Primer probe group, kit and detection method for detecting children diarrhea pathogens based on multiple PCR (Polymerase Chain Reaction) combined with time-of-flight mass spectrometry

Abstract

The invention provides a primer probe group, a kit and a detection method for detecting diarrhea pathogens of children based on multiple PCR (Polymerase Chain Reaction) combined with time-of-flight mass spectrometry. The primer probe group comprises specific primers and probes aiming at 10 diarrhea pathogens (5 bacteria and 5 viruses) and a human-derived reference gene. Specific primers and probes are designed in a pathogen gene conserved region, targeted detection is achieved, and specificity is high; 10 diarrhea-causing pathogens and a human-derived reference gene in an excrement sample can be simultaneously detected in the same PCR reaction system and the single-base extension system, the sensitivity and the accuracy are high, and the detection flux is improved by combining a full-automatic chip spotting instrument; the cost is low, the detection period is short, and reliable technical support is provided for diagnosis and treatment, epidemic situation monitoring and infection control of children with infectious diarrhea.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular, to the field of nucleic acid amplification technology; specifically, to a primer probe set, a kit and a detection method for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry. Background Art

[0002] Diarrhea refers to an abnormal increase in stool moisture and stool frequency, usually more than three times a day. Diarrhea is one of the most serious public health problems in the world and a leading cause of death among children. According to the latest data from the World Health Organization (WHO), diarrhea is the third leading cause of death among children under five, killing about 440,000 children under five and another 50,000 children aged 5 to 9 each year. Diarrhea is one of the main causes of illness and death among children in developing countries, and is also one of the main causes of malnutrition, growth retardation and cognitive development disorders among children.

[0003] Infectious diarrhea is a group of intestinal infectious diseases caused by a variety of bacteria and viruses. Accurate detection of diarrhea-related pathogens is of great significance in helping to promptly diagnose and treat children, quickly investigate clusters of diarrhea in children, and monitor and control the spread of infection.

[0004] The current routine detection schemes for diarrhea etiology in clinical laboratories mainly include bacterial isolation and culture and biochemical identification technology of stool samples, as well as pathogen nucleic acid detection technology based on real-time fluorescence quantitative PCR. Although these methods can provide microbiological evidence for clinical diagnosis, they have dual technical bottlenecks of long detection cycles and few types of detected pathogens. In the epidemic season of childhood diarrhea or in the treatment of sudden clustered cases, the existing detection system is difficult to quickly complete the simultaneous screening of multiple pathogens due to limited throughput and insufficient timeliness, which directly affects the efficiency of infection source tracing and the timeliness of the implementation of prevention and control measures, and may lead to an increased risk of epidemic spread and delays in clinical precision diagnosis and treatment.

[0005] The existing clinical etiological detection methods for childhood diarrhea mainly include the following:

[0006] (1) Immunological tests:

[0007] The most widely used immunological detection technology in clinical practice is the gold-label immunochromatography (gold-label method), which is mainly based on the specific binding of antigens and antibodies and the principle of color reaction to detect pathogen antigens such as rotavirus, norovirus, and Clostridium difficile in the feces of children with diarrhea. However, the gold-label method is affected by factors such as the intensity of the color reaction signal and the load of pathogens. Its sensitivity is lower than that of the conventional polymerase chain reaction (PCR), and it is usually a single pathogen test, which cannot provide a comprehensive diagnosis for children with complex infections.

[0008] (2) Cultivation method:

[0009] The culture method is to identify diarrhea-related bacteria and test their drug resistance through bacterial culture. However, the biggest disadvantage of the culture method is the long detection time. It usually takes several days to obtain the test results, which leads to a delay in the etiological diagnosis of children with acute diarrhea. In addition, the culture method has low sensitivity, and the results are affected by factors such as the use of antimicrobial drugs, pathogen load, and culture medium selectivity, and the manual operation steps are complicated.

[0010] (3) Conventional PCR:

[0011] Reverse transcription PCR (RT-PCR) and real-time fluorescence quantitative PCR (qRT-PCR) are widely used in laboratory diagnosis and research of infectious diarrhea in children, mainly for the identification of single pathogens such as rotavirus and norovirus and differentiation of subtypes. However, these methods can only detect pathogens that are highly suspected clinically, with a single detection target and the possibility of false negatives. In addition, organic solvent residues in the experiment have an inhibitory effect on DNA synthesis, which poses a risk of false negatives.

[0012] (4) High-throughput sequencing:

[0013] High-throughput sequencing is a technology that sequences thousands to billions of DNA fragments simultaneously. High-throughput sequencing (non-targeted sequencing) has the widest coverage, not only can it detect potential pathogens, but also can detect the drug resistance genes of pathogenic bacteria, and even detect mutations in the genetic genes of pathogens. However, high-throughput sequencing should not be used as the first choice in the detection of pathogens of infectious diarrhea in children, because it is easily affected by interfering genes such as humans, intestinal flora and food, and requires reference to more gene sequence data. At the same time, high-throughput sequencing has a long detection cycle and is expensive. It requires the support of platform construction and bioinformatics teams, which is not conducive to large-scale clinical applications. Therefore, high-throughput sequencing is more used in the detection of pathogens or pathogen gene research where the cause is unclear and cultivation is difficult. Summary of the invention

[0014] In view of this, the purpose of the present invention is to develop a new method based on multiplex PCR and nucleic acid mass spectrometry for detecting 5 bacteria, 5 viruses and internal reference genes related to infectious diarrhea in children, and to propose a primer probe set, a kit and a detection method for detecting pathogens of diarrhea in children based on multiplex PCR combined with time-of-flight mass spectrometry, which can simultaneously detect 5 diarrhea-related viruses and their subtypes including human rotavirus, norovirus GII type, astrovirus, enteric adenovirus, and saprovirus in stool specimens, as well as Escherichia coli O157:H7, Salmonella typhi, Shigella, Campylobacter jejuni, Vibrio parahaemolyticus and other pathogens. 5 common diarrhea-causing bacteria; specific primers and probes are designed in the conserved region of pathogen genes for targeted detection with strong specificity; 10 diarrhea-causing pathogens in stool specimens can be detected simultaneously in the same PCR reaction system and single-base extension system with high sensitivity and high accuracy, and combined with a 384-well fully automatic chip spotter to improve detection throughput; reduce costs and shorten detection time, solve the pain points of traditional PCR in detecting multiple pathogens in stool specimens at the same time, and overcome the difficulty of being easily interfered by inhibitors in stool specimen detection, providing reliable technical support for the diagnosis and treatment of children with infectious diarrhea, epidemic monitoring and infection control.

[0015] The present invention provides a primer probe set for detecting diarrhea pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry, which is used for simultaneously detecting 10 diarrhea pathogens of common diarrhea in children, wherein the 10 diarrhea pathogens include: 5 bacteria and 5 viruses; wherein the 5 bacteria include: Escherichia coli O157:H7, Salmonella typhi, Shigella, Campylobacter jejuni, Vibrio parahaemolyticus; the 5 viruses include: human rotavirus, norovirus GII type, astrovirus, enteric adenovirus and zaruvirus;

[0016] The primer-probe group includes specific primers and probes for the 10 diarrhea pathogens and 1 human internal reference gene; all primers and probes are in the same PCR reaction system.

[0017] The nucleotide sequences of the primers and probes are shown in Table 1:

[0018] Table 1

[0019]

[0020]

[0021] In Table 1, forward PCR primer and reverse PCR primer represent the upstream and downstream amplification primers of multiplex PCR (the bold font represents the tag sequence of 10 bases in length); UEP represents the single base extension primer.

[0022] Furthermore, the design regions of the primers and probes for the five bacteria and the primers and probes for the five viruses are in the conserved regions of pathogen genes, as shown in Table 2:

[0023] Table 2

[0024]

[0025]

[0026] The present invention also provides a kit for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry, comprising the primer probe set as described above.

[0027] The present invention also provides a detection method for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry, using the primer probe set for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry as described above and the kit for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry as described above, comprising the following steps:

[0028] S1. Nucleic acid extraction, including: using the Tianlong GeneRotex 96 fully automatic rotary nucleic acid extractor and matching magnetic bead extraction reagents to extract fecal nucleic acids including human and pathogen genomic nucleic acids, wherein the genomic nucleic acids include DNA and RNA;

[0029] S2. Use HiScript II One Step RT-PCR Kit to perform reverse transcription and PCR reaction in one step, and configure the reverse transcription-multiplex PCR reaction system; mix the upstream and downstream primers of 5 bacteria, 5 viruses and human internal reference genes, and configure the primer mix as shown in Table 3:

[0030] Table 3

[0031]

[0032] S3. Perform one-step RT-PCR reaction according to the following conditions:

[0033] Reaction program: 50°C for 30 min; 94°C for 3 min; followed by 45 cycles, each cycle including 94°C for 30 sec, 56°C for 30 sec, 72°C for 30 sec, and 72°C for 5 min after the end of the cycle;

[0034] S4, shrimp alkaline phosphatase reaction: shrimp alkaline phosphatase SAP was used to remove unreacted dNTPs, and SAP mix was prepared on ice according to Table 4; 2 μl SAP mix was dispensed into each 384-well plate containing 5 μl of the PCR reaction product of step S3; the 384-well plate was sealed with a membrane and centrifuged to collect the liquid at the bottom; the 384-well plate was gently shaken to mix, centrifuged instantaneously, and placed on a 384-well plate PCR instrument for incubation: 37°C for 40 min, 85°C for 5 min, and stored at 4°C; after the SAP reaction, the 384-well plate was taken out, centrifuged at high speed, the liquid was collected at the bottom of the well, and the next step of the reaction was continued;

[0035] Table 4

[0036]

[0037] S5. UEP single base extension reaction: prepare the UEP extension mix for each well of the 384-well plate according to Table 5;

[0038] Table 5

[0039]

[0040] From the 7 μl of PCR product after SAP reaction treatment in step S4, 2 μl of UEP extension mix was dispensed into each well of the 384-well plate, and after instant centrifugation, the mixture was gently shaken and centrifuged to collect the liquid at the bottom, and placed in a PCR instrument for the following temperature cycle:

[0041]

[0042] S6, resin purification: add 16 μl of water to each well of the reaction product of the 384-well plate in step S5, mix well and centrifuge; use a small spoon to take an appropriate amount of resin and fill it into the 6mg 384 plate, and use a plastic cover to repeatedly push the resin left and right to compact it so that the resin content in each well is uniform; let the resin stand and dry for 15 minutes; turn the 384-well plate upside down and press it on the 6mg 384 plate; turn the two plates upside down, with the 6mg 384 plate on top and the 384-well plate containing the single-base extension product on the bottom; at this time, the resin falls into the 384-well plate containing the single-base extension product; use a sealing film to seal the 384-well plate containing the single-base extension product and resin, and rotate and mix the 384-well plate at 15 rpm at room temperature for 15 minutes to disperse the resin in the product and fully purify it; centrifuge at 3200g for 5 minutes to collect the resin to the bottom of the plate;

[0043] The resin can remove excess salt ions that may cause false mass spectrometry peaks to appear during data acquisition, affecting the detection results.

[0044] S7, time-of-flight mass spectrometry detection: the product of step S6 is spotted onto the chip using an automatic spotter, and then the on-chip mass spectrometer performs detection;

[0045] S8. Perform multiplex PCR-time-of-flight mass spectrometry sensitivity verification on the primers and probes of the five bacteria and five viruses.

[0046] In the embodiment of the present invention, a recombinant plasmid containing gene fragments of five kinds of bacteria and five kinds of viruses is used as a standard, and multiplex PCR combined with time-of-flight mass spectrometry detection is performed according to the above steps S1-S8. The five kinds of bacteria and five kinds of viruses are specifically amplified and single-base primer extended only in their respective primers, indicating that the primers and probes of the detection system have good specificity and can be used for subsequent research.

[0047] In the embodiments of the present invention, the minimum detection limits (LOD) of the five bacterial plasmid DNA templates all reached 1 copy / reaction; among the five viruses, the detection limits LOD of norovirus GII type and enteric adenovirus reached 1 copy / reaction; the detection limits LOD of astrovirus, human rotavirus and saprovirus reached 10 copy / reaction.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The primer probe set, kit and detection method for detecting diarrhea pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry provided by the present invention can simultaneously detect five diarrhea-related viruses and their subtypes including human rotavirus, norovirus GII type, astrovirus, enteric adenovirus and zaruvirus, as well as five common diarrhea-causing bacteria including Escherichia coli O157:H7, Salmonella typhi, Shigella, Campylobacter jejuni and Vibrio parahaemolyticus in stool specimens; specific primers and probes are designed in the conserved region of pathogen genes for targeted detection with high specificity; 10 diarrhea-causing pathogens in stool specimens can be simultaneously detected in the same PCR reaction system and single base extension system, with a detection limit of 1-10 copy / reaction, high sensitivity, high accuracy, combined with a 384-well fully automatic chip spotter, improves detection throughput; greatly reduces costs, shortens detection time, solves the pain point that traditional PCR cannot detect multiple pathogens simultaneously in stool specimens, overcomes the difficulty of being easily interfered by inhibitors in stool specimen detection, and provides accurate and efficient technical support for the diagnosis and treatment of children with infectious diarrhea, epidemic monitoring and infection control, and helps clinicians quickly determine the type of pathogen, thereby formulating more targeted treatment plans and effectively shortening the course of the disease in children. The present invention has the advantages of high coverage of pathogen types, short detection time, high sensitivity, high tolerance, high accuracy, high throughput, and low cost, and the specificity of amplification primers and single-base extension primers is good, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the following detailed description of the preferred embodiment.The drawings are only for the purpose of illustrating the preferred embodiments and are not to be construed as limiting the invention.

[0051] In the attached picture:

[0052] Figure 1 This is a graph showing the detection results of detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry according to an embodiment of the present invention. DETAILED DESCRIPTION

[0053] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Instead, they are merely examples of devices and products consistent with some aspects of the present disclosure as detailed in the appended claims.

[0054] The terms used in this disclosure are for the purpose of describing specific embodiments only and are not intended to limit the disclosure. The singular forms of "a", "said" and "the" used in this disclosure and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0055] It should be understood that although the terms first, second, third, etc. may be used in the present disclosure to describe various information, such information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of the present disclosure, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Depending on the context, the word "if" as used herein may be interpreted as "at the time of" or "when" or "in response to determining".

[0056] The embodiments of the present invention are described in further detail below.

[0057] The embodiment of the present invention provides a primer probe set for detecting diarrhea pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry, which is used to simultaneously detect 10 diarrhea pathogens of common diarrhea in children, wherein the 10 diarrhea pathogens include: 5 bacteria and 5 viruses; wherein the 5 bacteria include: Escherichia coli O157:H7, Salmonella typhi, Shigella, Campylobacter jejuni, Vibrio parahaemolyticus; the 5 viruses include: human rotavirus, norovirus GII type, astrovirus, enteric adenovirus and zaruvirus;

[0058] The primer-probe set includes primers and probes specific to the 10 diarrheal pathogens and 1 human internal reference gene; all primers and probes are in the same PCR reaction system;

[0059] The nucleotide sequences of the primers and probes are shown in Table 1:

[0060] Table 1

[0061]

[0062]

[0063] In Table 1, forward PCR primer and reverse PCR primer represent the upstream and downstream amplification primers of multiplex PCR (the bold font represents the tag sequence of 10 bases in length); UEP represents the single base extension primer.

[0064] The design regions of the primers and probes for the five bacteria and the primers and probes for the five viruses are in the conserved regions of pathogen genes, as shown in Table 2:

[0065] Table 2

[0066]

[0067] The embodiment of the present invention also provides a kit for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry, comprising the primer probe set as described above.

[0068] The embodiment of the present invention also provides a detection method for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry, using the primer probe set for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry as described above and the kit for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry as described above, comprising the following steps:

[0069] S1. Nucleic acid extraction, including: using the Tianlong GeneRotex 96 fully automatic rotary nucleic acid extractor and matching magnetic bead extraction reagents to extract fecal nucleic acids including human and pathogen genomic nucleic acids, wherein the genomic nucleic acids include DNA and RNA;

[0070] S2. Use HiScript II One Step RT-PCR Kit to perform reverse transcription and PCR reaction in one step, and configure the reverse transcription-multiplex PCR reaction system; mix the upstream and downstream primers of 5 bacteria, 5 viruses and human internal reference genes, and configure the primer mix as shown in Table 3:

[0071] Table 3

[0072]

[0073] S3. Perform one-step RT-PCR reaction according to the following conditions:

[0074] Reaction program: 50°C for 30 min; 94°C for 3 min; followed by 45 cycles, each cycle including 94°C for 30 sec, 56°C for 30 sec, 72°C for 30 sec, and 72°C for 5 min after the end of the cycle;

[0075] S4, shrimp alkaline phosphatase reaction: shrimp alkaline phosphatase SAP was used to remove unreacted dNTPs, and SAP mix was prepared on ice according to Table 4; 2 μl SAP mix was dispensed into each 384-well plate containing 5 μl of the PCR reaction product of step S3; the 384-well plate was sealed with a membrane and centrifuged to collect the liquid at the bottom; the 384-well plate was gently shaken to mix, centrifuged instantaneously, and placed on a 384-well plate PCR instrument for incubation: 37°C for 40 min, 85°C for 5 min, and stored at 4°C; after the SAP reaction, the 384-well plate was taken out, centrifuged at high speed, the liquid was collected at the bottom of the well, and the next step of the reaction was continued;

[0076] Table 4

[0077]

[0078] S5. UEP single base extension reaction: prepare the UEP extension mix for each well of the 384-well plate according to Table 5;

[0079] Table 5

[0080]

[0081] From the 7 μl of PCR product after SAP reaction treatment in step S4, 2 μl of UEP extension mix was dispensed into each well of the 384-well plate, and after instant centrifugation, the mixture was gently shaken and centrifuged to collect the liquid at the bottom, and placed in a PCR instrument for the following temperature cycle:

[0082]

[0083] S6, resin purification: add 16 μl of water to each well of the reaction product of the 384-well plate in step S5, mix well and centrifuge; use a small spoon to take an appropriate amount of resin and fill it into the 6mg 384 plate, and use a plastic cover to repeatedly push the resin left and right to compact it so that the resin content in each well is uniform; let the resin stand and dry for 15 minutes; turn the 384-well plate upside down and press it on the 6mg 384 plate; turn the two plates upside down, with the 6mg 384 plate on top and the 384-well plate containing the single-base extension product on the bottom; at this time, the resin falls into the 384-well plate containing the single-base extension product; use a sealing film to seal the 384-well plate containing the single-base extension product and resin, and rotate and mix the 384-well plate at 15 rpm at room temperature for 15 minutes to disperse the resin in the product and fully purify it; centrifuge at 3200g for 5 minutes to collect the resin to the bottom of the plate;

[0084] S7, time-of-flight mass spectrometry detection: the product of step S6 is spotted onto the chip using an automatic spotter, and then the on-chip mass spectrometer performs detection;

[0085] S8. Perform multiplex PCR-time-of-flight mass spectrometry sensitivity verification on the primers and probes of the five bacteria and five viruses.

[0086] In this example, a recombinant plasmid containing gene fragments of five bacteria and five viruses was used as a standard product, and multiplex PCR combined with time-of-flight mass spectrometry detection was performed according to the above steps S1-S8. The detection results are as follows: Figure 1 As shown, 5 bacteria and 5 viruses were specifically amplified and single-base primer extended only in their respective primers, indicating that the primers and probes of this detection system had good specificity.

[0087] The minimum detection limits (LOD) of the five bacterial plasmid DNA templates all reached 1 copy / reaction; among the five viruses, the detection limits of norovirus GII type and enteric adenovirus reached 1 copy / reaction; the detection limits of astrovirus, human rotavirus and saprovirus reached 10 copy / reaction.

[0088] right Figure 1 The name symbols in are annotated as follows:

[0089] ST: Salmonella Typhi; Shigella: Shigella; C.jejuni: Campylobacter jejuni; VP: Vibrio parahaemolyticus; O157: Escherichia coli O157:H7;

[0090] NOV-GII: Norovirus GII; Astrovirus: Astrovirus; HRV: Human Rotavirus; Sapovirus: Sapovirus; EAV: Enteric Adenovirus.

[0091] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments, but it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A primer probe set for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry, characterized in that: Used to simultaneously detect 10 common diarrheal pathogens in children, the 10 diarrheal pathogens include: 5 bacteria and 5 viruses; wherein the 5 bacteria include: Escherichia coli O157:H7, Salmonella typhi, Shigella, Campylobacter jejuni, Vibrio parahaemolyticus; the 5 viruses include: human rotavirus, norovirus GII type, astrovirus, enteroadenovirus and zaruvirus; The primer-probe set includes specific primers and probes for the 10 diarrheal pathogens and a human internal reference gene; all primers and probes are in the same PCR reaction system; The nucleotide sequences of the primers and probes are shown in Table 1: Table 1 ; In Table 1, forward PCR primer and reverse PCR primer represent the upstream and downstream amplification primers of multiplex PCR, the bold words represent the tag sequence of 10 bases in length; UEP represents a single base extension primer.

2. A primer probe set for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry, characterized in that: The design regions of the primers and probes for the five bacteria and the primers and probes for the five viruses are in the conserved regions of pathogen genes, as shown in Table 2: Table 2 。 3. A kit for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry, characterized in that: Comprising the primer probe set as described in claim 1 or 2.

4. A method for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry, using the primer probe set for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry as described in claim 1 or 2 and the kit for detecting diarrheal pathogens in children based on multiplex PCR combined with time-of-flight mass spectrometry as described in claim 3, characterized in that: The following steps are involved: S1. Nucleic acid extraction, including: using the Tianlong GeneRotex 96 fully automatic rotary nucleic acid extractor and matching magnetic bead extraction reagents to extract fecal nucleic acids including human and pathogen genomic nucleic acids, wherein the genomic nucleic acids include DNA and RNA; S2. Use HiScript II One Step RT-PCR Kit to perform reverse transcription and PCR reaction in one step, and configure the reverse transcription-multiplex PCR reaction system; mix the upstream and downstream primers of 5 bacteria, 5 viruses and human internal reference genes, and configure the primer mix as shown in Table 3: Table 3 ; S3. Perform one-step RT-PCR reaction according to the following conditions: Reaction program: 50°C for 30 min; 94°C for 3 min; followed by 45 cycles, each cycle including 94°C for 30 sec, 56°C for 30 sec, 72°C for 30 sec, and 72°C for 5 min after the end of the cycle; S4, shrimp alkaline phosphatase reaction: shrimp alkaline phosphatase SAP was used to remove unreacted dNTPs, and SAP mix was prepared on ice according to Table 4; 2 μl SAP mix was dispensed into each 384-well plate containing 5 μl of the PCR reaction product of step S3; the 384-well plate was sealed with a membrane and centrifuged to collect the liquid at the bottom; the 384-well plate was gently shaken to mix, centrifuged instantaneously, and placed on a 384-well plate PCR instrument for incubation: 37°C for 40 min, 85°C for 5 min, and stored at 4°C; after the SAP reaction, the 384-well plate was taken out, centrifuged at high speed, the liquid was collected at the bottom of the well, and the next step of the reaction was continued; Table 4 ; S5. UEP single base extension reaction: prepare the UEP extension mix for each well of the 384-well plate according to Table 5; Table 5 ; From the 7 μl of PCR product after SAP reaction treatment in step S4, 2 μl of UEP extension mix was dispensed into each well of the 384-well plate, centrifuged briefly and then gently shaken to mix, the liquid was collected by centrifugation to the bottom, and placed in a PCR instrument for the following temperature cycle: ; S6, resin purification: add 16 μl of water to each well of the reaction product of the 384-well plate in step S5, mix well and centrifuge; use a small spoon to take an appropriate amount of resin and fill it into the 6mg 384 plate, use a plastic cover to repeatedly push the resin left and right to compact it so that the resin content in each well is uniform; let the resin stand and dry for 15 minutes; turn the 384-well plate upside down and press it on the 6mg 384 plate; turn the two plates upside down, with the 6mg 384 plate on top and the 384-well plate containing the single-base extension product on the bottom; at this time, the resin falls into the 384-well plate containing the single-base extension product; use a sealing film to seal the 384-well plate containing the single-base extension product and resin, and rotate and mix the 384-well plate at 15 rpm at room temperature for 15 minutes to disperse the resin in the product and fully purify it; centrifuge at 3200g for 5 minutes to collect the resin to the bottom of the plate; S7, time-of-flight mass spectrometry detection: the product of step S6 is spotted onto the chip using an automatic spotter, and then the on-chip mass spectrometer performs detection; S8. Perform multiplex PCR-time-of-flight mass spectrometry sensitivity verification on the primers and probes of the five bacteria and five viruses.

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