the identification result of the LNA-TaqMan multiplex fluorescence PCR method is consistent with the result obtained by the ITS sequencing method. Therefore, the LNA-TaqMan multiplex fluorescence PCR method provided by the invention has high accuracy, and compared with a gold standard method, the LNA-TaqMan multiplex fluorescence PCR method is simpler and faster and has the capability of real-time multiplex specificity judgment. Although the phenotype biochemical method is used as a common detection means in clinic, the subjectivity of the identification result is too strong due to higher requirements of phenotype judgment on operators, erroneous judgment or erroneous judgment is easy to occur when the real result is judged, the detection time of the phenotype biochemical method is too long, the sensitivity is not high, the accuracy of the phenotype biochemical method in the neighborhood on the disease of a patient is limited, and the treatment of the patient is delayed finally.

TABLE 7 analysis of results of non-concordant identification of LNA-TaqMan multiplex fluorescent PCR and phenotypic Biochemical methods

TABLE 8 analysis of results of non-concordant identification of LNA-TaqMan multiplex fluorescent PCR method and phenotypic Biochemical method

78 cases positive in PCR	Number of examples	78 cases negative to phenotype Biochemical method
			Candida albicans	60	-
Candida tropicalis	1	-
			Candida glabrata	9	-
Candida parapsilosis	3	-
			Candida krusei	5	-
11 cases negative to PCR	Number of examples	11 cases positive for phenotypic biochemistry
			-	5	Candida albicans
-	2	Candida parapsilosis
			-	2	Candida krusei
-	1	Candida glabrata
			-	1	Can not determine

。

The above description of the specific embodiments of the present application is not intended to limit the present application, and those skilled in the art may make various changes and modifications according to the present application without departing from the spirit of the present application, which is intended to fall within the scope of the appended claims.

Sequence listing

<110> Zhongzhong fast and excellent detection Biotechnology (Jiangsu) Co., Ltd

<120> LNA-Taqman-multiplex fluorescence PCR technology and application thereof in rapid detection of candida

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 487

<212> DNA

<213> Candida albicans (Candida albicans)

<400> 1

ctgatttgct taattgcacc acatgtgttt ttctttgaaa caaacttgct ttggcggtgg 60

gcccagcctg ccgccagagg tctaaactta caaccaattt tttatcaact tgtcacacca 120

gattattact taatagtcaa aactttcaac aacggatctc ttggttctcg catcgatgaa 180

gaacgcagcg aaatgcgata cgtaatatga attgcagata ttcgtgaatc atcgaatctt 240

tgaacgcaca ttgcgccctc tggtattccg gagggcatgc ctgtttgagc gtcgtttctc 300

cctcaaaccg ctgggtttgg tgttgagcaa tacgacttgg gtttgcttga aagacggtag 360

tggtaaggcg ggatcgcttt gacaatggct taggtctaac caaaaacatt gcttgcggcg 420

gtaacgtcca ccacgtatat cttcaaactt tgacctcaaa tcaggtagga ctacccgctg 480

aacttaa 487

<210> 2

<211> 707

<212> DNA

<213> Candida glabrata (Candida glabrata)

<400> 2

taagtgcgcg gttggtgggt gttctgcagt ggggggaggg agccgacaaa gacctgggag 60

tgtgcgtgga tctctctatt ccaaaggagg tgttttatca cacgactcga cactttctaa 120

ttactacaca cagtggagtt tactttacta ctattctttt gttcgttggg ggaacgctct 180

ctttcggggg ggagttctcc cagtggatgc aaacacaaac aaatattttt ttaaactaat 240

tcagtcaaca caagatttct tttagtagaa aacaacttca aaactttcaa caatggatct 300

cttggttctc gcatcgatga agaacgcagc gaaatgcgat acgtaatgtg aattgcagaa 360

ttccgtgaat catcgaatct ttgaacgcac attgcgccct ctggtattcc ggggggcatg 420

cctgtttgag cgtcatttcc ttctcaaaca cattgtgttt ggtagtgagt gatactcgtt 480

tttgagttaa cttgaaattg taggccatat cagtatgtgg gacacgagcg caagcttctc 540

tattaatctg ctgctcgttt gcgcgagcgg cgggggttaa tactgtatta ggttttacca 600

actcggtgtt gatctaggga gggataagtg agtgttttgt gcgtgctggg cagacagacg 660

tctttaagtt tgacctcaaa tcaggtaggg ttacccgctg aacttaa 707

<210> 3

<211> 493

<212> DNA

<213> Candida tropicalis (Candida tropicalis)

<400> 3

aaggatcatt actgatttgc ttaattgcac cacatgtgtt ttttattgaa caaatttctt 60

tggtggcggg agcaatccta ccgccagagg ttataactaa accaaacttt ttatttacag 120

tcaaacttga tttattatta caatagtcaa aactttcaac aacggatctc ttggttctcg 180

catcgatgaa gaacgcagcg aaatgcgata cgtaatatga attgcagata ttcgtgaatc 240

atcgaatctt tgaacgcaca ttgcgccctt tggtattcca aagggcatgc ctgtttgagc 300

gtcatttctc cctcaaaccc ccgggtttgg tgttgagcaa tacgctaggt ttgtttgaaa 360

gaatttaacg tggaaactta ttttaagcga cttaggttta tccaaaaacg cttattttgc 420

tagtggccac cacaatttat ttcataactt tgacctcaaa tcaggtagga ctacccgctg 480

aacttaagca tat 493

<210> 4

<211> 521

<212> DNA

<213> Candida parapsilosis

<400> 4

tccgtaggtg aacctgcgga aggatcatta cagaatgaaa agtgcttaac tgcatttttt 60

cttacacatg tgtttttctt tttttgaaaa ctttgctttg gtaggccttc tatatggggc 120

ctgccagaga ttaaactcaa ccaaatttta tttaatgtca accgattatt taatagtcaa 180

aactttcaac aacggatctc ttggttctcg catcgatgaa gaacgcagcg aaatgcgata 240

agtaatatga attgcagata ttcgtgaatc atcgaatctt tgaacgcaca ttgcgccctt 300

tggtattcca aagggcatgc ctgtttgagc gtcatttctc cctcaaaccc tcgggtttgg 360

tgttgagcga tacgctgggt ttgcttgaaa gaaaggcgga gtataaacta atggataggt 420

tttttccact cattggtaca aactccaaaa cttcttccaa attcgacctc aaatcaggta 480

ggactacccg ctgaacttaa gcatatcatt aagcggagga a 521

<210> 5

<211> 470

<212> DNA

<213> Candida Krusei (Candida)

<400> 5

aggatcatta ctgtgattta gtactacact gcgtgagcgg aacgaaaaca acaacaccta 60

aaatgtggaa tatagcatat agtcgacaag agaaatctac gaaaaacaaa caaaactttc 120

aacaacggat ctcttggttc tcgcatcgat gaagagcgca gcgaaatgcg atacctagtg 180

tgaattgcag ccatcgtgaa tcatcgagtt cttgaacgca cattgcgccc ctcggcattc 240

cggggggcat gcctgtttga gcgtcgtttc catcttgcgc gtgcgcagag ttgggggagc 300

ggagcggacg acgtgtaaag agcgtcggag ctgcgactcg cctgaaaggg agcgaagctg 360

gccgagcgaa ctagactttt tttcagggac gcttggcggc cgagagcgag tgttgcgaga 420

caacaaaaag ctcgacctca aatcaggtag gaatacccgc tgaacttaag 470

Claims

1. A PCR amplification primer and LNA-Taqman probe set, comprising: a primer of SEQ ID NO.1 and LNA-Taqman probe for Candida albicans, a primer of SEQ ID NO.2 and LNA-Taqman probe for Candida glabrata, a primer of SEQ ID NO.3 and LNA-Taqman probe for Candida tropicalis, a primer of SEQ ID NO.4 and LNA-Taqman probe for Candida parapsilosis, a primer of SEQ ID NO.5 and LNA-Taqman probe for Candida krusei, a reverse universal primer;

the sequences of the PCR amplification primer and the LNA-Taqman probe are as follows:

wherein, + represents a locked nucleic acid modification at the next base adjacent to "+".

2. A composition comprising the PCR amplification primers of claim 1 and a LNA-Taqman probe set.

3. A candida detection kit comprising the PCR amplification primer of claim 1 and a LNA-Taqman probe set.

4. The candida detection kit of claim 3, further comprising nucleic acid amplification reagents comprising a DNA polymerase and a PCR buffer.

5. The candida detection kit of claim 4, wherein the nucleic acid amplification reagent comprises water and the following final concentration components:

the kit also comprises a positive control and a negative control, wherein the positive control is a nucleic acid DNA equal ratio mixture of Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei.

6. The use of the PCR amplification primers and LNA-Taqman probe sets of claim 1 in the preparation of a detection kit for Candida.

7. The use according to claim 6, wherein said Candida is Candida in female genital secretion.

8. The use of claim 7, wherein the female genital tract secretions are from a vaginal swab.