CN-121975959-A - Primer combination, kit and method for detecting sepsis pathogen based on dPCR

CN121975959ACN 121975959 ACN121975959 ACN 121975959ACN-121975959-A

Abstract

The invention discloses a primer combination, a kit and a method for detecting sepsis pathogens based on dPCR. The invention discloses a primer composition for detecting sepsis pathogens, which comprises at least one group of primer groups of 28 sepsis pathogens such as Acinetobacter baumannii, candida, coagulase-negative staphylococcus, enterococcus, KPC genes and the like and/or common drug-resistant genes. The invention provides a primer combination for detecting sepsis pathogens based on dPCR, a nucleic acid detection kit containing the primer combination and a detection method, which can rapidly and accurately detect various sepsis pathogens, so as to make up the defects of time and labor consumption existing in the existing sepsis pathogen detection technology, improve the detection sensitivity and specificity, reduce the labor intensity and shorten the detection period.

Inventors

XU GAOLIAN
AI JINGWEN
LIN KE
GONG JIAN
FENG XIAOYAN
ZHANG YUEJIAN
ZHANG WENHONG

Assignees

上海感染与免疫科技创新中心
艾普拜生物科技(苏州)有限公司

Dates

Publication Date: 20260505
Application Date: 20260205

Claims (10)

1. A primer composition for detecting sepsis pathogens and/or drug-resistant genes is characterized in that the primer composition comprises a primer set of Acinetobacter baumannii, a primer set of candida, a primer set of coagulase-negative staphylococcus, a primer set of enterococcus, a primer set of escherichia coli, a primer set of klebsiella, a primer set of KPC gene, a primer set of mecA gene, a primer set of MecC genes, a primer set of NDM gene, a primer set of pseudomonas aeruginosa, a primer set of staphylococcus aureus, a primer set of stenotrophomonas maltophilia, a primer set of streptococcus, a primer set of Vana gene, a primer set of VanM gene, a primer set of bacteroides, a primer set of corynebacterium striatum, a primer set of Citrobacter, a primer set of CMV cytomegalovirus, a primer set of EBV, a primer set of Enterobacter cloacae, a primer set of herpes simplex virus 1/2, a primer set of IMP gene, a primer set of OXA-48 gene, a primer set of Salmonella, a primer set of at least one of Serratia, (1) The Acinetobacter baumannii primer set comprises primers shown in SEQ ID NO. 1 and SEQ ID NO. 2; (2) The candida primer group comprises primers shown in SEQ ID NO. 4 and SEQ ID NO. 5; (3) The coagulase negative staphylococcus primer group comprises primers shown in SEQ ID NO. 7 and SEQ ID NO. 8; (4) The enterococcus primer group comprises primers shown in SEQ ID NO. 10 and SEQ ID NO. 11; (5) The escherichia coli primer group comprises primers shown in SEQ ID NO. 13 and SEQ ID NO. 14; (6) The klebsiella primer group comprises primers shown in SEQ ID NO. 16 and SEQ ID NO. 17; (7) The KPC gene primer group comprises primers shown in SEQ ID NO. 19 and SEQ ID NO. 20; (8) The MecA gene primer group comprises primers shown in SEQ ID NO. 22 and SEQ ID NO. 23; (9) The MecC gene primer group comprises primers shown in SEQ ID NO. 25 and SEQ ID NO. 26; (10) The NDM gene primer group comprises primers shown in SEQ ID NO. 28 and SEQ ID NO. 29; (11) The pseudomonas aeruginosa primer group comprises primers shown in SEQ ID NO. 31 and SEQ ID NO. 32; (12) The staphylococcus aureus primer group comprises primers shown in SEQ ID NO. 34 and SEQ ID NO. 35; (13) The stenotrophomonas maltophilia primer group comprises primers shown in SEQ ID NO. 37 and SEQ ID NO. 38; (14) The streptococcus primer group comprises primers shown in SEQ ID NO. 40 and SEQ ID NO. 41; (15) The VanA gene primer group comprises primers shown in SEQ ID NO. 43 and SEQ ID NO. 44; (16) The VanM gene primer group comprises primers shown as SEQ ID NO. 46 and SEQ ID NO. 47; (17) The bacteroides primer group comprises primers shown as SEQ ID NO. 52 and SEQ ID NO. 53; (18) The corynebacterium striatum primer group comprises primers shown in SEQ ID NO. 55 and SEQ ID NO. 56; (19) The primer group of the genus Citrobacter comprises a primer shown in SEQ ID NO. 58 and a primer shown in SEQ ID NO. 59; (20) The CMV cytomegalovirus primer group comprises a primer shown as SEQ ID NO. 61 and a primer shown as SEQ ID NO. 62; (21) The EBV primer group comprises primers shown as SEQ ID NO. 64 and SEQ ID NO. 65; (22) The enterobacter cloacae primer group comprises primers shown as SEQ ID NO. 67 and SEQ ID NO. 68; (23) The herpes simplex virus 1/2 primer group comprises primers shown in SEQ ID NO. 70 and SEQ ID NO. 71; (24) The IMP gene primer group comprises primers shown in SEQ ID NO. 73 and SEQ ID NO. 74; (25) The OXA-48 gene primer group comprises primers shown in SEQ ID NO. 76 and SEQ ID NO. 77; (26) The Proteus mirabilis primer group comprises primers shown in SEQ ID NO. 79 and SEQ ID NO. 80; (27) The salmonella primer group comprises primers shown as SEQ ID NO. 82 and SEQ ID NO. 83; (28) The serratia marcescens primer group comprises primers shown as SEQ ID NO. 85 and SEQ ID NO. 86.
2. The primer composition for detecting a sepsis pathogen and/or drug resistance gene according to claim 1, further comprising a control primer set for amplifying the internal standard fragment, the control primer set comprising primers shown in SEQ ID No. 49, SEQ ID No. 50.
3. A detection reagent comprising the primer composition according to claim 1 or 2.
4. The detection reagent of claim 3, further comprising a probe selected from the group consisting of: (1) The probe of Acinetobacter baumannii is shown as SEQ ID NO. 3; (2) The probe of the candida primer group is shown as SEQ ID NO. 5; (3) The coagulase negative staphylococcus probe is shown as SEQ ID NO. 9; (4) The probe of enterococcus is shown as SEQ ID NO. 12; (5) The probe of the escherichia coli is shown as SEQ ID NO. 15; (6) The probe of the Klebsiella is shown as SEQ ID NO. 18; (7) The probe of the KPC gene is shown as SEQ ID NO. 21; (8) The probe of the MecA gene is shown as SEQ ID NO. 24; (9) The probe of MecC gene is shown as SEQ ID NO. 27; (10) The probe of the NDM gene is shown as SEQ ID NO. 30; (11) The probe of the pseudomonas aeruginosa is shown as SEQ ID NO. 33; (12) The staphylococcus aureus probe is shown as SEQ ID NO. 36; (13) The probe of the stenotrophomonas maltophilia is shown as SEQ ID NO. 39; (14) The streptococcus probe is shown as SEQ ID NO. 42; (15) The VanA gene probe is shown as SEQ ID NO. 45; (16) The probe of VanM gene is shown as SEQ ID NO. 48; (17) The probes of the bacteroides are shown as SEQ ID NO. 54; (18) The probe of the corynebacterium striatum is shown as SEQ ID NO. 57; (19) The probe of the Citrobacter is shown as SEQ ID NO. 60; (20) The CMV cytomegalovirus probe is shown as SEQ ID NO. 63; (21) The EBV probe is shown as SEQ ID NO. 66; (22) The probe of enterobacter cloacae is shown as SEQ ID NO. 69; (23) The probe of the herpes simplex virus 1/2 is shown as SEQ ID NO. 72; (24) The probe of the IMP gene is shown as SEQ ID NO. 75; (25) The probe of the OXA-48 gene is shown as SEQ ID NO. 78; (26) The probe of the Proteus mirabilis is shown as SEQ ID NO. 81; (27) The salmonella probe is shown as SEQ ID NO. 84; (28) The probe of Serratia marcescens is shown as SEQ ID NO. 87.
5. A detection system, the detection system comprising: (1) The primer composition according to claim 1 or 2, or the detection reagent according to claim 3 or 4; (2) Optionally, a nucleic acid extraction reagent; (3) Optionally, BSA and/or DMSO.
6. A digital PCR chip is characterized in that a digital PCR amplification reagent is embedded in a reaction hole, and the digital PCR amplification reagent comprises the primer composition according to claim 1 or 2 or the detection reagent according to claim 3 or 4.
7. A sepsis pathogen nucleic acid detection kit based on digital PCR, comprising: (a) The primer composition of claim 1 or 2, the detection reagent of claim 3 or 4, or the digital PCR chip of claim 6; (b) Nucleic acid extraction reagents; (c) Optionally, a probe.
8. Use of the primer composition of claim 1 or 2, the detection reagent of claim 3 or 4, or the digital PCR chip of claim 6 for preparing a kit for detecting sepsis pathogens and/or drug resistance genes.
9. A method for detecting sepsis pathogens and/or drug resistance genes, characterized in that the method uses the primer composition of claim 1 or 2, the detection reagent of claim 3 or 4, the detection system of claim 5, the digital PCR chip of claim 6, or the kit of claim 7.
10. The method according to claim 9, comprising the step of: (ii) Extracting and purifying nucleic acid in a sample; (iii) Digital PCR amplification; (iv) And (3) judging the result, correcting the fluorescence of each channel through the fluorescence crosstalk matrix, and simultaneously analyzing and reporting the data by combining the automatic interpretation function of the software.

Description

Primer combination, kit and method for detecting sepsis pathogen based on dPCR Technical Field The invention relates to the technical field of pathogen detection and diagnosis, in particular to a primer combination, a kit and a method for detecting sepsis pathogens based on dPCR. Background Sepsis is a deadly organ dysfunction caused by a body's uncontrolled response to infection, which is urgent, rapid in progression, and high in mortality, and has become a major public health problem worldwide threatening human health. It is counted that the global annual sepsis patients exceed 3000 ten thousand, the mortality rate is as high as 20% -50%, and the surviving patients often accompany sequelae such as long-term organ injury, cognitive dysfunction and the like, which brings heavy medical burden and economic pressure to families and society. Rapid recognition of pathogens is a key element in blocking disease progression during infection-induced sepsis. However, the conventional pathogen detection method has significant limitations in that the culture method takes 24-72 hours as a clinical gold standard, and has extremely low detection rate (only about 30% -40%) on causticized bacteria, slow-growing bacteria or used antibiotics, antigen-antibody detection can only target specific pathogens, has limited coverage, and common PCR technology can shorten time but is easily affected by pollution, and multiple pathogens are difficult to detect simultaneously. These defects result in about 60% of sepsis patients not having an definitive pathogen at the time of initial treatment, but only relying on empirical broad-spectrum antibiotic therapy. The blindness of empirical treatment further exacerbates the clinical dilemma in that, on the one hand, improper antibiotic use may delay effective treatment, resulting in rapid deterioration of the patient's organ function, and, on the other hand, excessive use of broad-spectrum antibiotics may significantly increase the risk of developing resistant bacteria (e.g., methicillin-resistant staphylococcus aureus, carbapenem-resistant enterobacteriaceae) resulting in a "infection-resistant-more difficult to control" vicious circle. It has been studied that the mortality rate of drug-resistant bacterial infections in sepsis patients is more than 30% higher than that of sensitive bacterial infections, and the global spread of drug-resistant bacteria has faced some patients with the "no drug available" deadline. In addition, the pathogen spectrum of sepsis is complex and diverse, including bacteria, fungi, viruses, etc., and the epidemic characteristics of different regions and different populations are different. For example, gram-negative bacteria (such as Escherichia coli and Klebsiella pneumoniae) account for higher proportion of nosocomial acquired sepsis, while community acquired sepsis is mainly caused by gram-positive bacteria (such as streptococcus and staphylococcus), and patients with low immune function are easy to be combined with fungal infection (such as candida), and sepsis caused by viruses (such as influenza virus and coronavirus) also has an ascending trend in specific seasons. The diversity and variability of pathogens place higher demands on the coverage and accuracy of detection techniques. The pathogen type and drug resistance characteristics are quickly determined, so that a clinician can be helped to adjust the treatment scheme at the first time, the treatment is changed from the empirical medication to the targeted antibiotic treatment, and the process of trial and error is reduced. Studies have shown that effective antimicrobial treatment is initiated within 1 hour after sepsis occurs, patient survival can be increased by more than 40% and mortality increased by 7.6% every 1 hour delay. Through accurate detection, not only can the therapeutic efficiency be promoted, but also the damage of invalid medication to the liver and kidney functions of a patient can be reduced, and the occurrence risk of multiple organ failure is reduced. The targeted use of narrow-spectrum antibiotics can obviously reduce the damage to normal flora and reduce the selective expression and transmission probability of drug-resistant genes. Pathogen detection combined with drug resistance gene analysis (such as beta-lactamase gene and vancomycin drug resistance gene) can provide clear basis for antibiotic selection, and avoid unreasonable drug use modes of wide coverage and large dosage. From the perspective of public health, the measure has important strategic significance for delaying the spread of the global drug-resistant bacteria and protecting the antibiotic reserve. In a word, the rapid and accurate detection of sepsis pathogens is a core link breaking through the bottleneck of the current clinical treatment, has significance in not only improving the treatment success rate of individual patients, but also suppressing drug-resistant bacteria spreading, optimizing medical resource all