CN118127195A - Mycoplasma genitalium nucleic acid detection kit - Google Patents

Abstract

The invention discloses a genital mycoplasma nucleic acid detection kit, which comprises: amplification primers of mycoplasma genitalium, amplification primers of internal reference genes, specific probes CES, specific probes LES, internal reference specific probes CES, internal reference specific probes LES, C-line chromogenic probes, gold probes and test strips. The invention adopts the method of isothermal amplification of pathogen RNA and combines gold probe chromatography, and the detection of the genital mycoplasma nucleic acid has the advantages of high sensitivity, strong specificity and simple operation, has low technical requirements on experimental staff, does not need special instruments and equipment, and is easy to popularize the genital mycoplasma nucleic acid detection to basic layers and remote rural medical institutions.

Description

Mycoplasma genitalium nucleic acid detection kit

Technical Field

The invention relates to the technical field of pathogen detection, in particular to a mycoplasma genitalium nucleic acid detection kit.

Background

Mycoplasma genitalium (Mycoplasrna genitalium, MG) belongs to the order Mycoplasma mollis, and is the 12 th Mycoplasma species found in humans. The mycoplasma genitalium mainly hosts on epithelial cells of human genitourinary tract, which adhere to the surface of host cells, absorb nutrition from the cells, release metabolic products harmful to the cells, cause damage to cell membranes, cause abnormal chromosomes of the epithelial cells, affect normal metabolism of the cells, and even cause necrosis of the cells. In recent years, researchers have conducted intensive studies on the pathogenicity of mycoplasma genitalium, confirming the important role of mycoplasma genitalium in genital tract diseases. The research shows that MG is one of the main pathogens causing nongonococcal urethritis and can cause cervicitis, pelvic inflammatory disease, prostatitis, epididymitis, arthritis, infertility of fallopian tubes and other symptoms. Early discovery and effective treatment of MG infection is therefore critical for preventing and controlling mycoplasma genitalium transmission.

At present, three methods for detecting mycoplasma genitalium are mainly adopted, namely a pathogen culture method, a serological method and a molecular biological method. Isolated culture of MG pathogens is the most traditional detection method, but the time required for identifying MG by using a culture method is too long (more than 50 days), and MG nutrition conditions are harsh, so that the isolated culture of MG pathogens is not easy to culture, and therefore, the isolated culture is difficult to apply to clinical diagnosis of MG. The diagnosis standard and time for detecting the MG antibody by the serum method are difficult to grasp, are difficult to detect at the early stage of infection, and have low sensitivity. Furthermore, the serological characteristics of MG and mycoplasma pneumoniae are widely crossed, and the specificity cannot be ensured. The current molecular biological method for MG detection is mainly a fluorescent quantitative PCR method, and has the advantages of simple operation and strong specificity. However, the PCR method requires expensive fluorescent probes and detection instruments with low matching value, has high detection cost, has high operation technical requirements on experimenters, is easily influenced by various limiting factors, such as single detection of one pathogen, and has the advantages of time and labor consumption, high detection cost and low PCR detection efficiency when detecting various pathogens; amplification product DNA contamination can lead to false positives in detection results, DNA contamination is very stable and difficult to eliminate, and so forth.

Therefore, it is necessary to continue to find a convenient and low cost diagnostic method for MG pathogens.

Disclosure of Invention

The invention aims to provide a Mycoplasma genitalium nucleic acid detection kit, which realizes the efficient detection of Mycoplasma genitalium nucleic acid by introducing a strategy of combining RNA isothermal amplification with gold probe chromatography. The method does not need high-end complex instruments and equipment, only needs ordinary laboratory conditions, and can be completed even by a simple method only by constant-temperature water bath. The method has the advantages of high sensitivity, strong specificity, simple and convenient operation and easy popularization and promotion. Meanwhile, as the amplified product is RNA molecule, the natural degradation speed is high, the environmental pollution is not easy to cause, and the wide application of detecting the mycoplasma nucleic acid is possible.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the invention provides a genital mycoplasma nucleic acid detection kit, which comprises:

An amplification primer for mycoplasma genitalium, comprising: MG-F primer shown as SEQ ID NO.1 and MG-R primer shown as SEQ ID NO. 2;

An amplification primer for a reference gene, comprising: an internal reference-F primer shown as SEQ ID NO.3 and an internal reference-R primer shown as SEQ ID NO. 4;

a specific probe CES, wherein the nucleotide sequence is at least one of SEQ ID NO.5 or SEQ ID NO. 6;

Specific probe LES, nucleotide sequence is selected from at least one of SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO. 9;

The internal reference specific probe CES has a nucleotide sequence selected from at least one of SEQ ID NO.10 or SEQ ID NO. 11;

the reference specific probe LES has a nucleotide sequence selected from at least one of SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO. 14;

the nucleotide sequence of the line C chromogenic probe is shown as SEQ ID NO. 15;

The nucleotide sequence of the gold probe is shown as SEQ ID NO. 16; and the 5' end of the gold probe is modified by sulfhydrylation;

The test strip comprises a bottom plate, a sample pad, an NC film and absorbent paper, wherein the sample pad is adhered to the bottom plate and sequentially overlapped, 3 detection lines are sequentially arranged on the NC film according to the chromatography direction, the detection lines are respectively an MG-T line, an internal reference-N line and a quality control-C line, the MG-T line is coated with an MG coating probe with a nucleotide sequence shown as SEQ ID NO.17, the internal reference-N line is coated with an internal reference coating probe with a nucleotide sequence shown as SEQ ID NO.18, and the quality control-C line is coated with a quality control coating probe with a nucleotide sequence shown as SEQ ID NO. 19.

Further, the kit further comprises an amplification reaction solution: tris-HCl (pH 8.0), mgCl ₂, KCl, DMSO, DTT, dNTPs, NTPs.

Further, the kit also comprises a negative control.

Further, the kit further comprises a cell lysate.

Further, the kit further comprises an RNA isothermal amplicon enzyme: reverse transcriptase, RNaseH, T7 RNA polymerase.

Further, the concentration of the MG-T line coated MG coating probe is 10+/-2 mu M, and the spraying amount is 2-3 mu L/cm.

Further, the concentration of the ginseng coating probe coated at the internal reference-N line is 10+/-2 mu M, and the film spraying amount is 2-3 mu L/cm.

Further, the concentration of the quality control coating probe coated at the quality control-C line is 10+/-2 mu M, and the film spraying amount is as follows: 2-3 mu L/cm.

One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:

1. The invention realizes the capability of amplifying two indexes of MG and internal reference genes simultaneously in the same tube by RNA isothermal amplification technology. The method not only improves the detection efficiency, but also ensures the stability and reliability of the amplified product. The constant-temperature amplification of RNA is carried out in an environment of 42 ℃, so that an experimental instrument is simplified to a constant-temperature water bath, the requirement on experimental equipment is greatly reduced, and the feasibility and the popularity of the experiment are improved. In addition, the amplification primers are designed and subjected to multiple rounds of tests and verification, so that each single primer has high amplification efficiency and no interference between the single primers.

2. The kit for detecting the genital mycoplasma nucleic acid has the advantages of strong specificity and high sensitivity:

In the invention, the specific probes CES and LES series which are carefully designed play a key role in the detection process. The method successfully combines the coating probe, the amplified product RNA and the gold probe in series through the action of a molecular bridge, thereby realizing the specific detection of the target RNA nucleic acid fragment. It is the design of the two sets of probes, so that any set of probes and the index nucleic acid amplified fragment cannot be successfully immobilized on the NC membrane due to hybridization failure, and positive detection results cannot occur, so that the detection specificity is ensured. More than two probes can be designed for each set of probes, and the design is beneficial to improving the sensitivity of the test strip.

3. The invention combines the advantages of the RNA isothermal amplification technology and the test strip chromatography technology, not only retains the advantage of low requirement on instruments by RNA isothermal amplification, but also plays the advantage of rapid detection of colloidal gold. The result of detecting nucleic acid by the test strip can be interpreted within about 5 minutes, the operation is simple and convenient, the technical requirement on experimental staff is low, special instruments and equipment are not needed, and the test strip is very suitable for popularization and use in basic level and remote rural medical institutions.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of isothermal amplification of RNA;

FIG. 2 is a schematic diagram of the color development of the test strip;

FIG. 3 is a schematic diagram of a test strip; 1. a bottom plate, 2, a sample pad, 3, NC films, 4 and absorbent paper;

FIG. 4 shows the detection of negative and positive conditions.

Detailed Description

The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.

Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present invention are commercially available or may be obtained by existing methods. The words "first," "second," and the like herein do not denote a order, and are to be construed as nouns.

The Mycoplasma genitalium nucleic acid detection kit of the present application will be described in detail with reference to examples and experimental data. Example 1 Mycoplasma genitalium nucleic acid kits and methods of use thereof

1. A mycoplasma genitalium nucleic acid kit comprising:

1. Amplification reaction solution: contains 40mM Tris-HCl (pH 8.0), 12mM MgCl ₂, 70mM KCl,15% DMSO,5mM DTT, 1mM each dNTP, 2mM each NTP, and 0.2. Mu.M each amplification primer. Wherein the amplification primers comprise two pairs of: mycoplasma genitalium and human internal reference genes. The method comprises the following steps:

(1) Amplification primers for MG:

MG-F primer (5 '-3'): CTGCAATTCGTCCTCATGAAG (SEQ ID NO. 1);

MG-R primer (5 '-3'): TAATACGACTCACTATAGGGAGACGGTGCTATCCTTGACAT GC (SEQ ID NO. 2);

(2) Amplification primers for internal reference gene (human 18SrRNA sequence of conserved region):

reference-F primer (5 '-3'): AGAAACGGCTACCACATCC (SEQ ID NO. 3);

reference-R primer (5 '-3'): TAATACGACTCACTATAGGGAGACACCAGACTTGCCCTCC A (SEQ ID NO. 4);

2. Amplification enzyme: is prepared from three enzymes including reverse transcriptase (such as AMV or M-MLV), T7 RNA polymerase and RNaseH.

3. Cell lysate: cells are lysed, releasing the nucleic acids.

4. Detection liquid: comprises a nucleic acid probe (gold probe) marked by colloidal gold particles and a specific probe salt solution of each index. Each index specific probe is divided into two types, namely CES series and LES series, wherein the CES series and the LES series can be designed into a plurality of pieces to adapt to hybridization combination with different parts of target fragment nucleic acid, so that the detection of the Mycoplasma genitalium nucleic acid is realized with high efficiency, sensitivity and strong specificity.

The detection kit of the present invention may contain both CES series (i.e., MG-CES1, MG-CES2, reference CES1, reference CES 2) and LES series (i.e., MG-LES1, MG-LES2, MG-LES3, reference LES1, reference LES2, reference LES 3), or may contain one of CES series and LES series. Wherein, in CES series, one or two of MG-CES1 and MG-CES2 are selected, and one or two of internal reference CES1 and internal reference CES2 are selected; one or two or three of MG-LES1, MG-LES2 and MG-LES3 in the LES series can be selected, and one or two or three of the reference LES1, the reference LES2 and the reference LES3 can be selected.

The method comprises the following steps:

(1) Specific probes for MG:

MG-CES1(5’-3’)：GGAATCACTAGTAATttttTATCTATAGCTGGTGT(SEQ ID NO.5)；

MG-CES2(5’-3’)：CGCGAATCAGCTATGTttttTATCTATAGCTGGTGT(SEQ ID NO.6)；

MG-LES1(5’-3’)：CGCGGTGAATACGTTCttttCGCAGTGCTCGAGCTCTGAGC(SE Q ID NO.7)；

MG-LES2(5’-3’)：TCGGGTCTTGTACACAttttCGCAGTGCTCGAGCTCTGAGC(SE Q ID NO.8)；

MG-LES3(5’-3’)：CCGCCCGTCAAACTATttttCGCAGTGCTCGAGCTCTGAGC(SE Q ID NO.9)；

(2) Reference specific probe

Internal reference CES1 (5 '-3'): AAGGAAGGCAGCAGGCTTTTATCTGTATAGTGTCTG (SEQ ID NO. 10);

internal reference CES2 (5 '-3'): GCGCAAATTACCCACTTTTTATCTGTATAGTGTCTG (SEQ ID NO. 11);

Internal reference LES1 (5 '-3'): CCCGACCCGGGGAGGTTTTTCGCAGTGCTCGAGCTCTGAGC (SEQ ID NO. 12);

internal reference LES2 (5 '-3'): AGTGACGAAAAATAACTTTTCGCAGTGCTCGAGCTCTGAGC (SEQ ID NO. 13);

Internal reference LES3 (5 '-3'): AATACAGGACTCTTTCTTTTCCGCAGTGCTCGAGCTCTGAGC (SEQ ID NO. 14);

(3) C line color development probe

TCAGATCACTATGTACttttCGCAGTGCTCGAGCTCTGAGC(SEQ ID NO.15)；

(4) Gold probe

The gold probe is modified by thiolation at the 5' end, and the sequence is (5 ' -3 '):

CCTACTCTGCAGTGCTCCATCGTACGTCTGTCATTTTTGCTCAGAGCTCGAGCACT GCG(SEQ ID NO.16)；

5. Test strip: the test strip is fixed on a PVC bottom plate, and a sample pad, an NC film and absorbent paper are arranged in sequence from bottom to top. Three detection lines on the NC film are MG-T line, internal reference-N line and quality control-C line (figure 3) from the sample pad to the absorbent paper. An MG coating probe is fixed at the MG-T line, an internal reference coating probe is fixed at the internal reference-N line, and a quality control coating probe is fixed at the quality control-C line. The specific sequence is as follows:

MG coating probe: ACACCAGCTATAGATATTTTACACCAGCTATAGATA (SEQ ID NO. 17);

internal reference coated probe: CAGACACTATACAGATTTTTCAGACACTATACAGAT (SEQ ID NO. 18);

C line coating probe: GTACATAGTGATCTGATTTTGTACATAGTGATCTGA (SEQ ID NO. 19);

2. the invention provides an MG nucleic acid detection kit adopting an RNA isothermal amplification-gold probe chromatography method, which comprises the following specific operation steps:

(1) Isothermal amplification of RNA

The invention realizes the amplification of two indexes of MG and the human internal reference gene in the same amplification tube. A pair of (F/R primers) amplification primers was designed for each index, wherein the 5' end of each R primer was introduced with a T7 RNA polymerase promoter sequence. During amplification, under the action of an R primer (SEQ ID NO.2 or SEQ ID NO. 4) with a T7 promoter sequence and reverse transcriptase, using a target region of RNA to be detected as a template, and performing reverse transcription to form an RNA-cDNA hybrid; digesting RNA in the RNA-cDNA hybrid by RNaseH in the amplifying enzyme to obtain single-stranded cDNA; under the action of the F primer (SEQ ID NO.1 or SEQ ID NO. 3) and the DNA polymerase function of reverse transcriptase, synthesizing a second strand by taking single-stranded cDNA as a template to form double-stranded DNA with a T7 promoter; double-stranded DNA with the T7 promoter is transcribed by T7 RNA polymerase to produce a large number of RNA molecular products (FIG. 1).

The detection of the internal reference genes of the human beings is not only helpful for ensuring the effectiveness of a sample collection and amplification system, but also provides an important reference for the detection results of pathogens such as MG and the like. Its main purpose is to monitor the effectiveness of the sample collection and amplification system. During the test, the acceptable sample collection should contain cells from the human body that contain the reference gene, thereby ensuring that the presence of the reference gene is detected during the test. The presence or absence of the reference gene is a key indicator for assessing the quality of the sample. When the MG detection result is negative, if the reference gene is positive at the same time, the method means that the sample collection and amplification system are effective, and the detection result is reliable. If the reference gene is negative, this may mean that there are problems in sample collection, such as poor sample quality, contamination during collection, or insufficient sample volume. At this time, in order to ensure the accuracy of the detection result, resampling and retesting are required.

(2) Gold probe chromatography

A. designing specific probe, gold probe and coated probe

Specific probes: for each detection index, two types of specific probes are typically designed: CES series and LES series. Each type of probe can be designed into a plurality of probes according to the requirements so as to adapt to different detection requirements. The CES probe has specific binding sites at both ends, one end capable of specifically binding to the amplification product of the target gene and the other end capable of binding to the coated probe on the NC membrane (nitrocellulose membrane). This binding allows the CES probe to act to immobilize the amplified product RNA during detection. After the amplified product RNA is bound to the CES probe, unbound impurities can be removed by a specific elution step, thereby improving the specificity and sensitivity of the detection. The LES probe also has binding sites at both ends, one end specifically binding to the amplification product of the target gene and the other end capable of binding to the gold probe. The combination mode enables the LES probe to play a role in the color development of the gold-linked probe in the detection process, thereby realizing the visual detection of the amplified product.

Gold probe: the 5' end of the gold probe is modified by sulfhydrylation, so that the sulfhydryl group can form a stable covalent bond with the colloidal gold particles, thereby realizing the labeling of the gold probe and the colloidal gold particles. The 3' end of the gold probe may be bound to one end of the specific probe LES.

Coating a probe: the NC film is fixed with a coated probe which can be combined with one end of a specific probe CES, thereby realizing the fixation effect.

The specific probes must be designed to ensure that different probes of the same index do not interfere with each other, and meanwhile, the CES series probes, the amplifying probes and the coating probes must not have cross reaction. Thereby ensuring the specificity of the detection.

The CES series and LES series of the specific probes are designed in a plurality to improve the immobilization efficiency and bind more gold probes, thereby enhancing the sensitivity of the detection.

B. Test strip detection

The test strip is provided with a detection line and a quality control line which are an MG-T line, an internal reference-N line and a quality control-C line respectively, wherein a coated probe on the MG-T line is specially combined with one end of an MG-CES series probe; the reference coated probe at the reference-N line is capable of specifically binding to one end of the reference CES series probe. The coated probe at the quality control-C line can be specifically combined with the C line chromogenic probe.

When the detection solution containing the specific probe CES, the specific probe LES and the gold probe is mixed with the specific amplified product of the nucleic acid to be detected and is dripped on a test strip for chromatography, if the detection line is colored, the existence of the nucleic acid to be detected is indicated; meanwhile, if the quality control line is also colored, the whole detection process is proved to be effective (as shown in fig. 2).

In combination with the principle, the working process of the method of the invention is described as follows:

(1) Sample collection

For detecting pathogens in the genital tract of suspected patients, methods have been employed to collect a sample of genital tract swabs, followed by lysing the cells using a cell lysate, thereby releasing the pathogen.

(2) Isothermal amplification of RNA

To 17. Mu.L of an amplification reaction solution (containing 40mM Tris-HCl (pH 8.0), 12mM MgCl ₂, 70mM KCl,15% DMSO,5mM DTT, 1mM each dNTP, 2mM each NTP, 0.2. Mu.M each amplification primer) containing the MG primer (including the MG-F primer shown in SEQ ID NO. 1) and the internal reference primer (including the internal reference-F primer shown in SEQ ID NO.3, the internal reference-R primer shown in SEQ ID NO. 4) was added 2. Mu.L of a sample cleavage extract, heated at 95℃for 2 minutes, and preheated at 42℃for 2 minutes. 1. Mu.L of the amplification enzyme was added thereto, and the mixture was amplified at a constant temperature of 42℃for 30 minutes. If MG pathogen exists in the sample to be detected, a large amount of amplified and enriched index RNA molecules can be obtained.

(3) Test strip chromatography

A. Prehybridization

After isothermal amplification of RNA, the amplified product was mixed with a detection solution (containing specific probes, including CES series, namely MG-CES1, MG-CES2, internal reference CES1, internal reference CES2, and LES series, namely MG-LES1, MG-LES2, MG-LES3, internal reference LES1, internal reference LES2, internal reference LES3, C-line chromogenic probe shown in SEQ ID NO.15, gold probe shown in SEQ ID NO. 16) and prehybridized at 42℃for 5 minutes to promote complementary paired binding between the amplified RNA target fragment and the specific probes (including CES series probes and LES series probes). One end of CES series probes is hybridized and complementarily paired with RNA molecules, and the other end of CES series probes is combined with coated probes on NC films; one end of the LES series probes are hybridized and complementarily paired with RNA molecules, and the other end of the LES series probes can be complementarily paired with gold probes for combination, and when the sample is MG positive and the amplified RNA target fragment exists, a CES probe-RNA target fragment-LES probe-gold probe complex can be formed.

B. Chromatographic detection

After the prehybridization step, the prehybridization product is dripped onto the sample pad of the test strip. Due to capillary action, the prehybridization solution can move along the NC membrane in a chromatographic manner towards the absorbent paper. During this process, if the RNA amplification product to be detected is present in the sample, a "CES probe-RNA molecule-LES probe-gold probe complex" is formed. When this complex is chromatographed onto the NC membrane with prehybridization solution, it is intercepted by the immobilized coating probe on the NC membrane. Because of the specific binding capacity of the coated probes to CES probes, the complex will be immobilized on the NC membrane, forming a visible band. The presence of this band indicates the presence of the RNA amplification product to be detected in the sample, and therefore the detection result is positive; in contrast, if the RNA amplification product to be detected is not present in the sample, then a "CES probe-RNA molecule-LES probe-gold probe complex" cannot be formed. In this case, the colloidal gold particles cannot be aggregated at the T line, and thus no visible band can be formed. This indicates that the detection result is negative (see FIG. 4).

Regardless of whether the RNA product to be detected is amplified or not, the C-line chromogenic probe will flow forward along the NC membrane by chromatographic action. When the C-line chromogenic probe reaches the C-line, it will bind to the specific sequence coated at the C-line, and since this binding is specific, the C-line chromogenic probe will remain at the C-line, forming a macroscopic colored band. The appearance of this colored band is an indicator that the experimental results are valid (see fig. 4).

Example 2 preparation of nucleic acid detection test strip

The main raw materials required in preparing the nucleic acid detection test strip are as follows: nitrocellulose membrane (NC membrane), sample pad, absorbent paper, PVC base plate, etc.

1. Spraying a film:

Detection line MG-T: CES sequence (10 μm) of the probe capable of capturing the binding MG-specific probe, amount of sprayed film: 2-3 mu L/cm;

Detection line internal reference-T: the CES sequence of the combined reference specific probe can be captured, (10 mu M), and the film spraying amount is as follows: 2-3 mu L/cm;

quality control line (C line): the binding C line chromogenic probe sequence can be captured, (10 mu M), and the film spraying amount is as follows: 2-3 mu L/cm;

After film spraying, the film is automatically crosslinked once in an ultraviolet crosslinking instrument, and the film is dried in a clean constant temperature box at 37 ℃ for 2 hours and stored in a dry environment for standby.

2. Test strip assembly

And respectively cutting water absorbing paper with the length of 2cm, a coated NC film and a sample pad, and sequentially fixing the water absorbing paper, the coated NC film and the sample pad on a PVC bottom plate from top to bottom to obtain the detection test paper strip. The assembly structure of the test strip is shown in FIG. 3.

Example 3 sensitivity test

Performing gradient dilution on the MG pathogen stock solution, calibrating by using a digital PCR method, and detecting MG nucleic acid by using the method to determine the minimum detection limit; 3-5 parts of each gradient of diluent are repeated, each part is repeatedly detected for 20 times, the virus level with the positive detection rate of 90-95% is taken as the lowest detection limit, and the detection result is as follows:

TABLE 1 test data for different titres of MG

Table 2MG minimum limit of detection experimental data

From the above, the lowest detection limit is: 2.0X10 ² copies/mL, the sensitivity of the human metapneumovirus nucleic acid detection kit provided by the invention is high.

Example 4 specificity verification

The method is used for detecting different pathogens and verifying the specificity of the primer and the probe in the kit. The results were as follows:

TABLE 3 specificity verification test data

As can be seen from Table 3, the detection results of the kit of the present invention on these microorganisms are all negative, which proves that the kit of the present invention has no cross reaction with other microorganisms, and shows that the kit has strong specificity for detecting pathogens.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (8)

1. A mycoplasma genitalium nucleic acid detection kit, characterized in that the kit comprises:

An amplification primer for mycoplasma genitalium, comprising: MG-F primer shown as SEQ ID NO.1 and MG-R primer shown as SEQ ID NO. 2;

An amplification primer for a reference gene, comprising: an internal reference-F primer shown as SEQ ID NO.3 and an internal reference-R primer shown as SEQ ID NO. 4;

a specific probe CES, wherein the nucleotide sequence is at least one of SEQ ID NO.5 or SEQ ID NO. 6;

Specific probe LES, nucleotide sequence is selected from at least one of SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO. 9;

The internal reference specific probe CES has a nucleotide sequence selected from at least one of SEQ ID NO.10 or SEQ ID NO. 11;

the reference specific probe LES has a nucleotide sequence selected from at least one of SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO. 14;

the nucleotide sequence of the line C chromogenic probe is shown as SEQ ID NO. 15;

The nucleotide sequence of the gold probe is shown as SEQ ID NO. 16; and the 5' end of the gold probe is modified by sulfhydrylation;

The test strip comprises a bottom plate, a sample pad, an NC film and absorbent paper, wherein the sample pad is adhered to the bottom plate and sequentially overlapped, 3 detection lines are sequentially arranged on the NC film according to the chromatography direction, the detection lines are respectively an MG-T line, an internal reference-N line and a quality control-C line, the MG-T line is coated with an MG coating probe with a nucleotide sequence shown as SEQ ID NO.17, the internal reference-N line is coated with an internal reference coating probe with a nucleotide sequence shown as SEQ ID NO.18, and the quality control-C line is coated with a quality control coating probe with a nucleotide sequence shown as SEQ ID NO. 19.

2. The mycoplasma genitalium nucleic acid detection kit according to claim 1, wherein the kit further comprises an amplification reaction solution: tris-HCl (pH 8.0), mgCl ₂, KCl, DMSO, DTT, dNTPs, NTPs.

3. The mycoplasma genitalium nucleic acid detection kit of claim 1, wherein the kit further comprises a cell lysate.

4. The mycoplasma genitalium nucleic acid detection kit of claim 1, wherein the kit further comprises a negative control: the negative quality control is physiological saline.

5. The mycoplasma genitalium nucleic acid detection kit of claim 1, wherein the kit further comprises an RNA isothermal amplification enzyme: reverse transcriptase, RNaseH, T7 RNA polymerase.

6. The kit for detecting genital mycoplasma nucleic acid according to claim 1, wherein the concentration of the MG-T line coated MG coating probe is 10+ -2 μm, and the spraying amount is 2-3. Mu.L/cm.

7. The kit for detecting genital mycoplasma nucleic acid according to claim 1, wherein the concentration of the coated probe coated at the internal reference-N line is 10+ -2 μm, and the amount of the sprayed film is 2-3. Mu.L/cm.

8. The kit for detecting genital mycoplasma nucleic acid according to claim 1, wherein the concentration of the quality control coating probe coated at the quality control-C line is 10±2 μm, and the amount of the coating: 2-3 mu L/cm.