NL2034274B1 - DETECTION METHOD OF MUTATION TYPE OF parC GENE OF MYCOPLASMA GENITALIUM AND KIT - Google Patents

Abstract

The present disclosure provides a detection method of mutation types of a parC gene of Mycoplasma genitalium and a kit. The detection method is used for detecting eight 5 mutation types of the parC gene of M. genitall'um, and detection targets of the eight mutation types are (l) ParC $831, (2) ParC S83C‚ (3) ParC S83N‚ (4) ParC S83R‚ (5) ParC D87G, (6) ParC D87N, (7) ParC D87H, and (8) ParC D87Y. The present disclosure further provides reaction primer sequences SEQ ID NO: 1 to SEQ ID NO: 9 for detecting the eight mutation types, respectively. According to the present disclosure, 10 the eight mutation types are detected by using high resolution melting (HRM) in combination with an unlabeled probe.

Description

DETECTION METHOD OF MUTATION TYPE OF parC GENE OF

MYCOPLASMA GENITALIUM AND KIT

TECHNICAL FIELD

[0001] The present disclosure belongs to the technical field of molecular biological detection, and relates to a detection method of eight mutation types of a par’ gene of

Mycoplasma genitalium, in particular to a method of eight mutation types of a part: gene of M. genitalium and a kit.

BACKGROUND

[0002] M. genitalium was first isolated from samples from two male patients with non- gonococcal urethritis in 1980. M. genitalium infection accounts for 10-35% of non- chlamydial non-gonococcal urethritis in men. In female patients, M. genitalium is associated with cervicitis and pelvic inflammatory disease (PID). However, in the 10 years following the pathogen's discovery, little progress has been made in determining the clinical importance of M. genitalium in bacteria due to the lack of reliable detection methods. M. genitalium is an extremely slow-growing and finicky bacterium, and new isolates were cultured and obtained after a series of techniques. Co-culture technique of clinical samples in Vero cells has been established. A. genitalium has a long culture cycle (up to six months), and the sensitivity of culture is poor. Therefore, the development of AM. genitalium-related culture techniques is crucial for epidemiological surveillance of drug resistance in M. genitalium and for understanding the genetic mechanisms behind it. In fact, studies have shown that nucleic acid amplification testings (NAATs) are more sensitive than culture methods.

[0003] M. genitalium infection is the main pathogen of non-chlamydial non- gonococcal urethritis in men and is associated with cervicitis and PID in women. At present, there is no effective vaccine against M. genitalium, and effective antimicrobial therapy is still the main means to treat and control Af. genitalium infection. Macrolide antibiotics (azithromycin) are first-line drugs recommended in the guideline for M. genitalium infection. However, due to the wide application and the high dosage of azithromycin, the\ resistance rate of M. genitalium infection in many areas has reached as high as 50%. Therefore, second-line recommended fluoroquinolone antibiotics (fluoroquinolone) have gradually become the main drug for M. genitalium infection in many areas. Unfortunately, clinical samples of fluoroquinolone-resistant M. genitalium have been reported in recent years, seriously threatening the currently recommended therapeutic regimens.

[0004] Enhanced surveillance of fluoroquinolone resistance in M. genitalium is necessary to control and predict resistance trends, in order to ensure the effectiveness of currently recommended therapeutic regimens. Conventional drug resistance detection methods are mainly based on bacterial isolation and culture methods. The isolates are obtained by pure culture, and the growth of the isolates at the corresponding antibiotic concentration is observed to evaluate the drug resistance of gonococci. However, because A4. genitalium 1s extremely difficult to culture, this method is difficult to implement in clinic and laboratory. Whole-genome sequencing (WGS) can analyze the carrying drug resistance by acquiring the whole genome sequence information of M. genitalium. WGS has been successfully applied in molecular epidemiological screening and drug resistance monitoring of A. genitalium.

The advantages of WGS are obvious, which can provide more comprehensive drug resistance information, track the genetic and evolutionary relationships of different drug-resistant strains, and analyze the distribution and variation of drug-resistant strains in specific populations and regions. However, WGS is expensive, requires specialized personnel for data analysis, and processes a limited number of samples at one time. Fortunately, in the past few decades, great progress has been made in studying the mechanisms of molecular resistance in A4. genitalium, making it possible to establish molecular screening methods for detecting specific resistance genes. A series of NAATSs have been rapidly established due to the advantages of short time, simple operation, and automation. NAATs replace culture methods and gradually become the first choice for detecting the drug resistance in M. genitalim.

Fluoroquinolone resistance is mainly mediated by mutations at loci 83 and 87 of the parC gene. Conventional NAATSs use these two loci as molecular targets for detecting fluoroquinolone resistance. However, due to the complexity of mutations at the above two loci (ParC S83L S83C, S83N, S83R, D87G, DS7N, D87H, and D87Y), conventional NAATs cannot cover all mutants. Existing real-time quantitative PCR (RT-qPCR) methods need to design multi-hole probes and multiple probes to cover all mutants, which substantially increase the costs of these methods in use.

[0005] High resolution melting (HRM) is a novel molecular diagnostic technique that rises in recent years and combines saturated fluorescent dyes, unlabeled probes and

RT-qPCR to detect gene mutations and genotyping. On the basis of RT-qPCR, HRM adds saturated fluorescent dyes to the system, and uses high-precision instruments to monitor the DNA unwinding process in real time through HRM of PCR products, and analyze small differences in DNA sequence according to the characteristic changes of melting curves. HRM is widely used in sequence analysis, genotyping, mutation site scanning, single nucleotide polymorphism analysis and clinical testing due to the advantages of rapidness, accuracy, high throughput, strong specificity, high sensitivity, low cost, and realization of true closed tube operation. In addition, based on HRM, an unlabeled probe is innovatively added, because the probe has a shorter sequence, which will amplify the slight temperature difference caused by the mutation of the same base at different loci, further improving the genotyping ability of the method.

Since the added probe does not need fluorescence labeling, the detection cost is substantially reduced. After the reaction is completed, the experimental results can be quickly and sensitively analyzed through the HRM curve, and a variety of mutation information can be acquired.

SUMMARY

[0006] Special primers for detecting eight mutation types of a parC gene of M. genitalium are provided. Detection targets of the eight mutation types of the par(’ gene are: (1) ParC S831, (2) ParC S83C, (3) ParC S83N, (4) ParC S83R, (5) ParC D87G, (6)

ParC D87N, (7) ParC D87H, and (8) ParC D87Y; and the primers have sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9.

[0007] The primers have four primer sets: a first primer set consists of three primers, one is a forward primer, one is a reverse primer, and a last one is a 3'-phosphorylated primer; each of the remaining three primer sets consists of two primers, one is a forward primer and the other is a reverse primer, respectively for the eight mutation types of the part! gene, and corresponding relationships thereof are shown in Table 1.

[0008] The present disclosure further provides a kit for detecting eight mutation types of a parC’ gene of M. genitalium, including the primers provided by the present disclosure.

[0009] The kit provided by the present disclosure further includes others necessary reagents or items during detection. For example, sampling tubes, a crude extraction reagent Lysis Buffer, a reaction component EvaGreen Master Mix (an amplification enzyme, an amplification buffer, dNTP, and EvaGreen Dye), positive controls, and a negative control are included, the positive controls are wild-type positive samples of each detection target, and the negative control is ddH;0. The detection reagents or items included in the present disclosure may serve one or more people, and the more people may be 2-1,000 people.

[0010] The present disclosure further provides a detection method of eight mutation types of a part gene of M. genitalium, including the step of using the kit provided by the present disclosure. For example:

[0011] step 1, completing the extraction of genomic DNA of a sample using a method of the kit or a lysis method;

[0012] step 2, with the genomic DNA of an unknown sample as a template, preparing a high resolution melting (HRM) amplification reaction system for specific amplification under the guidance of the foregoing special primer sets; and

[0013] step 3, in a polymerase chain reaction (PCR), due to the fact that sequence specificity of a target sequence leads to a difference in base content of different PCR products, heating PCR amplicons under the action of saturated dyes according to properties of DNA, subjecting detection results to data integration and plotting to generate melting curves of the PCR products through real-time monitoring of changes in fluorescence intensity in the heating process, and determining differences of DNA sequences of the PCR products according to different melting curves.

[0014] Preferably, the detection method provided by the present disclosure includes the following steps:

[0015] step 1, collecting reproductive tract secretions or a urine sample of a patient using a sample collection tube provided by the kit;

[0016] step 2, centrifuging the urine sample at 8,000 rpm for 10 min, discarding a supernatant, and adding a volume of the Lysis Buffer to the sample collection tube; for a swab sample of secretions, adding a volume of the crude extraction reagent Lysis

Buffer to the sample collection tube, stirring, and soaking a swab in the Lysis Buffer for 5 min;

[0017] step 3, placing the sample collection tube in a metal bath or water bath, heating the sample collection tube at 95°C for 10 min, and letting the sample collection tube stand at room temperature to complete the extraction of genomic DNA of the sample;

[0018] step 4, with the genomic DNA of the sample obtained in the foregoing step as a template, realizing the detection of the eight mutation types of the parC gene of M. genitalium in primer sets for two assays, where a 20 pL reaction system includes: 10 uL of EvaGreen Master Mix (optimal amplification concentrations of all primers in the assays are shown in Table 1), 2 pL of the genomic DNA of the sample, and ddH:0 being made up to 20 pL, and positive control and negative control reaction tubes of the primers are used in each assay simultaneously;

[0019] step 5, conducting an amplification reaction and an HRM analysis on a

QuantStudio 6 Flex Real-Time PCR System, where the amplification reaction follows the following program: incubation at 95°C for 10 min, followed by a total of 30 cycles of annealing at 95°C for 15 s and extension at 60°C for 1 min for the amplification reaction, incubation at 40°C for 1 min, slowly heating to 95°C at a rate of 0.025°C/s, and continuously collecting fluorescence signals; and

[0020] step 6, after the reaction, making an analysis using QuantStudio 6 and 7 Flex

Real-Time PCR software v1.0, where the software automatically generates a melting curve and a Tm value corresponding to an amplicon; and determining a result by comparison with positive controls of different mutation types, where the melting curve does not show a shape change on condition that a par(’ mutation site of an unknown sample has the same mutation type as a control sample, and the melting curve shows a shape change correspondingly on condition that the unknown sample is different from the control sample and a mutation occurs.

[0021] The special primer set provided by the present disclosure may accurately distinguish an optimal primer pair of wild type and mutant type.

[0022] The special primer set for multiple detection of eight mutation types of a part! gene of M. gentitalium provided by the present disclosure selects the parC’ gene related to second-line drug (fluoroquinolone) resistance as a target gene for detection. First, gene sequences of all representative strains of M. genitalium that have been fully annotated as reference sequences are downloaded from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/), the reference sequences are aligned with the

NCBI nr database for BLAST of nucleic acid sequences (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and aligned results are downloaded to obtain more sequences of target genes for detection. All downloaded sequences are subjected to multiple alignment analysis, specific amplification primers are designed for mgpa,

HBB and parC’ genes of both sides of loci 83 and 87 using Beacon Designer 8.0 software, and the specificity of the primers is verified using an online primer tool on

NCBI (https://www.ncbi.nlm.nih.gov/tools/primer blast’). Melting temperatures (Tm) of PCR products are predicted using oligocalc (http://biotools.nubic.norwestern.edu/oligocalc.html) and UMELT online software

(https://www.dna.utah.edu/umelt/umelt.html). Specific primers can amplify the loci 83 to 87 of the part gene to form a main product peak with a higher Tm value, and this fragment of the sequence is preliminarily genotyped. In addition, a probe is designed for the loci 83 to 87 of the part’ gene. The probe should perfectly match a sequence of a ParC S831 mutant. The probe is 3'-phosphorylated to prevent probe extension. Six primer pairs are designed for mgpa and HBB, and par(’ genes of both sides of loci 83 and 87, and optimal primer pairs that can accurately distinguish wild types and mutant types are selected. Four probes of different lengths are designed for testing, and a probe with the greatest difference in Tm among different mutants is selected. Finally, an optimal primer pair and a probe that can accurately distinguish the wild types and a plurality of mutant types are selected to form a final multiplex HRM analysis system. The mgpa target is selected as the identification and confirmation of

M. genitalium species and the HBB as the quality control of nucleic acid extraction.

The detection loci are as follows:

[0023] Table 1 Information of primer sequences

[0024]

Assay | SEQ | Target | Primer sequence Concentration

ID | gene (uM)

NO:

SEQ | mgpa | MGpa F: CTTGAGCCTTTCTAACCGCTGCACT 0.25

ID MGpa_R: CAAGTCCAAGGGGTTAAGGTTTCAT 0.25

NO: 1

SEQ

ID

NO: 2

SEQ | HBB HBB _F: AGTGCTCGGTGCCTTTAGTGAT 0.2

HBB R: TGGCAAAGGTGCCCTTGA 0.2

NO: 3

Assay

SEQ

ID

NO: 4

SEQ | parC | ParC D87 F:CCCATGGTGATAGTTCCATTTAT 05

ID ParC_D87_R:AGCTTTGGGACATTCTGATAATTG 0.5

NO:

SEQ

ID

NO: 6

SEQ | parC ParC S83 F: GGGAGATCATGGGGAAATACC 0.0375

ID ParC_S83 R: CAGCTTTGGGACATTCTGATA 0.025

NO: ParC_S83_P: CCCCCATGGTGATATTTCCATTTATDRTGCAA* | 1 7

SEQ

ID

Assay2

NO:

SEQ

ID

NO: 9

[0025] NOTE: *3'-Phosphorylation

[0026] The method provided by the present disclosure can rapidly detect the eight mutation types of the par(’ gene of M. genifalium. Compared with other technologies and similar technologies for detecting the mutant types of the parC gene of M.

genitalium, the technical solutions of the present disclosure have the following advantages:

[0027] Compared with conventional PCR or other molecular detection technologies, the HRM technology is a high-throughput gene screening technology that analyzes

PCR products by monitoring the melting curve changes in real time, without limitations of mutation sites and types of detection targets. There is no need to synthesize expensive sequence-specific probes, substantially reducing detection costs.

After the reaction is completed, experimental results can be quickly and sensitively analyzed through the HRM curve, and mutation information of drug resistance-related loci can be acquired.

[0028] In addition, this method innovatively combines an unlabeled probe-based HRM technology, which can simultaneously detect the eight mutation types of the part gene of M. genitalium with only one probe and a pair of amplification primers, covering all significant fluoroquinolone resistance-related mutations of M. genitalium. And because the probe sequence completely matches the sequence of the ParC S831 mutant, the probe peak of the ParC S831 shows the highest Tm value, and the ParC S831 mutant can be quickly determined by the probe, which is the most common type in fluoroquinolone-resistant A. genitalium. The method has high sensitivity (20 copies/reaction) and high specificity, and can be directly applied to clinical samples.

The method is an important technical supplement for the detection of parC’ gene mutation in M. genitalium that cannot be subjected to pure culture. And because the probe does not need fluorescence labeling, the cost is much lower than that of conventional fluorescent quantitative PCR.

[0029] The present disclosure uses saturated dye EvaGreen for HRM analysis experiment. Since the saturated dye EvaGreen does not inhibit the PCR, EvaGreen can be directly added to the PCR system to participate in the PCR process before the reaction starts. The HRM analysis is performed directly without transferring the dye into other analysis equipment or uncapping to add the dye after the reaction. This method truly realizes closed tube operation, avoids the pollution that may be introduced by uncapping to cause false positive results, and improves the accuracy and reliability of the experimental results; finally, heating and cooling in the analysis process will not cause destructive damage to the DNA structure, subsequent cooling can renature DNA, and renatured DNA can be directly used for subsequent research

(such as sequencing for verification of results), which substantially saves time, manpower and material resources, and avoids unnecessary waste.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 is a schematic diagram of the results corresponding to Assay;

[0031] FIG. 2 is a schematic diagram of the results corresponding to Assay2;

[0032] FIG. 3 illustrates a determination process of results corresponding to Assayl; and

[0033] FIG. 4 illustrates a determination process of results corresponding to Assay2.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] The example is implemented on the premise of the present disclosure, and detailed implementations and specific operation processes are provided. The specific implementations and operation processes described herein are only intended to explain the present disclosure, but the protection scope of the present disclosure 1s not limited to the following example. The implementations and specific operation processes of the example of clinical testing of eight mutation types of a parC’ gene of M. genitalium in hospitals will be described below.

[0035] Example 1

[0036] Step 1, secretions or a urine sample of a patient was collected by using a sample collection tube provided by the kit.

[0037] Step 2, the urine sample was centrifuged at 8,000 rpm for 10 min, a supernatant was discarded, and a volume of the Lysis Buffer was added to the sample collection tube; for a swab sample of secretions, a volume of the crude extraction reagent Lysis

Buffer was added to the sample collection tube and stirred, and the swab was soaked in the Lysis Buffer for 5 min.

[0038] Step 3, the sample collection tube was placed in a metal bath or water bath, heated at 95°C for 10 min, and let stand at room temperature to complete the extraction of genomic DNA of the sample. (Based on the above step, the extraction of genomic

DNA could be completed using other nucleic acid extraction kits or methods)

[0039] Step 4, with the genomic DNA of the sample obtained in the foregoing step as a template, the detection of the eight mutation types the par’ gene of M. genitalium was realized in primer sets for two assays. Herein, a 20 uL reaction system included: 10 uL of EvaGreen Master Mix (optimal amplification concentrations of all primers in the assays are shown in Table 1), 2 uL of the genomic DNA of the sample, and ddH;0 being made up to 20 pL. Positive control and negative control reaction tubes of the primers were used in each assay simultaneously.

[0040] Step 5, an amplification reaction and an HRM analysis were conducted on a

QuantStudio 6 Flex Real-Time PCR System. The amplification reaction followed the following program: incubation at 95°C for 10 min, followed by a total of 40 cycles of annealing at 95°C for 15 s and extension at 60°C for 1 min for the amplification reaction, incubation at 40°C for 1 min, slowly heating to 95°C at a rate of 0.025°C/s, and continuously collecting fluorescence signals.

[0041] Step 6, after the reaction, an analysis was made using QuantStudio 6 and 7 Flex

Real-Time PCR software v1.0, where the software automatically generated a melting curve and a Tm value corresponding to an amplicon. The result interpretation was divided into three steps: step a, it was necessary to ensure that all samples were positive for M. genitalium (mgpa positive) and confirm that the nucleic acid extraction was successful (HBB positive). Step b, the main product types were preliminarily determined according to the melting curves of the main products in Assay2. There were three main product types: Typel, Type2, and Type3. Step 3, the parC gene was further genotyped by the probe peak amplified by the unlabeled probe. The primer set of ParC D87 in Assayl was used to assist in interpreting the results in the case of impure samples. Notably, since the probe perfectly matched the S831 sequence, the

S831 mutant showed a unique peak shape and the highest probe Tm, which allowed the inventors to quickly and directly interpret S831. The results were determined by comparing with positive controls of different mutants. (FIG. 4).

[0042] Sequence Listing

[0043] <110> Institute of Medical Biology, Chinese Academy of Medical

Sciences

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LLG <INSDSeq moltype>DNA</INSDSeg moltype> iiD <INSDSeq divislion»PAT</INSDSeqg division» 1a <INSDSeg feature~tablex iël <INSDFeature> 122 <IN3DFeature keysmisc feature /INSDFeature key» 1273 <IN3DFeature location>»l..18</INSDFeaturs locations 124 <INSDFsature qualsg>

EAS <INSDQualifier id="g7*> lat CINSDQualifisr name>note</INSDQvali fier name> 147 <INSDQualifier valus>Reverse primer sequence of target gene HBB</IN3DQualifier value» 128 </INSDQualifier> u 12% </IN3DFeature guals> 130 </INSDFeacture>

Lj <“INSDFesrure»>

LS <INSDFeature key>source</INSDFeature keys

133 <INSDFeature location>l..18</IN3DFeature locations 134 <INSDFealurse guals> 135 <INSDOQualifier» ijn <IN3DQualifier name>mol type“/INSDQualifier name> ia <INSDQualifier value>other DNA</IN3DGualifier value> 138 </INSDQuali fier» 13% <INSDQuaiifler id="g8*> 140 <INSDOQualifier namerorganism</INSDQualifier name> 14% <INSDQualifisr value>synthetic construct </INSDQualifisr valus> 142 </INSDOualifier> 143 </INBDFeature gvals> 144 </THSDFeatura> 145 </INSDSegy featurs-table> 148 <INSDSeq sequencertggcaaaggtgccettga“/INSDSeg sequence» 14 </INSDSeg> ids </SequenceData> 140 <Hedgquencelata segusnceliNumec=NS"> isd <INSDSeqg> 151 <IN3DSeq length»23</INSDSeq length» i582 ZINSDSegq molitypes>DNAC/INSDSeq moltyper> 1573 <IN3DSeq divisior>PAT</INSDIeqg division» 154 <INSDSeq feature-table>

Ton <INS3DFesature>

LIE <INSDFeaturs Keyrmisc feature</INSDFzature key> 137 <INSDFeature location>l..23</IN3DFeature location» 158 <INSDhFeature quels» 159 CINSDOualifisr id="g95>

Lan <IN3DQualifier name>note</INSDQualifier name>

Lel <INSDQualifiler valuerForward primer sequence of target gene Parc D87</1INSDSualifier value»

LEL «</INSDOQualifier»> its </THSDFeaturs guals> 164 </INSDFeature> 18h <INSDFeaturer» 14a <IN3DFeature key>source</IiN3DFeature key» ia <IN3DFeature lowation>l..23</INSDFeaturs location» 185 <INSDFeature guals>

Las <INSDOualifier>

LEG <INSDOQualifier name>mol type</INSDQualifier name>

LF <INSDgualifier valuerother DNA</INSDQualifier value»

LE </INSDQualifiers> iz <INSDOualifier id="qL0%> ijd <IN3DQualifier namevorganism“/INSDQualifier name> ih <INSDQualifier value>synthetic construct </INgDQualifier value»

LTE </INSDQualifier>

LEY </INSDFeature quals>

Lj </IN3DFeature> u ijn “/INSDSeqg fesature-table> iëN <IN3D3eq sequence»cccatggtgatagttccatttat-/INSDSeg seuuence> iel </INSDSegqs i182 </SeguencaData> 1873 <SequenceData sequenceIDNumber="6n>

Led <INSDSeqr

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LS <INSDF Feature location>l..24</INSDFeaturs location

Led <INSDFsature qualsg>

Las <INSDQualifier id="gilx>

Ld CINSDQualifisr name>note</INSDQvali fier name> 195 <INSDQualifier valus>Reverse primer sequence of target gene ParC D87</INSDQualifier valued 136 </INSDOualifier> 7 18 </IN3DFeature guals> 198 </INSDFeature>

Lee <“INSDFesrure»> 200 <INSDFeature key>source</INSDFeature key> aL <INSDFeature location»l..24</INSDFeature locations 207 <INSDFealurse guals>

ZO <INSDOQualifier» 20d <IN3DQualifier namedmol type</INSDQualifisr name> 205 <INSDQualifier value>other DNA</IN3DGualifier value> 206 </INSDQuali fier» 207 <INSDQuaiifier id="gid’x> 208 <INSDOQualifier namerorganism</INSDQualifier name> ain <INSDOualifier value>synthetic construct </INSDQualifisr valus> zin </INSDOualifier> 21d </IN3DFeature guala> 212 </THSDFeatura> u 213 </INSDSegy featurs-table> 244 <INSDSeq seguence>agetttgggacattctgataattg</INSD3eq sequence» 215 </INSDSeg> ais </SequenceData> 217 “<SequenceData segusnceliNumec=MN}N> z2ië <INSDSeqg> 219 <IN3DSeq length»21</INSDSeq length» 220 ZINSDSegq molitypes>DNAC/INSDSeq moltyper> 221 <IN3DSeq divisior>PAT</INSDIeqg division» zal <INSDSeq feature-table>

ZES <INS3DFesature>

PEs <INSDFeaturs Keyrmisc feature</INSDFzature key>

ZED <INSDFeature location>l..21</IN3DFeature location»

Zin <INSDhFeature quels» 227 <INSDQualifier id="gi3"> 228 <IN3DQualifier name>note</INSDQualifier name> 228 <INSDQualifiler valuerForward primer sequence of target gene ParC S83“/1INSDSualifier value» 2G </INSDOualifier> «St </THSDFeaturs guals> 232 </INSDFeatures zis <INSDFeature>» 224 <IN3DFeature key>source</IiN3DFeature key» 235 <IN3DFeature lowation>l..21</INSDFeaturs location» 238 <INSDFeature guals> 237 <INSDOualifier> 238 <INSDOQualifier name>mol type</INSDQualifier name> ws <INSDgualifier valuerother DNA</INSDQualifier value»

ZAG </INSDOualifier> zál <INSDOQualifier id="qlá®> 242 <IN3DQualifier namevorganism“/INSDQualifier name> 243 <INSDQualifier value>synthetic construct </INgDQualifier value» 244 </INSDQualifier>

TAL </INSDFeature quals> 248 </IN&DFeaturer

ZAT? “/INSDSeqg fesature-table> 248 <IN3D3eq sequence>gggagatcatggggaaatace</INSDSegq sequencer 249 </INSDSeq> u 250 </SeguencaData> 251 <SequenceData zaquencalDNumben=ngn> 25% <INSDSeqr 203 <INSDSeq length»>21</INSDSeqg length»

LA <INSDSeq moltype>DNA</INSDSeg moltype> 255 <INSDSeq divislion»PAT</INSDSeqg division»

EEN <INSDSeg feature~tablex 257 <INSDFeature> 258 <IN3DFeature keysmisc feature“/INSDFearure key» 258 <IN3DFeature location>1..21</INSDFeature locations 280 <INSDFsature qualsg>

Ze <INSDQualifier id="gij> gE CINSDQualifisr name>note</INSDQvali fier name> 263 <INSDQualifier valus>Reverse primer sequence of target gene ParC S83</INSDQualifier valued 264 </INSDOualifier> 7 285 </INSDFeature guals> 268 </INSDFealure> 287 <INSDFeature>

Zes <INSDFeature key>source</INSDFeature keys

“Eh <INSDFeature location>l..21</INSDFeature locations zij <INSDFealurse guals>

ZL <INSDOQualifier» 272 <IN3DQualifier name>mol type“/INSDQualifier name> 273 <INSDQualifier value>other DNA</IN3DGualifier value> 274 </INSDQuali fier» 205 <INSDQuaiifier id="gië’x>

FES <INSDOQualifier namerorganism</INSDQualifier name>

PA <INSDQualifisr value>synthetic construct </INSDQuali fier value» 278 </INSDOualifier> 278 </IN3DFeature guals> ann </THSDFeatura> u 281 </INSDSegy featurs-table>

ZEE <INSDSeq seguencercagctttgggacattctgata-/INSDSeq sequence»

SES </INSDSeg> “54 </SequenceData> zö5 “<SequenceData segusnceliNumec=NS8"> 288 <INSDSeq> 287 <IN3DSeq length»32</INSDSeq length» 288 ZINSDSegq molitypes>DNAC/INSDSeq moltyper> 288 <IN3DSeq divisior>PAT</INSDIeqg division» 280 <INSDSeq feature-table> 291 <INSDFeature> ad <INSDFeaturs key>mise feature</INSDF=ature key> <INSDFeature location>l..32</IN3DFeature location» 23d <INSDhFeature quels» 235 <INSDQualifier id="gijn> 236 <IN3DQualifier name>note</INSDQualifier name> 287 <INSDQualifiler valuerProbe sequence for target gene

ParC S83</INSDoualifier valued 243 </INSDOualifier>

Ache] </THSDFeaturs guals> 300 </INSDFeatures

S01 <INSDPeature»> 202 <IN3DFeature keyrsource</INSDFeature key> 303 <IN3DFeature lovation>l..32</INSDFeature Locations» 304 <INSDFeature guals> 30% <INSDOualifier> 306 <INSDQualifier name>mol type</INSDQualifier name>

S07 <INSDQualifler valverother DNA</INSDQualifier value» 308 </INSDOualifier> 303 <INSDOQualifier id="qL8%>

SLD <IN3DQualifier namevorganism“/INSDQualifier name>

GR <INSDQualifier value>synthetic construct </INgDQualifier value»

BEE <{INSDQualifier»

SLS </INSDFeature quals> 314 </IN3DFeature> u 315 </INSDSeg feature-iablex> is <IN3D3eq sequencer»cccccatggtgatatttccatttatdrtgcaa/INSDSeg sequenced 217 </INSDSeq> 7

GRE </SeguencaData> 3LS </ST26SeguencelListing> 3248

Claims (9)

Conclusions l. Primers in particular for detecting eight mutation types of a parC: gene of Mycoplasma genitalium, where the detection targets of the eight mutation types of the par(' gene are the following: (1) ParC S831, (2) ParC S83C, (3) ParC S83N, (4) ParC S83R, (5) ParC D87G, (6) ParC D87N, (7) ParC D87H, and (8) ParC D87Y; and the primers have sequences as in SEQ ID NO:1-SEQ ID NO:9 will be displayed. Primers according to claim 1, wherein the primers have four primer sets: wherein a first primer set comprises three primers, one being a forward primer, one being a reverse primer and one last being a 3' phosphorylated primer; wherein each of the remaining three primer sets comprises two primers, one being a forward primer and the other being a reverse primer, for the eight mutation types of the parC gene respectively, and corresponding ratios thereof are shown in Table 1. A kit for detecting eight mutation types of a parC' gene of Mycoplasma genitalium comprising the primers according to claim 1. Kit according to claim 3, further comprising required reagents or components during detection. The kit of claim 4, wherein the reagents or components in the kit include: a sampling tube, a crude extraction reagent Lysis Buffer, a reaction component EvaGreen Master Mix (an amplification enzyme, an amplification buffer, dNTP and EvaGreen Dye), positive controls and a negative control, where the positive controls are wild-type positive samples from each detection target and the negative control is ddH>O. The kit of claim 4, wherein the included detection reagents or components serve one or more persons, and the plurality of persons is 2-1,000 persons. A method of detection of eight mutation types of a parC gene of Mycoplasma genitalium comprising the step of using the kit according to claims 3-6. A detection method according to claim 7, comprising the following steps: step 1, complete extraction of genomic DNA from a sample using a method or the kit or a lysis method; step 2, using the genomic DNA of an unknown sample as a template, preparing a high resolution melting, HRM, amplification system for specific amplification under the guidance of the previous special primer sets; and step 3, in a polymerase chain reaction, PCR, due to the fact that the sequence specificity of a target sequence leads to a difference in the base content of different PCR products, the heating of PCR amplicons under the action of saturated dyes according to the properties of DNA, subjecting the detection results to data integration and mapping to generate melting curves of the PCR products by real-time monitoring of changes in fluorescence intensity in the heating process and determining differences in DNA sequences of the PCR products according to different melting curves. The detection method of claim 7, comprising the steps of: step 1, collecting reproductive tract secretions or a urine sample from a patient using a sample collection tube provided by the kit; step 2, centrifuging the urine sample at 8,000 rpm for 10 min, discarding a supernatant and adding a volume of the Lysis Buffer to the sample collection tube; for a smear of secretions, adding a volume of the crude extraction reagent Lysis Buffer to the sample collection tube, stirring, and soaking a smear in the Lysis Buffer for 5 min; step 3, placing the sample collection tube in a metal bath or water bath, heating the sample collection tube at 95°C for 10 min, and allowing the sample collection tube to stand at room temperature to complete the extraction of genomic DNA from the sample; step 4, using the genomic DNA of the sample obtained in the previous step as a template, realizing the detection of the eight mutation types of the parC gene of Mycoplasma genifalium in primer sets for two tests, using a 20 pL reaction system with the following includes: 10 uL EvaGreen Master Mix (optimal amplification concentrations of all primers in the assays are shown in Table 1), 2 uL of the sample's genomic DNA and ddH:O is made up to 20 uL and positive control and negative control reaction tubes of the sample primers are used simultaneously in each test; step 5, performing an amplification reaction and HRM analysis on a QuantStudio 6 Flex Real-Time PCR System, where the amplification reaction follows the following program: incubation at 95°C for 10 min, followed by a total of 30 cycles of binding at 95°C for 15 s and extension at 60°C for 1 min for the amplification reaction, incubation at 40°C for 1 min, slow heating to 95°C at a rate of 0.025°C/s, and continuous collection of fluorescent signals; and step 6, after the reaction, making an analysis using QuantStudio 6 and 7 Flex Real-Time PCR software v1.0, where the software automatically generates a melting curve and a Tm value corresponding to an amplicon; and determining a result by comparing with positive controls of different mutation types, wherein the melting curve shows no change in shape provided that a par(' mutation site from an unknown sample with the same mutation types as a control sample, and the melting curve shows a change in shape that corresponds to the condition that the unknown sample is different from the control sample and a mutation occurs.