Primer for detecting drug-resistant genes of genital tract pathogenic microorganisms and design method thereof

Abstract

According to the primer for detecting the drug-resistant genes of the genital tract pathogenic microorganisms and the design method of the primer, the obtained primer group can cover main drug-resistant genes and drug-resistant sites of various main genital tract pathogenic microorganisms, and multi-target rapid detection of the drug-resistant genes and mutation sites is achieved; the optimally designed primer avoids non-specific amplification, improves the detection reliability, and is suitable for various detection platforms such as targeted pathogen sequencing, real-time fluorescent PCR, digital PCR and the like.

Description

Technical Field

[0001] The invention relates to a primer for detecting drug-resistant genes of reproductive tract pathogenic microorganisms and a design method thereof, belonging to the field of biotechnology. Background Art

[0002] The female reproductive tract microecology is relatively complex. Female reproductive tract infectious diseases may be caused by a variety of pathogenic microorganisms (such as bacterial vaginosis, candidiasis, gonorrhea, etc.), and complex infections may occur. In addition, during the treatment process, the problem of drug resistance is becoming increasingly serious, such as resistance to antibiotics such as penicillin, tetracycline, vancomycin, and quinolones. Traditional microbial culture and drug sensitivity tests are time-consuming and low in sensitivity. Molecular detection technology has gradually attracted attention due to its rapid and efficient characteristics. However, existing molecular diagnostic technology still has shortcomings in the following aspects. The types of pathogenic microorganisms in the reproductive tract are complex, but most tests are only for a single target with low efficiency, and it is impossible to detect diversified pathogens; some drug resistance is closely related to point mutations, and it is difficult for existing primers to take into account both specificity and sensitivity. There are limitations in the detection of drug-resistant mutation sites; primer dimers and nonspecific amplification problems are prone to occur in multi-target detection.

[0003] Therefore, it is necessary to develop a primer and design method that can quickly, efficiently and multiplexly detect the resistance genes and mutation sites of reproductive tract pathogens, so as to achieve the simultaneous detection of multiple resistance genes and mutation sites of multiple pathogens. This can be suitable for rapid clinical diagnosis, reduce experimental time and cost, and has important clinical and social significance. Summary of the invention

[0004] The purpose of the present invention is to provide a specific primer design method and primer set for female reproductive tract pathogenic microorganisms, which can achieve efficient detection of drug-resistant genes and their mutation sites at the same time, and has both high sensitivity and high specificity. The present invention adopts the following technical solutions: A primer for detecting drug-resistant genes of reproductive tract pathogenic microorganisms, comprising: Primers for detecting the 23SrRNA, parC, and 16SrRNA resistance genes of Mycoplasma genitalium; Primers for detecting gonococcal penA, ftsX and 23SrRNA resistance genes; Primers for detecting PBP2x, gyrA, and parC resistance genes of S. agalactiae; Primers for detecting 23SrRNA, grlA, Gras and rpoB resistance genes of Staphylococcus aureus; Primers for detecting the parC resistance gene of Ureaplasma urealyticum; Primers for detecting the parC resistance gene of Ureaplasma parvum; Primers for detecting the gyrA and parC resistance genes of Mycoplasma hominis; Primers for detecting the ntr6 resistance gene of Trichomonas vaginalis; Primers for detecting the ERG11 drug resistance gene of Candida albicans; Primers for detecting the ERG11 and UPC2 resistance genes of Candida tropicalis; Primers used to detect the ERG11 and ERE6 resistance genes of Candida glabrata.

[0005] Wherein, the primers for detecting Mycoplasma genitalium 23SrRNA, parC and 16SrRNA resistance genes include: Primers for detecting the A2058C / G / T (nucleic acid) site and the A2059C / G / T (nucleic acid) site of the 23SrRNA drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2; Primers for detecting the A247C&T249G (S83R) site and the G248T / A (S83I / N) site of the parC drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4; The upstream primer nucleotide sequence of the primers used to detect the G259A / T (D87N / Y) site and the A260G (D87G) site 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; Primers for detecting the C1192T (nucleic acid) site of the 16SrRNA drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8; The primers for detecting gonococcal penA, ftsX and 23SrRNA drug resistance genes include: Primers for detecting the C932T (nucleic acid) site of the penA drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.10; 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; Primers for detecting the R251H (CGC751-753) site of the ftsX drug 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; Primers for detecting the A256G (nucleic acid) site of the 23SrRNA drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.15, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.16; Primers for detecting the C2611T (nucleic acid) site of the 23SrRNA drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.17, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.18; The primers for detecting the drug-resistant genes of Streptococcus agalactiae PBP2x, gyrA and parC include: Primers for detecting the V405A (GTT1213-1215) site of the PBP2x drug 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 (CAA1669-1671) site of the PBP2x drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.21, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.22; Primers for detecting the S81L (TCA241-243) site of the gyrA drug 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 (TCC235-237) 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 for detecting Staphylococcus aureus 23SrRNA, grlA, Gras and rpoB drug resistance genes include: Primers for detecting the G2576T (nucleic acid) site of the 23SrRNA 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; 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; 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; 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; 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; The primers for detecting the ureaplasma urealyticum parC drug resistance gene include: 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; Primers for detecting the E87Q (GAA259-261) and E87K (GAA259-261) sites of the parC drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.39, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.40; The primers for detecting the parC drug resistance gene of Ureaplasma parC include: 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; The primers for detecting the gyrA and parC drug resistance genes of Mycoplasma hominis include: Primers for detecting the S153L (TCA457-459) site of the gyrA drug 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 (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; The primers for detecting the drug-resistant gene of Trichomonas vaginalis ntr6 include: 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; The primers for detecting the Candida albicans ERG11 drug resistance gene include: Primers for detecting the F126L (TTC376-378) site of the ERG11 drug 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 (TAT394-396) site of the ERG11 drug 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 (TCT1213-1215) site of the ERG11 drug 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 (TTT1345-1347) site of the ERG11 drug 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 (AGA1399-1401) site of the ERG11 drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.57, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.58; Primers for detecting the I471T (ATT1411-1413) site of the ERG11 drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.59, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.60; The primers for detecting tropical Candida ERG11 and UPC2 drug resistance genes include: Primers for detecting the ERG11 drug resistance gene V125A (GTT373-375) and Y132F (TAT394-396 sites, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.61, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.62; Primers for detecting the S154F (TCT460-462) site of the ERG11 drug 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 (TAT661-663) site of the ERG11 drug 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 (AGA733-735) site of the ERG11 drug 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 (TAC769-771) site of the ERG11 drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.69, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.70; Primers for detecting the V362I (GTT1084-1086) site of the ERG11 drug 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 (GGT1390-1392) site of the ERG11 drug 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 (CAA1018-1020) site of the UPC2 drug 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 (ACT1141-1143) site of the UPC2 drug 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; Primers for detecting the ERG11 and ERE6 resistance genes of Candida glabrata; Primers for detecting the Y192 (TAC574-576UAA / UAG / UGA) site of the ERE6 drug 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 (TGG856-858UAA / UAG / UGA) site of the ERE6 drug 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 (CTA1021-1023UAA / UAG / UGA) site of the ERE6 drug 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; The primers used to detect the G315D (GGT943-945) site of the ERG11 drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.85, and the downstream primer nucleotide sequence is shown in SEQ ID NO.86.

[0006] The present invention also proposes the use of the above primers in preparing products for detecting drug-resistant genes of reproductive tract pathogenic microorganisms.

[0007] The present invention also provides a kit, comprising the above primers.

[0008] The present invention also proposes a method for designing primers for drug-resistant genes of reproductive tract pathogenic microorganisms, comprising the following steps: Step 1) Obtain information on drug-resistant genes and mutation sites of reproductive tract pathogenic microorganisms; Step 2) obtaining a drug-resistant gene reference sequence according to the drug-resistant gene information obtained in step 1), and then filtering the obtained drug-resistant gene reference sequence to obtain a high-quality drug-resistant gene reference sequence; Step 3) obtaining the conserved region sequence of the drug-resistant gene based on the high-quality drug-resistant gene reference sequence obtained in step 2); Step 4) designing primers based on the conserved region sequences obtained in step 3); Step five) screening the primers obtained in step four) to obtain primers for drug resistance genes of reproductive tract pathogenic microorganisms.

[0009] Optionally, in the step 2), according to the drug-resistant gene information obtained in step 1), drug-resistant gene reference sequences are obtained from the NCBI, CARD and ENA databases, and according to whether the obtained drug-resistant gene reference sequences contain N bases, the drug-resistant gene reference sequences containing N bases are eliminated to obtain high-quality drug-resistant gene reference sequences.

[0010] Optionally, after obtaining the high-quality drug-resistant gene reference sequence in step 3), use Clone Manager or MAFFT software to obtain the conserved region sequence of the drug-resistant gene.

[0011] Optionally, the screening conditions in step five) are: primer length 18-25bp, GC content 30%-70%, annealing temperature 57-63°C, specificity and coverage values ​​both greater than 90, and no primer dimers and / or hairpin structures are generated. If the primer fails the screening, return to step four for redesign.

[0012] Optionally, the method for calculating the specificity in step 5) includes: aligning the primer sequence to the CARD database using BLAST software; the specificity value is the percentage of the number of corresponding drug-resistant gene sequences aligned by the primer to the number of sequences aligned by the primer in the CARD database; The calculation method of the coverage value includes: aligning the primer sequence to the high-quality drug-resistant gene reference sequence obtained in step 2) using BLAST software; the coverage value is the percentage of the number of corresponding drug-resistant gene sequences on the primer alignment to the number of corresponding drug-resistant genes in the high-quality drug-resistant gene reference sequence.

[0013] Optionally, in step 5), mfeprimer software is used to check whether the primers will generate primer dimers and / or hairpin structures.

[0014] Compared with the prior art, the present invention has the following technical effects: The present invention screens and obtains high-quality drug-resistant gene sequences based on the information of the main drug-resistant genes and drug-resistant sites of the main pathogenic microorganisms related to reproductive tract infections, and obtains the conservative region sequences of the drug-resistant genes on this basis. According to the conservative region sequences, primers are designed to obtain corresponding primers, and then the final primer set is screened according to conditions such as primer length, annealing temperature, and specificity. The primer set obtained by this method can cover the main drug-resistant genes and drug-resistant sites of various main reproductive tract pathogenic microorganisms, realize multi-target rapid detection of drug-resistant genes and mutation sites, and the optimized designed primers avoid non-specific amplification and improve the reliability of detection. At the same time, it is suitable for multiple detection platforms such as targeted pathogen sequencing, real-time fluorescence PCR, and digital PCR. Compared with existing primers and technologies, it has obvious innovation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for the specific implementation methods or the prior art are briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is the primer design process of the present invention. DETAILED DESCRIPTION

[0017] The present invention is further elaborated in detail below in conjunction with specific embodiments. The following embodiments are provided for a better understanding of the present invention, but are not limited to the best implementation mode, and do not limit the content and protection scope of the present invention. Any product that is the same or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the present invention with the features of other prior arts falls within the protection scope of the present invention.

[0018] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the art can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially. The raw materials used are not specifically specified, and they are all commercially available products that can be routinely obtained by those skilled in the art.

[0019] Example 1 A method for designing primers for drug-resistant genes of reproductive tract pathogenic microorganisms, comprising the following steps, the process is as follows Figure 1 As shown: Step 1) Obtain information on drug-resistant genes and mutation sites of pathogenic microorganisms related to the female reproductive tract; Step 2) obtaining as many drug-resistant gene reference sequences as possible based on the drug-resistant gene information obtained in step 1, and then filtering the obtained drug-resistant gene reference sequences to obtain high-quality drug-resistant gene reference sequences; Step 3) Obtaining the conserved region sequence of the drug-resistant gene based on the high-quality drug-resistant gene reference sequence obtained in step 2; Step 4) Designing primers based on the conserved region sequences obtained in step 3; Step 5) The primers obtained in step 4 were screened according to the standard. The screening conditions were primer length 18-25 bp, GC content 30%-70%, annealing temperature 57-63°C, specificity and coverage value greater than 90, and avoidance of primer dimers and hairpin structures to obtain the final primer set.

[0020] Among them, the information on drug-resistant genes and mutation sites of pathogenic microorganisms related to the female reproductive tract in the step 1) is obtained mainly by consulting the latest literature, expert consensus and clinical data on websites such as Google Scholar and CNKI, and finally determining the main drug-resistant genes and drug-resistant site information of pathogenic microorganisms related to the main female reproductive tract infections used to design primers; In the step 2), based on the drug-resistant gene information obtained in the step 1, as many drug-resistant gene reference sequences as possible are obtained from databases such as NCBI, CARD and ENA, so as to provide more drug-resistant gene reference sequences for subsequent prediction of conservative regions and calculation of coverage values; based on whether the obtained drug-resistant gene reference sequences contain N bases, the drug-resistant gene reference sequences containing N bases are eliminated to obtain high-quality drug-resistant gene reference sequences, so that the results are more reliable.

[0021] After obtaining the high-quality drug-resistant gene reference sequence in step 3), the conservative region sequence of the drug-resistant gene can be obtained using software such as Clone Manager and MAFFT.

[0022] Optionally, the primers obtained in step five) are screened, and the specificity is calculated by aligning the primer sequences obtained with the CARD database using BLAST software, and the specificity is equal to the number of matched drug-resistant gene sequences themselves divided by the number of all matched CARD database sequences multiplied by 100; the coverage value is calculated by aligning the primer sequences obtained with the high-quality drug-resistant gene reference sequences obtained in step two using BLAST software, and the coverage value is equal to the number of matched drug-resistant gene sequences themselves divided by the number of all high-quality drug-resistant gene reference sequences themselves 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) to redesign the primers.

[0023] A primer for drug resistance genes and mutation sites of reproductive tract pathogenic microorganisms. The nucleotide sequences and primer information of these primers are finally obtained as shown in Table 1, and a total of 43 pairs of primers are obtained. These primers can be used to simultaneously detect drug resistance genes and their corresponding drug resistance sites. These primers include 11 species of major female reproductive tract pathogenic microorganisms: Mycoplasma genitalium, gonococci, Streptococcus agalactiae, Staphylococcus aureus, Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, Trichomonas vaginalis, Candida albicans, Candida tropicalis, and Candida glabrata. There are 14 drug resistance genes in total: 23SrRNA, parC, 16SrRNA, 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 3-735), 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), L341 (CTA1021-1023UAA / UAG / UGA), a total of 48 resistance sites.

[0024] Table 1 Primers corresponding to drug resistance genes

[0025] Example 2 Based on the primer design method for a drug-resistant gene of a reproductive tract pathogenic microorganism in Example 1, taking the R251H (CGC751-753) drug-resistant site of the drug-resistant gene ftsX of gonococci as an example, primers are designed according to each step, and the primer information is shown in Table 2 below: Table 2 Primer information of gonococcal resistance genes

[0026] The amplicon sequence of the primer is TTTGCGGTTGGCTGCTCTctgccgtgcgccccttggtcgatgccatcttcaaaccCTACGGCTTGAACATCGGCT. The amplicon sequence uses the sequence of the drug resistance gene ftsX of gonococci with GeneID 66754195 in the Gene sub-database of the NCBI database as a position reference. The amplification position is 725-799, and the drug resistance site is 751-753, which corresponds to the 27th-29th position of the above amplicon sequence. It was found that the primers designed by this method can simultaneously amplify the drug-resistant gene and its corresponding drug-resistant site. Assuming that the primers are applied to targeted pathogen sequencing, no matter the sequencing strategy of SE50, SE100, SE150, PE50, PE100, or PE150 is used, because the length of the amplicon is 75bp and the drug-resistant site is at the 27th-29th position of the amplicon sequence, the sequencing fragments at the front and back ends will include the site, so the drug-resistant gene and its drug-resistant site can be detected simultaneously. This primer helps to provide technical support for precise diagnosis and treatment and has important clinical application value.

[0027] The primers were applied to 6 actual clinical samples using targeted pathogen sequencing analysis. The sequencing analysis results are shown in Table 3, indicating that the drug-resistant gene could be detected in all 6 samples, with a detection rate of up to 100%.

[0028] Table 3 Sample test results

[0029] Comparative Example 1 Using common primer design software, Oligo 7, primer design was performed without reference sequence quality filtering, conserved region prediction, and primer screening. The primer sequences and information obtained are shown in Table 4: Table 4 Oligo 7 designed primer information

[0030] The amplicon sequence is ACTACTTCTCGCTGCACGTCgaatccgcgcgctccgcactcaaacagcttctgcgccaacccttcGGCACACTGCTTACCCTCAT. The sequence of the drug-resistant gene ftsX of gonococci with GeneID 66754195 is used as a position reference. The amplification position is 14-98, and only the drug-resistant gene can be detected but the drug-resistant site cannot be detected. The primer is applied to the targeted pathogen sequencing analysis of the same 6 actual clinical samples as in Example 1. The sequencing analysis results are shown in Table 5, indicating that only 4 of the 6 samples can detect the drug-resistant gene, the detection rate is only 66.67%, and the number of reads detected by the corresponding sample is not as high as that in Example 1, and the primer effect is poor.

[0031] Table 5 Comparative Example Test Results

[0032] Example 3 The primers designed in Example 1 of the present invention can be applied to a variety of detection platforms such as targeted pathogen sequencing, real-time fluorescence PCR, and digital PCR. In this example, multiple gynecological clinical genital secretion samples were mixed to obtain a mixed sample containing all 43 drug-resistant gene sites mentioned above, and then the primers in Example 1 were applied to the targeted pathogen genome sequencing of the mixed secretion samples for testing. The results showed that all 43 pairs of primers were detected, as shown in Table 6, indicating that the reliability of the primers designed by the present invention is as high as 100%.

[0033] Table 6 Test results

[0034] Example 4 In order to further verify the applicability of the designed primers in actual samples, we extracted DNA from a variety of genital tract infection samples collected clinically, used the primers designed in Example 1 for detection, and analyzed the detection of drug-resistant genes and sites. This experiment used two platforms, real-time fluorescence PCR and targeted pathogen genome sequencing, for detection to evaluate the sensitivity and accuracy of the method. Vaginal secretions, urine or urethral secretions samples from 100 patients suspected of genital tract infection were collected. These samples came from patients of different ages, genders and infection backgrounds.

[0035] 100 samples were tested by qPCR, and the positive results are shown in Table 7 below: Table 7 qPCR test results

[0036] The qPCR positive products of 100 samples were subjected to targeted pathogen sequencing, and the distribution and detection rate of drug resistance genes and sites are shown in Table 8 below: Table 8 Targeted pathogen sequencing results

[0037] According to the above test results, in actual samples, the primers designed by the present invention can efficiently amplify target drug-resistant genes and sites, and the consistency of the detection results of qPCR and targeted sequencing is as high as more than 90%. The positive detection rate of drug-resistant gene sites of various species is high, showing the high sensitivity and specificity of the method. The number of reads of targeted pathogen genome sequencing shows that the designed primers have good coverage and amplification efficiency. The reliability and applicability of the primers designed by the present invention in the detection of actual samples have been fully verified, providing an effective tool for the detection of drug resistance of reproductive pathogenic microorganisms, and having important clinical application value.

[0038] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A primer for detecting drug resistance genes of reproductive tract pathogenic microorganisms, characterized in that: include: Primers for detecting the 23SrRNA, parC, and 16SrRNA resistance genes of Mycoplasma genitalium; Primers for detecting gonococcal penA, ftsX and 23SrRNA resistance genes; Primers for detecting PBP2x, gyrA, and parC resistance genes of S. agalactiae; Primers for detecting 23SrRNA, grlA, Gras and rpoB resistance genes of Staphylococcus aureus; Primers for detecting the parC resistance gene of Ureaplasma urealyticum; Primers for detecting the parC resistance gene of Ureaplasma parvum; Primers for detecting the gyrA and parC resistance genes of Mycoplasma hominis; Primers for detecting the ntr6 resistance gene of Trichomonas vaginalis; Primers for detecting the ERG11 resistance gene of Candida albicans; Primers for detecting the ERG11 and UPC2 resistance genes of Candida tropicalis; Primers for detecting the ERG11 and ERE6 resistance genes of Candida glabrata; Wherein, the primers for detecting Mycoplasma genitalium 23SrRNA, parC and 16SrRNA resistance genes include: Primers for detecting the A2058C / G / T (nucleic acid) site and the A2059C / G / T (nucleic acid) site of the 23SrRNA drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2; Primers for detecting the A247C&T249G (S83R) site and the G248T / A (S83I / N) site of the parC drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4; The upstream primer nucleotide sequence of the primers used to detect the G259A / T (D87N / Y) site and the A260G (D87G) site 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; Primers for detecting the C1192T (nucleic acid) site of the 16SrRNA drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8; The primers for detecting gonococcal penA, ftsX and 23SrRNA drug resistance genes include: Primers for detecting the C932T (nucleic acid) site of the penA drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.10; Primers for detecting the A501T (GCG1501-1503) site of the penA resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.11, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.12; Primers for detecting the R251H (CGC751-753) site of the ftsX drug 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; Primers for detecting the A256G (nucleic acid) site of the 23SrRNA drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.15, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.16; Primers for detecting the 23SrRNA drug resistance gene C2611T (nucleic acid) site, 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; The primers for detecting the drug-resistant genes of Streptococcus agalactiae PBP2x, gyrA and parC include: Primers for detecting the V405A (GTT1213-1215) site of the PBP2x drug 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 (CAA1669-1671) site of the PBP2x drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.21, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.22; Primers for detecting the S81L (TCA241-243) site of the gyrA drug 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 (TCC235-237) 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 for detecting Staphylococcus aureus 23SrRNA, grlA, Gras and rpoB drug resistance genes include: Primers for detecting the G2576T (nucleic acid) site of the 23SrRNA 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; 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; 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; 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; 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; The primers for detecting the ureaplasma urealyticum parC drug resistance gene include: Primers for detecting the D82N (GAT244-246) site of the parC drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.37, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.38; Primers for detecting the E87Q (GAA259-261) and E87K (GAA259-261) sites of the parC drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.39, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.40; The primers for detecting the parC drug resistance gene of Ureaplasma parC include: 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; The primers for detecting the gyrA and parC drug resistance genes of Mycoplasma hominis include: Primers for detecting the S153L (TCA457-459) site of the gyrA drug 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 (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; The primers for detecting the drug-resistant gene of Trichomonas vaginalis ntr6 include: 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; The primers for detecting the Candida albicans ERG11 drug resistance gene include: Primers for detecting the F126L (TTC376-378) site of the ERG11 drug 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 (TAT394-396) site of the ERG11 drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.51, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.52; Primers for detecting the S405F (TCT1213-1215) site of the ERG11 drug 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 (TTT1345-1347) site of the ERG11 drug 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 (AGA1399-1401) site of the ERG11 drug 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 (ATT1411-1413) site of the ERG11 drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.59, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.60; The primers for detecting tropical Candida ERG11 and UPC2 drug resistance genes include: Primers for detecting the ERG11 drug resistance gene V125A (GTT373-375) and Y132F (TAT394-396 sites, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.61, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.62; Primers for detecting the S154F (TCT460-462) site of the ERG11 drug 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 (TAT661-663) site of the ERG11 drug 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 (AGA733-735) site of the ERG11 drug 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 (TAC769-771) site of the ERG11 drug 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 (GTT1084-1086) site of the ERG11 drug resistance gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.71, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.72; Primers for detecting the G464S (GGT1390-1392) site of the ERG11 drug 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 (CAA1018-1020) site of the UPC2 drug 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 (ACT1141-1143) site of the UPC2 drug 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; Primers for detecting the ERG11 and ERE6 resistance genes of Candida glabrata; Primers for detecting the Y192 (TAC574-576UAA / UAG / UGA) site of the ERE6 drug 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 (TGG856-858UAA / UAG / UGA) site of the ERE6 drug 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 (CTA1021-1023UAA / UAG / UGA) site of the ERE6 drug 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; The primers used to detect the G315D (GGT943-945) site of the ERG11 drug resistance gene, the upstream primer nucleotide sequence is shown in SEQ ID NO.85, and the downstream primer nucleotide sequence is shown in SEQ ID NO.

86.

2. Use of the primers according to claim 1 in the preparation of products for detecting drug-resistant genes of reproductive tract pathogenic microorganisms.

3. A kit, characterized in that: Comprising the primer of claim 1.

4. The method for designing primers for drug-resistant genes of reproductive tract pathogenic microorganisms according to claim 1, characterized in that: The steps include: Step 1) Obtain information on drug-resistant genes and mutation sites of reproductive tract pathogenic microorganisms; Step 2) obtaining a drug-resistant gene reference sequence according to the drug-resistant gene information obtained in step 1), and then filtering the obtained drug-resistant gene reference sequence to obtain a high-quality drug-resistant gene reference sequence; Step 3) obtaining the conserved region sequence of the drug-resistant gene based on the high-quality drug-resistant gene reference sequence obtained in step 2); Step 4) designing primers based on the conserved region sequences obtained in step 3); Step five) Screening the primers obtained in step four) to obtain primers for drug resistance genes of reproductive tract pathogenic microorganisms.

5. The method for designing primers for drug-resistant genes of reproductive tract pathogenic microorganisms according to claim 4, characterized in that: In the step 2), according to the drug-resistant gene information obtained in step 1), drug-resistant gene reference sequences are obtained from the NCBI, CARD and ENA databases, and according to whether the obtained drug-resistant gene reference sequences contain N bases, the drug-resistant gene reference sequences containing N bases are eliminated to obtain high-quality drug-resistant gene reference sequences.

6. The method for designing primers for drug-resistant genes of reproductive tract pathogenic microorganisms according to claim 4, characterized in that: After obtaining the high-quality drug-resistant gene reference sequence in step 3), use Clone Manager or MAFFT software to obtain the conservative region sequence of the drug-resistant gene.

7. The method for designing primers for drug-resistant genes of reproductive tract pathogenic microorganisms according to claim 4, characterized in that: The screening conditions in step 5) are: primer length 18-25 bp, GC content 30%-70%, annealing temperature 57-63°C, specificity and coverage values ​​both greater than 90, and no primer dimers and / or hairpin structures are generated. If the primer does not pass the screening conditions, return to step 4) for redesign.

8. The method for designing primers for drug-resistant genes of reproductive tract pathogenic microorganisms according to claim 7, characterized in that: The specificity calculation method in step 5) includes: aligning the primer sequence to the CARD database using BLAST software; the specificity value is the percentage of the number of corresponding drug-resistant gene sequences aligned by the primer to the number of sequences aligned by the primer in the CARD database.

9. The method for designing primers for drug-resistant genes of reproductive tract pathogenic microorganisms according to claim 7, characterized in that: The calculation method of the coverage value in step five) 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 corresponding drug-resistant gene sequences on the primer alignment to the number of corresponding drug-resistant genes in the high-quality drug-resistant gene reference sequence.

10. The method for designing primers for drug-resistant genes of reproductive tract pathogenic microorganisms according to claim 7, characterized in that: In the step 5), the mfeprimer software is used to check whether the primers will generate primer dimers and / or hairpin structures.

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