Primers for detecting drug resistance genes of reproductive tract pathogens and their design method

Abstract

This invention proposes primers and their design method for detecting drug resistance genes of reproductive tract pathogens. The primer set obtained by this invention can cover the major drug resistance genes and drug resistance sites of a variety of major reproductive tract pathogens, enabling rapid multi-target detection of drug resistance genes and mutation sites. The optimized primer design avoids non-specific amplification and improves the reliability of detection. It is also applicable to various detection platforms such as targeted pathogen sequencing, real-time fluorescence PCR, and digital PCR.

Description

Technical Field

[0001] The invention relates to a primer for detecting drug resistance genes of pathogenic microorganisms in the reproductive tract and its design method, belonging to the field of biotechnology. Background Technology

[0002] The female reproductive tract microecology is complex, and infections can be caused by multiple pathogens (such as bacterial vaginosis, candidiasis, and gonorrhea), leading to co-infections. Furthermore, drug resistance is becoming increasingly serious during treatment, for example, resistance to antibiotics such as penicillin, tetracycline, vancomycin, and quinolones. Traditional microbial culture and drug sensitivity testing are time-consuming and have low sensitivity, while molecular detection technologies are gaining attention due to their speed and efficiency. However, current molecular diagnostic technologies still have shortcomings in the following aspects: The reproductive tract is home to a complex array of pathogens, but most detection methods target only a single target, resulting in low efficiency and an inability to detect diverse pathogens; some drug resistance is closely related to point mutations, and existing primers struggle to balance specificity and sensitivity, limiting the detection of drug-resistant mutation sites; and multi-target detection is prone to primer dimerization and non-specific amplification.

[0003] Therefore, developing primers and design methods for rapid, efficient, and multiplex detection of drug resistance genes and mutation sites of reproductive tract pathogens is of great clinical and social significance. This would enable the simultaneous detection of multiple pathogens, multiple drug resistance genes, and their mutation sites, making it suitable for rapid clinical diagnosis, reducing experimental time and costs. Summary of the Invention

[0004] The purpose of this invention is to provide a specific primer design method and primer set for pathogenic microorganisms in the female reproductive tract, enabling efficient simultaneous detection of drug resistance genes and their mutation sites, with both high sensitivity and high specificity. The technical solution adopted in this invention is as follows:

[0005] A primer for detecting drug resistance genes of reproductive tract pathogens, comprising:

[0006] Primers for detecting drug resistance genes of Mycoplasma genitalium 23S rRNA, parC and 16S rRNA;

[0007] Primers used to detect the penA, ftsX and 23S rRNA resistance genes of Neisseria gonorrhoeae;

[0008] Primers for detecting the PBP2x, gyrA, and parC resistance genes in Streptococcus agalactiae;

[0009] Primers for detecting Staphylococcus aureus 23S rRNA, grlA, Gras and rpoB resistance genes;

[0010] Primers used to detect the parC resistance gene in Ureaplasma urealyticum;

[0011] Primers used to detect the parC resistance gene of Ureaplasma parvovirus;

[0012] Primers used to detect the gyrA and parC drug resistance genes in Mycoplasma hominis;

[0013] Primers used to detect the NTR6 resistance gene in Trichomonas vaginalis;

[0014] Primers used to detect the ERG11 resistance gene in Candida albicans;

[0015] Primers used to detect the ERG11 and UPC2 resistance genes in Candida tropicalis;

[0016] Primers used to detect the ERG11 and ERE6 resistance genes in Candida glabrata.

[0017] The primers used to detect the drug resistance genes of Mycoplasma genitalium 23S rRNA, parC, and 16S rRNA include:

[0018] Primers for detecting the A2058C / G / T (nucleic acid) and A2059C / G / T (nucleic acid) sites of the 23S rRNA drug resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.1 and the downstream primer nucleotide sequence shown in SEQ ID NO.2;

[0019] Primers for detecting the A247C&T249G(S83R) and G248T / A(S83I / N) sites of the parC drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4;

[0020] The upstream primer nucleotide sequence for detecting the G259A / T (D87N / Y) and A260G (D87G) sites of the parC drug resistance gene is shown in SEQ ID NO.5, and the downstream primer nucleotide sequence is shown in SEQ ID NO.6.

[0021] Primers for detecting the C1192T (nucleic acid) site of the 16S rRNA drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.7, and the downstream primer nucleotide sequence is shown in SEQ ID NO.8;

[0022] The primers used to detect the penA, ftsX, and 23S rRNA resistance genes of Neisseria gonorrhoeae include:

[0023] Primers for detecting the C932T (nucleic acid) site of the penA resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.9, and the downstream primer nucleotide sequence is shown in SEQ ID NO.10;

[0024] Primers for detecting the A501T (GCG1501-1503) site of the penA resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.11, and the downstream primer nucleotide sequence is shown in SEQ ID NO.12;

[0025] Primers for detecting the R251H (CGC751-753) site of the ftsX resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.13, and the downstream primer nucleotide sequence is shown in SEQ ID NO.14;

[0026] Primers for detecting the A256G (nucleic acid) site of the 23S rRNA drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.15, and the downstream primer nucleotide sequence is shown in SEQ ID NO.16;

[0027] Primers for detecting the C2611T (nucleic acid) site of the 23S rRNA drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.17, and the downstream primer nucleotide sequence is shown in SEQ ID NO.18;

[0028] The primers used to detect the PBP2x, gyrA, and parC resistance genes in Streptococcus agalactiae include:

[0029] Primers for detecting the V405A (GTT1213-1215) site of the PBP2x resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.19 and the downstream primer nucleotide sequence shown in SEQ ID NO.20;

[0030] Primers for detecting the Q557E (CAA1669-1671) site of the PBP2x resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.21, and the downstream primer nucleotide sequence is shown in SEQ ID NO.22;

[0031] Primers for detecting the S81L (TCA241-243) site of the gyrA resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.23 and the downstream primer nucleotide sequence shown in SEQ ID NO.24;

[0032] Primers for detecting the S79K (TCC235-237) site of the parC resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.25, and the downstream primer nucleotide sequence is shown in SEQ ID NO.26;

[0033] The primers used to detect Staphylococcus aureus 23S rRNA, grlA, Gras and rpoB resistance genes include:

[0034] Primers for detecting the G2576T (nucleic acid) site of the 23S rRNA drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.27, and the downstream primer nucleotide sequence is shown in SEQ ID NO.28;

[0035] Primers for detecting the S80F (TCC238-240) site of the grlA drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.29, and the downstream primer nucleotide sequence is shown in SEQ ID NO.30;

[0036] Primers for detecting the E84K (GAA250-252) site of the grlA drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.31, and the downstream primer nucleotide sequence is shown in SEQ ID NO.32;

[0037] Primers for detecting the T136I (ACA406-408) site of the Gras resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.33, and the downstream primer nucleotide sequence is shown in SEQ ID NO.34;

[0038] Primers for detecting the H481Y (CAT1441-1443) site of the rpoB resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.35, and the downstream primer nucleotide sequence is shown in SEQ ID NO.36;

[0039] The primers used to detect the parC resistance gene of Ureaplasma urealyticum include:

[0040] Primers for detecting the D82N (GAT244-246) site of the parC drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.37, and the downstream primer nucleotide sequence is shown in SEQ ID NO.38;

[0041] Primers for detecting the E87Q (GAA259-261) and E87K (GAA259-261) sites of the parC drug resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.39 and the downstream primer nucleotide sequence shown in SEQ ID NO.40;

[0042] The primers used to detect the parC resistance gene of Ureaplasma parvovirus include:

[0043] Primers for detecting the S83L (TCA247-249) site of the parC drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.41, and the downstream primer nucleotide sequence is shown in SEQ ID NO.42;

[0044] The primers used to detect the mycoplasma hominis gyrA and parC drug resistance genes include:

[0045] Primers for detecting the S153L (TCA457-459) site of the gyrA resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.43 and the downstream primer nucleotide sequence shown in SEQ ID NO.44;

[0046] Primers for detecting the S91I (AGT271-273) site of the parC drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.45, and the downstream primer nucleotide sequence is shown in SEQ ID NO.46;

[0047] The primers used to detect the NTR6 resistance gene of Trichomonas vaginalis include:

[0048] Primers for detecting the K80S (AAG238-240) site of the ntr6 drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.47, and the downstream primer nucleotide sequence is shown in SEQ ID NO.48;

[0049] The primers used to detect the ERG11 resistance gene in Candida albicans include:

[0050] Primers for detecting the F126L (TTC376-378) site of the ERG11 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.49 and the downstream primer nucleotide sequence shown in SEQ ID NO.50;

[0051] Primers for detecting the Y132H (TAT394-396) site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.51, and the downstream primer nucleotide sequence is shown in SEQ ID NO.52;

[0052] Primers for detecting the S405F (TCT1213-1215) site of the ERG11 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.53 and the downstream primer nucleotide sequence shown in SEQ ID NO.54;

[0053] Primers for detecting the F449S / V (TTT1345-1347) site of the ERG11 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.55 and the downstream primer nucleotide sequence shown in SEQ ID NO.56;

[0054] Primers for detecting the R467K (AGA1399-1401) site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.57, and the downstream primer nucleotide sequence is shown in SEQ ID NO.58;

[0055] Primers for detecting the I471T (ATT1411-1413) site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.59, and the downstream primer nucleotide sequence is shown in SEQ ID NO.60;

[0056] The primers used to detect the ERG11 and UPC2 resistance genes in Candida tropicalis include:

[0057] Primers for detecting the V125A (GTT373-375) and Y132F (TAT394-396) sites of the ERG11 resistance gene are shown in SEQ ID NO.61 for the upstream primer and SEQ ID NO.62 for the downstream primer.

[0058] Primers for detecting the S154F (TCT460-462) site of the ERG11 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.63 and the downstream primer nucleotide sequence shown in SEQ ID NO.64;

[0059] Primers for detecting the Y221F (TAT661-663) site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.65, and the downstream primer nucleotide sequence is shown in SEQ ID NO.66;

[0060] Primers for detecting the R245K (AGA733-735) site of the ERG11 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.67 and the downstream primer nucleotide sequence shown in SEQ ID NO.68;

[0061] Primers for detecting the Y257H (TAC769-771) site of the ERG11 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.69 and the downstream primer nucleotide sequence shown in SEQ ID NO.70;

[0062] Primers for detecting the V362I (GTT1084-1086) site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.71, and the downstream primer nucleotide sequence is shown in SEQ ID NO.72;

[0063] Primers for detecting the G464S (GGT1390-1392) site of the ERG11 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.73 and the downstream primer nucleotide sequence shown in SEQ ID NO.74;

[0064] Primers for detecting the Q340H (CAA1018-1020) site of the UPC2 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.75 and the downstream primer nucleotide sequence shown in SEQ ID NO.76;

[0065] Primers for detecting the T381S (ACT1141-1143) site of the UPC2 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.77 and the downstream primer nucleotide sequence shown in SEQ ID NO.78;

[0066] Primers used to detect the ERG11 and ERE6 resistance genes in Candida glabrata;

[0067] Primers for detecting the Y192 (TAC574-576UAA / UAG / UGA) site of the ERE6 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.79 and the downstream primer nucleotide sequence shown in SEQ ID NO.80;

[0068] Primers for detecting the W286 (TGG856-858UAA / UAG / UGA) site of the ERE6 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.81 and the downstream primer nucleotide sequence shown in SEQ ID NO.82;

[0069] Primers for detecting the L341 (CTA1021-1023UAA / UAG / UGA) site of the ERE6 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.83 and the downstream primer nucleotide sequence shown in SEQ ID NO.84;

[0070] Primers for detecting the G315D (GGT943-945) site of the ERG11 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.85 and the downstream primer nucleotide sequence shown in SEQ ID NO.86.

[0071] The present invention also proposes the application of the above primers in the preparation of products for detecting drug resistance genes of reproductive tract pathogens.

[0072] The present invention also proposes a kit comprising the primers described above.

[0073] This invention also proposes a primer design method for drug resistance genes of reproductive tract pathogens, comprising the following steps:

[0074] Step 1) Obtain information on drug resistance genes and mutation sites of pathogenic microorganisms in the reproductive tract;

[0075] Step 2) Obtain the drug resistance gene reference sequence based on the drug resistance gene information obtained in Step 1), and then filter the obtained drug resistance gene reference sequence to obtain a high-quality drug resistance gene reference sequence.

[0076] Step 3) Obtain the conserved region sequence of the drug resistance gene based on the high-quality drug resistance gene reference sequence obtained in Step 2);

[0077] Step 4) Design primers based on the conserved region sequences obtained in Step 3);

[0078] Step 5) Screen the primers obtained in Step 4) to obtain primers for drug resistance genes of reproductive tract pathogens.

[0079] Optionally, in step two), based on the drug resistance gene information obtained in step one, drug resistance gene reference sequences are obtained from the NCBI, CARD, and ENA databases. Based on whether the obtained drug resistance gene reference sequences contain N bases, drug resistance gene reference sequences containing N bases are removed to obtain high-quality drug resistance gene reference sequences.

[0080] Optionally, after obtaining the high-quality drug resistance gene reference sequence in step three), the conserved region sequence of the drug resistance gene is obtained using Clone Manager or MAFFT software.

[0081] Optionally, the screening conditions in step five are: primer length 18-25bp, GC content 30%-70%, annealing temperature 57-63℃, specificity and coverage value both greater than 90, and no primer dimers and / or hairpin structures are generated. If the primers fail the screening, return to step four to redesign.

[0082] Optionally, the method for calculating specificity in step five includes: aligning the primer sequences to the CARD database using BLAST software; the specificity value is the percentage of the number of corresponding drug resistance gene sequences aligned with the primers relative to the number of sequences aligned with the primers in the CARD database.

[0083] The calculation method for the coverage value includes: aligning the primer sequence to the high-quality drug-resistant gene reference sequence obtained in step two) using BLAST software; the coverage value is the percentage of the number of drug-resistant gene sequences aligned with the primers relative to the number of drug-resistant genes in the high-quality drug-resistant gene reference sequence.

[0084] Optionally, in step five, the mfeprimer software is used to check whether the primers will generate primer dimers and / or hairpin structures.

[0085] Compared with the prior art, the present invention has the following technical effects:

[0086] This invention screens high-quality drug-resistant gene sequences based on information about major drug resistance genes and resistance sites of major pathogenic microorganisms associated with reproductive tract infections. Based on these sequences, conserved regions of the drug resistance genes are obtained. Primers are designed according to these conserved regions, and then the final primer set is obtained by screening based on primer length, annealing temperature, specificity, and other conditions. The primer set obtained by this method can cover the major drug resistance genes and resistance sites of various major reproductive tract pathogenic microorganisms, enabling rapid multi-target detection of drug resistance genes and mutation sites. The optimized primer design avoids non-specific amplification and improves the reliability of detection. It is also applicable to various detection platforms such as targeted pathogen sequencing, real-time fluorescence PCR, and digital PCR, demonstrating significant innovation compared to existing primers and technologies. Attached Figure Description

[0087] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required for the specific embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0088] Figure 1 This is the primer design process of the present invention. Detailed Implementation

[0089] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0090] Where specific experimental steps or conditions are not specified in the examples, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available products. Unless otherwise specified, all raw materials used are commercially available products that are routinely obtainable by those skilled in the art.

[0091] Example 1

[0092] A primer design method for drug resistance genes of reproductive tract pathogens, comprising the following steps, the process of which is as follows: Figure 1 As shown:

[0093] Step 1) Obtain information on drug resistance genes and mutation sites of pathogenic microorganisms related to the female reproductive tract;

[0094] Step 2) Obtain as many drug resistance gene reference sequences as possible based on the drug resistance gene information obtained in Step 1, and then filter the obtained drug resistance gene reference sequences to obtain high-quality drug resistance gene reference sequences.

[0095] Step 3) Obtain the conserved region sequence of the drug resistance gene based on the high-quality drug resistance gene reference sequence obtained in Step 2;

[0096] Step 4) Design primers based on the conserved region sequences obtained in Step 3;

[0097] Step 5) Screen the primers obtained in Step 4 according to the standard. The screening conditions are: primer length 18-25bp, GC content 30%-70%, annealing temperature 57-63℃, specificity and coverage value greater than 90, and avoidance of primer dimers and hairpin structures, to obtain the final primer set.

[0098] In step one), the information on drug resistance genes and mutation sites of pathogenic microorganisms related to the female reproductive tract is obtained by consulting the latest literature, expert consensus and clinical data from websites such as Google Scholar and CNKI, and finally determining the main drug resistance genes and drug resistance sites of the main pathogenic microorganisms that are mainly infected in the female reproductive tract for primer design.

[0099] In step two, based on the drug resistance gene information obtained in step one, as many drug resistance gene reference sequences as possible are obtained from databases such as NCBI, CARD, and ENA. This provides more drug resistance gene reference sequences for subsequent prediction of conserved regions and calculation of coverage values. Based on whether the obtained drug resistance gene reference sequences contain N bases, drug resistance gene reference sequences containing N bases are removed to obtain high-quality drug resistance gene reference sequences, making the results more reliable.

[0100] After obtaining the high-quality drug resistance gene reference sequence in step three), software such as Clone Manager and MAFFT can be used to obtain the conserved region sequence of the drug resistance gene.

[0101] Optionally, in step five), the obtained primers are screened. Specificity is calculated by aligning the obtained primer sequences to the CARD database using BLAST software. Specificity equals the number of aligned drug-resistant gene sequences divided by the total number of aligned sequences to the CARD database, multiplied by 100. Coverage is calculated by aligning the obtained primer sequences to the high-quality drug-resistant gene reference sequences obtained in step two using BLAST software. Coverage equals the number of aligned drug-resistant gene sequences divided by the total number of high-quality drug-resistant gene reference sequences, multiplied by 100. Primer dimers and hairpin structures can be viewed using software such as mfeprimer. If primer dimers and hairpin structures are found, return to step four and redesign primers.

[0102] Primers for drug resistance genes and mutation sites of reproductive tract pathogens were developed. The nucleotide sequences and primer information of these primers are shown in Table 1, totaling 43 pairs. These primers can be used to simultaneously detect drug resistance genes and their corresponding resistance sites. These primers include 11 species of major female reproductive tract pathogens: *Mycoplasma genitalium*, *Neisseria gonorrhoeae*, *Streptococcus agalactiae*, *Staphylococcus aureus*, *Ureaplasma urealyticum*, *Ureaplasma microsporum*, *Mycoplasma hominis*, *Trichomonas vaginalis*, *Candida albicans*, *Candida tropicalis*, and *Candida glabrata*. Fourteen drug resistance genes were identified: 23S rRNA, parC, 16S rRNA, penA, ftsX, PBP2x, gyrA, grlA, Gras, rpoB, ntr6, ERG11, UPC2, and ERE6.A2058C / G / T (nucleic acid), A2059C / G / T (nucleic acid), A247C&T249G (S83R), G248T / A (S83I / N), G259A / T (D87N / Y), A260G (D87G), C1192T (nucleic acid), C932T (nucleic acid), A501T (GCG1501-1503), R251H (CGC751-753), A256G (nucleic acid), C2611T (nucleic acid), V405A (GTT1213-1215), Q557E (CAA1669-1671), S81L (TCA241-243), S79K(TCC235-237), G2576T (nucleic acid), S80F(TCC238-240), E84K(GAA250-252), T136I(ACA406-408), H481Y(CAT1441-1443), D82N(GAT244-246), E87Q(GAA259-261), E87K(GAA259-261), S83L(TCA247-249), S153L(TCA457-459), S91I(AGT271-273), K 80S(AAG238-240), F126L(TTC376-378), Y132H(TAT394-396), S405F(TCT1213-1215), F449S / V(TTT1345-1347), R467K(AGA1399- 1401), I471T (ATT1411-1413), V125A (GTT373-375), Y132F (TAT394-396), S154F (TCT460-462), Y221F (TAT661-663), R245K (AGA73 A total of 48 drug resistance sites were identified, including Y257H (TAC769-771), V362I (GTT1084-1086), G464S (GGT1390-1392), G315D (GGT943-945), Q340H (CAA1018-1020), T381S (ACT1141-1143), Y192 (TAC574-576UAA / UAG / UGA), W286 (TGG856-858UAA / UAG / UGA), and L341 (CTA1021-1023UAA / UAG / UGA).

[0103] Table 1 Primers corresponding to drug resistance genes

[0104]

[0105] Example 2

[0106] Based on the primer design method for a drug resistance gene of a reproductive tract pathogen in Example 1, taking the R251H (CGC751-753) resistance site of the ftsX drug resistance gene of Neisseria gonorrhoeae as an example, primers were designed according to each step, and the primer information is shown in Table 2 below:

[0107] Table 2 Primer information for Neisseria gonorrhoeae drug resistance genes

[0108]

[0109] The amplicon sequence of this primer is TTTGCGGTTGGCTGCTCTctgccgtgcgccccttggtcgatgccatcttcaaaccCTACGGCTTGAACATCGGCT. Its amplicon sequence uses the sequence of the ftsX drug resistance gene of Neisseria gonorrhoeae with GeneID 66754195 from the Gene sub-database of the NCBI database as a positional reference. The amplification positions are 725-799, and the resistance sites are 751-753, corresponding to positions 27-29 of the aforementioned amplicon sequence. The primers designed using this method can simultaneously amplify drug resistance genes and their corresponding drug resistance sites. Assuming this primer is applied to targeted pathogen sequencing, regardless of the sequencing strategy used (SE50, SE100, SE150, PE50, PE100, PE150), because the amplicon length is 75 bp and the drug resistance site is located at positions 27-29 of the amplicon sequence, both the preceding and following sequencing fragments will contain this site. Therefore, simultaneous detection of drug resistance genes and their drug resistance sites can be achieved. This primer helps provide technical support for accurate diagnosis and treatment and has significant clinical application value.

[0110] Using targeted pathogen sequencing analysis, the primers were applied to six actual clinical samples. The sequencing analysis results are shown in Table 3, indicating that the drug resistance gene could be detected in all six samples, with a detection rate as high as 100%.

[0111] Table 3 Sample test results

[0112]

[0113] Comparative Example 1

[0114] Primers were designed using Oligo 7, a common primer design software, without reference sequence quality filtering, conserved region prediction, or primer screening. The resulting primer sequences and information are shown in Table 4.

[0115] Table 4. Primer design information for Oligo 7

[0116]

[0117] The amplicon sequence was ACTACTTCTCGCTGCACGTCgaatccgcgcgctccgcactcaaacagcttctgcgccaacccttcGGCACACTGCTTACCCTCAT. Using the ftsX sequence of the gonococcal resistance gene (GeneID 66754195) as a positional reference, the amplification position was 14-98. Only the resistance gene was detected, but the resistance site itself was not. This primer was applied to targeted pathogen sequencing analysis of six actual clinical samples, similar to Example 1. The sequencing results are shown in Table 5, indicating that only four of the six samples tested positive for the resistance gene, a detection rate of only 66.67%. Furthermore, the number of reads detected in the corresponding samples was lower than in Example 1, indicating poor primer performance.

[0118] Table 5 Comparative test results

[0119]

[0120] Example 3

[0121] The primers designed in Example 1 of this invention are applicable to various detection platforms such as targeted pathogen sequencing, real-time fluorescence PCR, and digital PCR. In this example, multiple gynecological clinical reproductive tract secretion samples were mixed to obtain a mixed sample containing all 43 drug resistance gene loci mentioned above. Then, the primers of Example 1 were applied to the targeted pathogen genome sequencing of the mixed secretion sample for testing. The results showed that all 43 primer pairs were detected, as shown in Table 6, indicating that the primers designed in this invention have a reliability of up to 100%.

[0122] Table 6 Test Results

[0123]

[0124] Example 4

[0125] To further verify the applicability of the designed primers in real samples, we extracted DNA from various genital tract infection samples collected clinically, and used the primers designed in Example 1 for detection, analyzing the detection of drug resistance genes and sites. This experiment employed both real-time fluorescence PCR and targeted pathogen genome sequencing platforms to evaluate the sensitivity and accuracy of the methods. Vaginal secretions, urine, or urethral secretions were collected from 100 patients suspected of having genital tract infections. These samples came from patients of different ages, sexes, and infection backgrounds.

[0126] qPCR testing was performed on 100 samples, and the positive detection results are shown in Table 7 below:

[0127] Table 7 qPCR Detection Results

[0128]

[0129] The qPCR positive products from 100 samples were subjected to targeted pathogen sequencing. The distribution and detection rate of drug resistance genes and sites are shown in Table 8 below.

[0130] Table 8 Targeted pathogen sequencing results

[0131]

[0132] Based on the above test results, in actual samples, the primers designed in this invention can efficiently amplify target drug resistance genes and sites, with the consistency between qPCR and targeted sequencing detection results exceeding 90%. The positive detection rate of drug resistance gene sites in various species is high, demonstrating the high sensitivity and specificity of the method. The number of reads from targeted pathogen genome sequencing indicates that the designed primers have good coverage and amplification efficiency. The reliability and applicability of the primers designed in this invention in detecting actual samples have been fully verified, providing an effective tool for detecting drug resistance in reproductive tract pathogens and possessing significant clinical application value.

[0133] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A primer set for detecting drug resistance genes of pathogenic microorganisms in the reproductive tract, characterized in that, include: Primers used to detect the parC resistance gene in Mycoplasma genitalium; Primers used to detect the penA and ftsX resistance genes of Neisseria gonorrhoeae; Primers for detecting the PBP2x, gyrA, and parC resistance genes in Streptococcus agalactiae; Primers for detecting Staphylococcus aureus grlA, Gras and rpoB resistance genes; Primers used to detect the parC resistance gene in Ureaplasma urealyticum; Primers used to detect the parC resistance gene of Ureaplasma parvovirus; Primers used to detect the gyrA and parC drug resistance genes in Mycoplasma hominis; Primers used to detect the NTR6 resistance gene in Trichomonas vaginalis; Primers used to detect the ERG11 resistance gene in Candida albicans; Primers used to detect the ERG11 and UPC2 resistance genes in Candida tropicalis; Primers used to detect the ERG11 and ERE6 resistance genes in Candida glabrata; The primers used to detect the parC resistance gene in Mycoplasma genitalium include: Primers for detecting the A247C&T249G and G248T / A sites of the parC resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4; The upstream primer nucleotide sequence for detecting the G259A / T and A260G sites of the parC drug resistance gene is shown in SEQ ID NO.5, and the downstream primer nucleotide sequence is shown in SEQ ID NO.

6. The primers used to detect the penA and ftsX resistance genes of Neisseria gonorrhoeae include: Primers for detecting the C932T site of the penA resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.9, and the downstream primer nucleotide sequence is shown in SEQ ID NO.10; Primers for detecting the A501T site of the penA resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.11, and the downstream primer nucleotide sequence is shown in SEQ ID NO.12; Primers for detecting the R251H site of the ftsX resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.13, and the downstream primer nucleotide sequence is shown in SEQ ID NO.14; The primers used to detect the PBP2x, gyrA, and parC resistance genes in Streptococcus agalactiae include: Primers for detecting the V405A site of the PBP2x resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.19, and the downstream primer nucleotide sequence is shown in SEQ ID NO.20; Primers for detecting the Q557E site of the PBP2x resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.21, and the downstream primer nucleotide sequence is shown in SEQ ID NO.22; Primers for detecting the S81L site of the gyrA resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.23, and the downstream primer nucleotide sequence is shown in SEQ ID NO.24; Primers for detecting the S79K site of the parC drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.25, and the downstream primer nucleotide sequence is shown in SEQ ID NO.26; The primers used to detect Staphylococcus aureus grlA, Gras and rpoB resistance genes include: Primers for detecting the S80F site of the grlA drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.29, and the downstream primer nucleotide sequence is shown in SEQ ID NO.30; Primers for detecting the E84K site of the grlA drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.31, and the downstream primer nucleotide sequence is shown in SEQ ID NO.32; Primers for detecting the T136I site of the Gras resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.33, and the downstream primer nucleotide sequence is shown in SEQ ID NO.34; Primers for detecting the H481Y site of the rpoB resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.35, and the downstream primer nucleotide sequence is shown in SEQ ID NO.36; The primers used to detect the parC resistance gene of Ureaplasma urealyticum include: The primers used to detect the D82N site of the parC drug resistance gene have the following nucleotide sequences: upstream primer as shown in SEQ ID NO.37 and downstream primer as shown in SEQ ID NO.

38. Primers for detecting the E87Q and E87K sites of the parC drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.39, and the downstream primer nucleotide sequence is shown in SEQ ID NO.40; The primers used to detect the parC resistance gene of Ureaplasma parvovirus include: Primers for detecting the S83L site of the parC drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.41, and the downstream primer nucleotide sequence is shown in SEQ ID NO.42; The primers used to detect the mycoplasma hominis gyrA and parC drug resistance genes include: Primers for detecting the S153L site of the gyrA resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.43, and the downstream primer nucleotide sequence is shown in SEQ ID NO.44; Primers for detecting the S91I site of the parC drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.45, and the downstream primer nucleotide sequence is shown in SEQ ID NO.46; The primers used to detect the NTR6 resistance gene of Trichomonas vaginalis include: Primers for detecting the K80S site of the ntr6 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.47, and the downstream primer nucleotide sequence is shown in SEQ ID NO.48; The primers used to detect the ERG11 resistance gene in Candida albicans include: Primers for detecting the F126L site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.49, and the downstream primer nucleotide sequence is shown in SEQ ID NO.50; Primers for detecting the Y132H site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.51, and the downstream primer nucleotide sequence is shown in SEQ ID NO.52; Primers for detecting the S405F site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.53, and the downstream primer nucleotide sequence is shown in SEQ ID NO.54; Primers for detecting the F449S / V site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO. 55, and the downstream primer nucleotide sequence is shown in SEQ ID NO. 56; Primers for detecting the R467K site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.57, and the downstream primer nucleotide sequence is shown in SEQ ID NO.58; Primers for detecting the I471T site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.59, and the downstream primer nucleotide sequence is shown in SEQ ID NO.60; The primers used to detect the ERG11 and UPC2 resistance genes in Candida tropicalis include: Primers for detecting the V125A and Y132F sites of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO. 61, and the downstream primer nucleotide sequence is shown in SEQ ID NO. 62; Primers for detecting the S154F site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.63, and the downstream primer nucleotide sequence is shown in SEQ ID NO.64; Primers for detecting the Y221F site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.65, and the downstream primer nucleotide sequence is shown in SEQ ID NO.66; Primers for detecting the R245K site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.67, and the downstream primer nucleotide sequence is shown in SEQ ID NO.68; Primers for detecting the Y257H site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.69, and the downstream primer nucleotide sequence is shown in SEQ ID NO.70; Primers for detecting the V362I site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.71, and the downstream primer nucleotide sequence is shown in SEQ ID NO.72; Primers for detecting the G464S site of the ERG11 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.73, and the downstream primer nucleotide sequence is shown in SEQ ID NO.74; Primers for detecting the Q340H site of the UPC2 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.75, and the downstream primer nucleotide sequence is shown in SEQ ID NO.76; Primers for detecting the T381S site of the UPC2 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.77, and the downstream primer nucleotide sequence is shown in SEQ ID NO.78; The primers used to detect the ERG11 and ERE6 resistance genes of Candida glabrata include: Primers for detecting the Y192 site of the ERE6 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.79, and the downstream primer nucleotide sequence is shown in SEQ ID NO.80; Primers for detecting the W286 site of the ERE6 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.81, and the downstream primer nucleotide sequence is shown in SEQ ID NO.82; Primers for detecting the L341 site of the ERE6 resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.83, and the downstream primer nucleotide sequence is shown in SEQ ID NO.84; Primers for detecting the G315D site of the ERG11 resistance gene, with the upstream primer nucleotide sequence shown in SEQ ID NO.85 and the downstream primer nucleotide sequence shown in SEQ ID NO.

86.

2. The use of the primer set according to claim 1 in the preparation of products for detecting drug resistance genes of reproductive tract pathogens.

3. A reagent kit, characterized in that, Includes the primer set as described in claim 1.

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