A primer set and kit for multiplex amplification targeted sequencing for detecting multiple pathogens

Abstract

The present application relates to the technical field of molecular biology, and particularly relates to a primer group and a kit for detecting multiple pathogens through multiplex amplification targeted sequencing; the primer group comprises primers with nucleotide sequences shown in SQE ID NO. 1-SQE ID NO. 442, which are designed according to highly conserved and specific genomic regions of each pathogen, so as to ensure the specificity of amplification and the coverage of different variant strains; the primer group is suitable for prenatal TORCH screening, detection of pathogenic microorganisms of pregnant women with premature birth indications, screening of pathogenic microorganisms of suspected intrauterine infections, and detection of pathogenic microorganisms of unexplained abortion, and can simultaneously detect pathogenic microorganisms such as viruses, bacteria, fungi and parasites, and has high sensitivity and specificity.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to a primer set and kit for multiplex amplification targeted sequencing for detecting various pathogens. Background Technology

[0002] Infectious diseases are a significant threat to human health. Among them, intra-amniotic infection (IAI) is a serious perinatal complication that can directly lead to chorioamnionitis, premature birth, fetal distress, neonatal sepsis, and neurological sequelae (such as cerebral palsy), and even perinatal death. The spectrum of pathogens causing IAI is extremely broad, including but not limited to Group B Streptococcus agalactiae, Escherichia coli, Listeria monocytogenes, Mycoplasma hominis, Ureaplasma urealyticum, Chlamydia trachomatis, human cytomegalovirus (CMV), human parvovirus B19, and many other bacteria, mycoplasma / chlamydia, and viruses.

[0003] Currently, the etiological diagnosis of intrauterine amniotic fluid infection faces significant challenges. Commonly used clinical methods have significant limitations: (1) Microbial culture: It is considered the "gold standard", but it takes a long time (24-72 hours) and the detection rate of pathogens (such as mycoplasma and anaerobic bacteria) and viruses with strict nutritional requirements is extremely low, and false negatives are easy to occur.

[0004] (2) Gram staining microscopy: fast but with poor sensitivity, unable to achieve species-level identification of pathogens.

[0005] (3) Single PCR technology based on specific pathogens: Although it has high sensitivity, it has extremely low throughput and can only be used to "blindly detect" a few clinically suspected pathogens. It cannot cope with the complex and diverse spectrum of IAI pathogens and is very likely to cause mixed infections or missed detection of rare pathogens.

[0006] In recent years, metagenomic next-generation sequencing (mNGS) technology has theoretically shown potential in discovering unknown pathogens by directly sequencing all nucleic acids in amniotic fluid samples without any pre-defined parameters. However, amniotic fluid samples are predominantly composed of host (maternal and fetal) cells and cell-free DNA, with extremely low levels of pathogen nucleic acids. In such samples with a high host background, mNGS technology suffers from severely insufficient sensitivity in detecting low-biomass pathogens. Furthermore, the large volume of sequencing data, high cost, and complex bioinformatics analysis make it difficult to routinely promote and apply in clinical settings, especially in critical intraoperative diagnostic (IAI) scenarios.

[0007] Therefore, there is an urgent need in this field for a detection technology that can overcome the aforementioned shortcomings, is suitable for samples with low pathogen content such as amniotic fluid, and can provide a single, rapid, highly sensitive, and highly specific detection of a wide range of pathogens associated with intrauterine infection. Developing a targeted multiplex sequencing technology is of paramount importance for achieving accurate and comprehensive etiological diagnosis of intrauterine infection in amniotic fluid, guiding rational clinical drug use, and improving pregnancy outcomes. Summary of the Invention

[0008] To address the shortcomings of the existing technologies, this invention aims to provide a primer set and kit for multiplex amplification targeted sequencing to detect a variety of pathogens, specifically targeting multiple key clinical pathogens in amniotic fluid intrauterine infections.

[0009] To solve the above problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a primer set for multiplex amplification targeted sequencing for detecting a variety of pathogens, the primer set comprising primers with nucleotide sequences as shown in SQE ID NO.1-SQE ID NO.442.

[0010] Furthermore, the primer set includes primers with DNA pathogen nucleotide sequences as shown in SQE ID NO.1-SQE ID NO.370 and primers with RNA pathogen nucleotide sequences as shown in SQE ID NO.371-SQE ID NO.442.

[0011] Furthermore, the 5' end of the primer is connected to a universal adapter sequence.

[0012] Furthermore, the aforementioned pathogens include human herpesvirus type 1, human herpesvirus type 2, human herpesvirus type 3, human herpesvirus type 4, human herpesvirus type 5, human herpesvirus type 6, human herpesvirus type 7, human herpesvirus type 8, human parvovirus B19, human papillomavirus-16, human papillomavirus-18, human papillomavirus-26, human papillomavirus-31, human papillomavirus-33, human papillomavirus-35, human papillomavirus-39, and human papillomavirus-45. Human papillomavirus (HPV)-51, HPV-52, HPV-53, HPV-56, HPV-58, HPV-59, HPV-61, HPV-66, HPV-73, HPV-82, HPV-6, HPV-11, HPV-68, HPV-42, HPV-43, HPV-44, Hepatitis B virus, Toxoplasma gondii, blackening Prevotella, Salmonella enterica, Escherichia coli, Listeria monocytogenes, Timonehoyles, Haemophilus ducreyi, Prevotella divaricata, Klebsiella pneumoniae, Mycoplasma pneumoniae, Molecularcurvularia, Ureaplasma urealyticum, Staphylococcus aureus, Molecularcurvularia kohlii, Neisseria gonorrhoeae, Treponema pallidum, Proteus vulgaris, Proteus mirabilis, Prevotella humanis, Mycoplasma hominis, Chlamydia trachomatis, Mycoplasma genitalium, Prevotella bifidum, Ureaplasma microplasma, Streptococcus agalactiae, Prevotella amniotic fluid, Vaginal adenophora Topsporium, Gardnerella vaginalis, Prevotella intermedia, Candida albicans and Movula hymniferum, as well as hepatitis C virus, rubella virus, influenza A virus, influenza A virus H3N2, influenza A virus H1, influenza A virus H1N1, influenza A virus H3, influenza A virus H7, common influenza A virus, mumps virus, human parainfluenza virus type 2, human parainfluenza virus type 4, human immunodeficiency virus type 1, human immunodeficiency virus type 2, SARS-CoV-2 and Zika virus.

[0013] Secondly, the present invention provides a kit for detecting multiple pathogens using multiplex amplification targeted sequencing, comprising the aforementioned primer set.

[0014] The beneficial effects of this invention are as follows: This invention can detect multiple key clinical pathogens at once, with a coverage far exceeding that of conventional detection methods. Based on targeted amplification, it effectively enriches low-abundance pathogen sequences, avoiding interference from the host background. Because the primers are designed for conserved regions, even if the pathogens undergo partial mutations, they can still be effectively detected. Compared with mNGS, targeted sequencing has a smaller data volume and is simpler to analyze, greatly reducing costs and detection cycles. It is suitable for prenatal TORCH screening, pathogen detection in pregnant women with indications for preterm labor, pathogen screening for suspected intrauterine infections, and pathogen detection for unexplained miscarriages. It can simultaneously detect pathogens such as viruses, bacteria, fungi, and parasites, and has high sensitivity and specificity. Attached Figure Description

[0015] Figure 1 This is a flowchart of the testing process.

[0016] Figure 2 The graph shows the sequence number results for different enzyme combinations. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to specific embodiments.

[0018] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0019] Example 1 1. Target selection and primer design: Download the whole genome sequences of all pathogens listed in Table 1 from public databases (such as GenBank and RefSeq). Using multiple sequence alignment tools (such as MAFF), identify highly conserved and species-specific regions within each pathogen and designate them as target sequences. For these target sequences, design PCR primer pairs using specialized primer design software. The design parameters are as follows: primer length 18-25 bp, Tm value 58-64℃, and amplicon length 80-200 bp.

[0020] In terms of bioinformatics, primer pairs that cross-react with non-target sequences or easily form dimers were screened out using in silico PCR and alignment analysis. For each species, a pool of 5-10 specific primer pairs was used for testing, and primers with low amplification efficiency were removed. A final multiplex primer set containing hundreds of primer pairs was obtained, as shown in Table 1. All primers had a universal adapter sequence pre-added to their 5' ends for sequencing on the Illumina platform.

[0021] Table 1. Pathogens and Primers Corresponding to Pathogenic Microorganisms

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029] The primer pairs are designed for highly conserved and specific genomic regions of each pathogen (such as highly conserved gene regions of viruses, housekeeping genes of bacteria, or specific multicopy genes), ensuring amplification specificity and coverage of different variants. Through careful bioinformatics design and experimental optimization, the primer set of this invention can efficiently and uniformly amplify the target sequences of all target pathogens simultaneously in a two-tube (DNA / RNA) multiplex PCR reaction.

[0030] Compared to previous 16S methods that struggled to distinguish closely related species, this invention achieves precise species-level identification of pathogens, significantly improving detection specificity and clinical accuracy, while avoiding data waste and saving sequencing costs. Cross-reactivity experiments confirmed that this invention possesses good specificity and high accuracy.

[0031] The experimental procedure is as follows: Fifteen groups of different standards were mixed to verify the cross-reactivity and competitive reaction of the kit. All species contained in this reagent were correctly detected; standards lacking these species were undetectable, thus meeting the requirements. Detailed information on the standards is shown in Table 2. Standards beginning with BNCC were sourced from Beina Biotechnology Co., Ltd., standards beginning with ATCC were sourced from Ningbo Mingzhou Biotechnology Co., Ltd., and standards beginning with BDS were sourced from Bondsheng Technology Co., Ltd. Specific results are shown in Tables 3-4.

[0032] For detailed steps, please refer to Figure 1 : Sample pretreatment: Clinical samples were optimized for cell wall disruption. Simultaneously, the samples were separated into layers by centrifugation, and only the precipitate was subjected to cell wall disruption. The cell wall disruption parameters were optimized to ensure the integrity of viral nucleic acids and also to preserve the nucleic acids of fungi and Gram-positive bacteria species that are difficult to disrupt.

[0033] Fifteen sets of standards were added to negative amniotic fluid samples to simulate clinical samples. Total nucleic acids were extracted from the simulated clinical samples. The extracted nucleic acid was used as a template and added to the DNA primer pool and RNA primer pool respectively for multiplex PCR amplification to enrich the target pathogen sequence. The RNA amplification system consisted of 5 μL RNA amplification reagent, 0.6 μL primer (2.8 μM), and 10.4 μL nuclease-free water. The DNA amplification system consisted of 15 μL DNA amplification reagent, 4 μL primer (0.5 μM), and 2 μL nuclease-free water. The amplification program was as follows: 55℃ for 15 min; 95℃ for 3 min; 95℃ for 30 s; 60℃ for 1 min; 72℃ for 30 s, for 30 cycles; 72℃ for 3 min; and 4℃ for hold. DNA and RNA products were mixed and purified using 1.2X magnetic beads, followed by direct amplification with the magnetic beads, and then proceeded to the second round of PCR (ligation of sequencing adapters). The amplification reagent consisted of 20 μL, index 2 μL, and nuclease-free water 18 μL. The amplification program was: 98℃, 45s; 95℃, 20s; 60℃, 30s; 72℃, 30s, 30 cycles; 72℃, 5min; 10℃, hold. Sequencing libraries were then constructed. High-throughput sequencing was performed on the library; Bioinformatics methods are used to compare sequencing sequences with pathogen reference databases, and the pathogens present in the samples are determined based on indicators such as sequence number and coverage.

[0034] Table 2. Information on Accuracy and Specificity Standard Samples

[0035]

[0036]

[0037] Note: The species in bold are those included in this invention.

[0038] Table 3. Accuracy and Amplification Results of Each Primer

[0039]

[0040] Table 4 Specificity Results

[0041]

[0042]

[0043] 2. Comparison of different grinding parameters During the experiment, different grinding parameters were meticulously compared to optimize sample processing. The effects of adjusting key parameters such as grinding time, grinding speed, and grinding media on the quality of nucleic acid extraction and subsequent sequencing results were observed. After multiple rounds of experimental verification, the optimal combination of grinding parameters was finally determined, effectively improving the integrity and purity of nucleic acid extraction and providing a high-quality library construction foundation for subsequent high-throughput sequencing. Specific experimental procedures: Equal volumes of Staphylococcus aureus, Streptococcus agalactiae, Candida albicans, Escherichia coli, Klebsiella pneumoniae, and Mycoplasma pneumoniae standards were mixed and diluted 10-fold, serving as templates. Experiments were conducted using five different cell disruption parameters (Table 5), and the Ct values ​​of each species were finally detected using qPCR. A lower Ct value indicates higher extraction efficiency. Scheme 2 was ultimately selected; detailed results are shown in Table 6.

[0044] Table 5 Different cell wall breaking parameters

[0045] Table 6. Ct values ​​for different cell wall breaking parameters

[0046] 3. Comparison of amplification efficiency of different enzyme combinations In this invention, the library construction process involved the design of a DNA amplification enzyme, an RT-PCR enzyme, and a second-round high-fidelity amplification enzyme. To screen for suitable amplification systems, four combinations (A, B, C, and D) were set up. The enzymes included in each combination are detailed in Table 7, and the amplification reaction systems are shown in Tables 8, 9, and 10. The following four enzyme systems were used to detect influenza A virus, parainfluenza virus type 2, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus, with each species tested twice, and the average value was calculated. The mean number of detected reads was rounded to 1M. Scheme 4 was ultimately selected. See the results table for details. Figure 2 .

[0047] Table 7. Enzyme details for different amplification combinations.

[0048] Table 8. RNA Pathogen Reverse Transcription Reaction Mixture

[0049] Table 9 DNA First Round PCR Reaction Mixture

[0050] Table 10 Second Round PCR Reaction Mixture

[0051] 4. Sequencing and Data Analysis: The optimized multiplex PCR products were purified and Illumina sequencing libraries were constructed according to standard procedures. Single-end sequencing was then performed on the MiSeq or NextSeq platform. The data were analyzed using the following bioinformatics workflow.

[0052] First, the raw sequencing data (Raw Reads) are preprocessed by precisely removing adapter sequences to obtain high-quality Clean Reads. Then, denoising and clustering analysis are performed on the Clean Reads to form a standardized Data2 dataset. Based on this, the BLAST algorithm is used to perform homology comparisons between the Data2 dataset and specific pathogen databases to deeply mine potential pathogen information. Finally, an automated analysis workflow integrates the comparison results to generate a professional analysis report containing pathogen species, abundance, and variation characteristics, providing accurate and systematic interpretation of sequencing data for clinical pathogen detection and scientific research analysis.

[0053] Positive determination: The pathogen test result is determined to be positive based on the preset thresholds (coverage>96%, identity>98%).

[0054] Example 2 The primer combination in this invention was used to perform tNGS detection on different samples from 130 clinical patients. The final detection results were verified by qPCR, which showed that they were consistent with the qPCR results. The specific detection results are shown in Table 11.

[0055] Table 11 Clinical Sample Test Results

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[0057]

[0058]

[0059]

[0060] The results showed that the kit of the present invention not only accurately detected all HPV types (including 18, 31, 35, 53, 56, etc.) reported by commercial kits, but also additionally detected co-infecting pathogens such as HPV and microurea in some samples, which was highly consistent with the patients' clinical symptoms. This indicates that the present invention has significant advantages in detecting mixed infections.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A primer set for multiplex amplification targeted sequencing to detect multiple pathogens, characterized in that, The primer set includes primers with nucleotide sequences as shown in SQE ID NO.1-SQE ID NO.

442.

2. The primer set according to claim 1, characterized in that, The primer set includes primers with DNA pathogen nucleotide sequences as shown in SQE ID NO.1-SQE ID NO.370 and primers with RNA pathogen nucleotide sequences as shown in SQE ID NO.371-SQE ID NO.

442.

3. The primer set according to claim 1, characterized in that, The 5' end of the primer is connected to a universal adapter sequence.

4. The primer set according to claim 2, characterized in that, The aforementioned pathogens include human herpesvirus type 1, human herpesvirus type 2, human herpesvirus type 3, human herpesvirus type 4, human herpesvirus type 5, human herpesvirus type 6, human herpesvirus type 7, human herpesvirus type 8, human parvovirus B19, human papillomavirus-16, human papillomavirus-18, human papillomavirus-26, human papillomavirus-31, human papillomavirus-33, human papillomavirus-35, human papillomavirus-39, human papillomavirus-45, and human papillomavirus-45. Human papillomavirus-51, Human papillomavirus-52, Human papillomavirus-53, Human papillomavirus-56, Human papillomavirus-58, Human papillomavirus-59, Human papillomavirus-61, Human papillomavirus-66, Human papillomavirus-73, Human papillomavirus-82, Human papillomavirus-6, Human papillomavirus-11, Human papillomavirus-68, Human papillomavirus-42, Human papillomavirus-43, Human papillomavirus-44, Hepatitis B virus, Toxoplasma gondii, and Prevotella melanogaster. Salmonella enterica, Escherichia coli, Listeria monocytogenes, Timonehoyles, Haemophilus ducreyi, Prevotella bisporus, Klebsiella pneumoniae, Mycoplasma pneumoniae, Molecularcurvularia, Ureaplasma urealyticum, Staphylococcus aureus, Molecularcurvularia, Neisseria gonorrhoeae, Treponema pallidum, Proteus vulgaris, Proteus mirabilis, Prevotella humanis, Mycoplasma hominis, Chlamydia trachomatis, Mycoplasma genitalium, Prevotella bifidum, Ureaplasma microplasma, Streptococcus agalactiae, Prevotella amniotic fluid, Atropine vaginalis Prevotella, Gardnerella vaginalis, Prevotella intermedia, Candida albicans and Movula hymniferum, as well as hepatitis C virus, rubella virus, influenza A virus, influenza A virus H3N2, influenza A virus H1, influenza A virus H1N1, influenza A virus H3, influenza A virus H7, common influenza A virus, mumps virus, human parainfluenza virus type 2, human parainfluenza virus type 4, human immunodeficiency virus type 1, human immunodeficiency virus type 2, SARS-CoV-2 and Zika virus.

5. A kit for detecting multiplex amplification targeted sequencing of various pathogens, characterized in that, Includes the primer set as described in claim 1.

6. The reagent kit according to claim 5, characterized in that, It includes an RNA amplification system and a DNA amplification system; the RNA amplification system includes 5 μL of Novizan UNA321, 0.6 μL of primers, and 10.4 μL of nuclease-free water; the DNA amplification system includes 15 μL of Roche KK5801, 4 μL of primers, and 2 μL of nuclease-free water.

7. The reagent kit according to claim 6, characterized in that, The amplification programs for the RNA and DNA amplification systems are as follows: 55℃, 15 min; 95℃, 3 min; 95℃, 30 s; 60℃, 1 min; 72℃, 30 s, repeated 30 times; 72℃, 3 min; 4℃, hold.

8. The reagent kit according to claim 7, characterized in that, After amplification in the RNA and DNA amplification systems, the amplification products are mixed for a second amplification. The reagents for the second amplification include 20 μL of MEB3065 from Mingyi Intelligent Manufacturing, 2 μL of index, and 18 μL of nuclease-free water. The amplification program for the second amplification is as follows: 98℃, 45s; 95℃, 20s; 60℃, 30s; 72℃, 30s, for 30 cycles; 72℃, 5min; 4℃, hold.

9. The reagent kit according to claim 6, characterized in that, The kit is used to detect samples such as amniotic fluid, vaginal secretions, or abortion tissue that have undergone cell wall disruption.

10. The reagent kit according to claim 9, characterized in that, The cell disruption process includes centrifuging the sample to collect the precipitate, and then performing four cycles of cell disruption on the precipitate at a rotation speed of 5.5 m / s, a cell disruption time of 40 s / cycle, and a cell disruption interval of 1 min.

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