Composition for detecting meningitis pathogens, kit and application

Abstract

The invention relates to the technical field of biological detection, and discloses a composition for detecting meningitis pathogens, a kit and application. The composition comprises detection primers and selectable probes for 14 pathogens causing meningitis, multiple pathogens can be detected at the same time through the multiple PCR technology, the use amount of samples is reduced, and meanwhile the composition has the advantages of being high in detection speed, efficiency, accuracy and sensitivity and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a composition, a kit and application for detecting a meningitis pathogen. BACKGROUND

[0002] Meningitis is a serious central nervous system disease, and if it cannot be treated in time, it can seriously damage the brain function of the patient and even cause death. The main cause of meningitis is pathogen infection, and the types of pathogens that can cause meningitis are very diverse, including bacteria, fungi, viruses, protozoa, etc. Therefore, accurately and quickly diagnosing the pathogen is the key to timely treatment and reducing mortality and disability rate.

[0003] However, the symptoms of meningitis caused by different pathogens are relatively similar, and the early symptoms of meningitis are similar to cold. The difficulty in distinguishing symptoms and the existence of similar symptoms with common diseases such as cold are one of the main reasons for the delay in diagnosis and treatment. Traditional diagnostic methods include pathogen culture, nucleic acid sequencing, etc. These detection methods have defects such as long time consumption, high cost, high requirements for equipment and professional level of detection personnel. Although the application of nucleic acid detection technology in clinical practice has improved the efficiency of pathogen detection, the clinical sample for diagnosing meningitis is usually cerebrospinal fluid, and the cerebrospinal fluid sample obtained in clinic is often limited. Whether it is a traditional diagnostic method or a single pathogen nucleic acid detection, it is easy to cause sample waste and may miss detection due to insufficient sample amount.

[0004] In summary, in view of the problems of multiple types of meningitis pathogens, limited amount of clinical samples, insufficient detection efficiency, accuracy and sensitivity of the existing technology, and the urgent need of patients for rapid and accurate diagnostic results, it is necessary to further develop new meningitis pathogen detection technology and related products. SUMMARY

[0005] The present application aims to overcome the above-mentioned problems existing in the prior art, and provides a composition, a kit and an application for detecting meningitis pathogens. Specifically, the present application relates to a detection method and related products of 14 common pathogens causing meningitis, such as Coxsackievirus A10, Neisseria meningitidis, Japanese encephalitis virus, herpes simplex virus type 1, herpes simplex virus type 2, human herpesvirus 6, human bocavirus, Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Listeria monocytogenes, Klebsiella pneumoniae, Cryptococcus neoformans and group B streptococcus. The composition provided by the present application can be applied to multiplex PCR detection technology, so that multiple pathogens can be detected simultaneously by using one sample, the requirements for sample amount and the professional level of the operator are reduced, and the composition is more suitable for clinical detection, especially for the application and popularization of primary clinical detection.

[0006] In order to achieve the above-mentioned purpose, in one aspect, the present application provides a composition for detecting meningitis pathogens, the composition comprising primers and optional probes for amplifying target sequences of the pathogens, wherein the meningitis pathogens comprise at least one of Coxsackievirus A10, Neisseria meningitidis, Japanese encephalitis virus, herpes simplex virus type 1, herpes simplex virus type 2, human herpesvirus 6, human bocavirus, Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Listeria monocytogenes, Klebsiella pneumoniae, Cryptococcus neoformans and group B streptococcus; the target sequence of Coxsackievirus A10 has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 46; the target sequence of Neisseria meningitidis has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 47; the target sequence of Japanese encephalitis virus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 48; the target sequence of herpes simplex virus type 1 has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 49; the target sequence of herpes simplex virus type 2 has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 50; the target sequence of human herpesvirus 6 has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 51; the target sequence of human bocavirus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 52; the target sequence of Streptococcus pneumoniae has at least 80% identity with the nucleotide sequence shown in SEQ ID NO: 53; The target sequence of Haemophilus influenzae has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:54; The target sequence of Mycobacterium tuberculosis has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:55; The target sequence of Listeria monocytogenes has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:56; The target sequence of Klebsiella pneumoniae has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:57; The target sequence of Cryptococcus neoformans has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:58; The target sequence of Group B Streptococcus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:59.

[0007] A second aspect of the present invention provides a kit comprising the composition described in the first aspect.

[0008] A third aspect of the present invention provides a method for non-diagnostic detection of meningitis pathogens in a sample, the method comprising in vitro amplification of nucleic acids in the sample to be tested using the composition of the first aspect or the kit of the second aspect.

[0009] Through the above technical solution, the present invention can achieve at least the following beneficial effects: (1) The composition provided by the present invention contains detection primers and (optionally) probes for 14 common meningitis pathogens. These primers and probes can be used in combination to achieve joint detection of different pathogens as needed, or to perform rapid diagnosis and pathogen identification of meningitis in a targeted manner based on the regional prevalence of meningitis.

[0010] (2) The composition of the present invention can simultaneously detect and identify multiple meningitis pathogens present in a sample using multiplex PCR technology. Combined with fluorescence detection and other technologies, it can also achieve quantitative detection. Compared with conventional detection methods such as genome sequencing and pathogen culture, the composition of the present invention can greatly simplify the detection process, reduce the amount of sample required, shorten the detection time, and reduce the requirements for detection personnel and equipment, making it suitable for widespread application in grassroots detection.

[0011] (3) The primer pairs and probes contained in the composition provided by the present invention do not aggregate with each other and do not interfere with each other. They have good simultaneous detection effect on multiple targets in the sample, have good specificity, high detection sensitivity, and are easy to operate, making them suitable for large-scale promotion and application. Attached Figure Description

[0012] Figures 1A-1B This is a graph showing the results of the combined detection of simulated samples with mixed target sequences of 14 pathogens using the kit of this invention.

[0013] Figures 2A-2B This is a graph showing the specificity test results of the kit of the present invention.

[0014] Figures 3A-3B The figures show the results of using Comparative Kit I in Comparative Example 1 of this invention to detect Cryptococcus neoformans alone and to detect a simulated sample containing a mixed target sequence of 14 pathogens.

[0015] Figures 4A-4B The figures show the results of using the comparative kit II in Comparative Example 1 of this invention to detect Japanese encephalitis virus alone and to detect a simulated sample containing a mixed target sequence of 14 pathogens. Detailed Implementation

[0016] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0017] Unless otherwise specified, the sequences involved in this invention are provided in the order of 5'→3'.

[0018] The inventors of this invention have ingeniously designed a series of PCR primers and probes based on specific target sequences in 14 common meningitis pathogens. These primers and probes do not produce non-specific binding or dimers, can coexist in the reaction system, and do not have adverse effects on each other when used together. By utilizing multiplex PCR technology and fluorescent labeling, the types of pathogens in the sample can be easily and quickly distinguished and identified, and the detection of meningitis pathogens can be accurately achieved while reducing the amount of sample used.

[0019] Based on this, a first aspect of the present invention provides a composition for detecting meningitis pathogens, the composition comprising primers for amplifying a target sequence of the pathogen and optional probes, wherein the meningitis pathogens include Coxsackievirus A10 (CV A10), Neisseria meningitides (Nm), Japanese Encephalitis Virus (JEV), Herpes simplex virus type 1 (HSV 1), Herpes simplex virus type 2 (HSV 2), Human herpes virus type 6 (HHV 6), Human Parechoviruses (HPeVs), and Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae Haemophilus influenzae ( ) Haemophilus influenzae ), Mycobacterium tuberculosis ( Mycobacterium tuberculosis Listeria monocytogenes ( ) Listeria monocytogenes Listeria monocytogenes (abbreviated as Listeria) and Klebsiella pneumoniae ( Klebsiella Pneumoniae Cryptococcus neoformans ( Crytococcus Neoformans ) and Group B Streptococcus ( Streptococcus agalactiae At least one of (GBS); The target sequence of Coxsackievirus A10 has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:46; The target sequence of Neisseria meningitidis has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:47; The target sequence of Japanese encephalitis virus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:48; The target sequence of herpes simplex virus type 1 has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:49; The target sequence of herpes simplex virus type 2 has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:50; The target sequence of human herpesvirus type 6 has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:51; The target sequence of human diicovirus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:52; The target sequence of Streptococcus pneumoniae has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:53; The target sequence of Haemophilus influenzae has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:54; The target sequence of Mycobacterium tuberculosis has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:55; The target sequence of Listeria monocytogenes has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:56; The target sequence of Klebsiella pneumoniae has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:57; The target sequence of Cryptococcus neoformans has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:58; The target sequence of Group B Streptococcus has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:59.

[0020] In this invention, "optional probe" means that the probe is not a necessary component in the composition provided by this invention, and the composition may or may not contain a probe as needed. Without a probe, the nucleic acid in the sample to be tested can be amplified using the composition of this invention, and the amplified products can be detected and identified using methods such as electrophoresis separation and sequencing. With a probe, the amplified products can be identified using the labeling molecules carried on the probe.

[0021] Preferably, the target sequence of Coxsackievirus A10 has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:46. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0022] Preferably, the target sequence of Neisseria meningitidis has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:47. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0023] Preferably, the target sequence of the Japanese encephalitis virus has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:48. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0024] Preferably, the target sequence of herpes simplex virus type 1 has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:49. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value in that range.

[0025] Preferably, the target sequence of herpes simplex virus type 2 has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:50. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value in that range.

[0026] Preferably, the target sequence of human herpesvirus type 6 has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:51. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value in that range.

[0027] Preferably, the target sequence of the human diicovirus has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:52. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0028] Preferably, the target sequence of Streptococcus pneumoniae has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:53. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0029] Preferably, the target sequence of Haemophilus influenzae has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:54. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value in that range.

[0030] Preferably, the target sequence of Mycobacterium tuberculosis has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:55. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0031] Preferably, the target sequence of Listeria monocytogenes has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:56. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0032] Preferably, the target sequence of Klebsiella pneumoniae has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:57. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value in that range.

[0033] Preferably, the target sequence of Cryptococcus neoformans has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:58. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value in that range.

[0034] Preferably, the target sequence of Group B Streptococcus has at least 90% identity with the nucleotide sequence shown in SEQ ID NO:59. For example, it has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0035] In this invention, "identity" refers to the percentage of identical sequences between two sequences. For example, 90% identity between sequence a and sequence b means that 90% of sequence a is identical to sequence b, or 90% of sequence b is identical to sequence a. Differences between the two sequences can be due to nucleotide deletions, additions, or substitutions. For instance, sequence b, obtained by deleting 20 consecutive nucleotides from the 5' end of sequence a (containing 100 nucleotides), has 80% identity with sequence a, while sequence b has 100% identity with sequence a.

[0036] The compositions provided by this invention can be freely combined according to actual needs. For example, the composition may contain primers and probes targeting all of the above-mentioned pathogens, or it may contain primers and probes targeting only a portion of the pathogens.

[0037] According to a preferred embodiment of the present invention, the primers for detecting Coxsackievirus A10 contain the nucleotide sequences shown in SEQ ID NO:1-2, and the probes for detecting Coxsackievirus A10 contain the nucleotide sequence shown in SEQ ID NO:3.

[0038] According to a preferred embodiment of the present invention, the primers for detecting Neisseria meningitidis contain the nucleotide sequences shown in SEQ ID NO:4-5, and the probes for detecting Neisseria meningitidis contain the nucleotide sequence shown in SEQ ID NO:6.

[0039] According to a preferred embodiment of the present invention, the primers for detecting Japanese encephalitis virus contain the nucleotide sequences shown in SEQ ID NO:7-8, and the probes for detecting Japanese encephalitis virus contain the nucleotide sequence shown in SEQ ID NO:9.

[0040] According to a preferred embodiment of the present invention, the primer for detecting herpes simplex virus type 1 contains the nucleotide sequence shown in SEQ ID NO: 10-11, and the probe for detecting herpes simplex virus type 1 contains the nucleotide sequence shown in SEQ ID NO: 12.

[0041] According to a preferred embodiment of the present invention, the primer for detecting herpes simplex virus type 2 contains the nucleotide sequence shown in SEQ ID NO: 13-14, and the probe for detecting herpes simplex virus type 2 contains the nucleotide sequence shown in SEQ ID NO: 15.

[0042] According to a preferred embodiment of the present invention, the primer for detecting human herpesvirus 6 comprises the nucleotide sequence shown in SEQ ID NO:16-17, and the probe for detecting human herpesvirus 6 comprises the nucleotide sequence shown in SEQ ID NO:18.

[0043] According to a preferred embodiment of the present invention, the primers for detecting human diicovirus contain the nucleotide sequences shown in SEQ ID NO:19-20, and the probes for detecting human diicovirus contain the nucleotide sequence shown in SEQ ID NO:21.

[0044] According to a preferred embodiment of the present invention, the primer for detecting Streptococcus pneumoniae comprises the nucleotide sequence shown in SEQ ID NO:25-26, and the probe for detecting Streptococcus pneumoniae comprises the nucleotide sequence shown in SEQ ID NO:27.

[0045] According to a preferred embodiment of the present invention, the primers for detecting Haemophilus influenzae contain the nucleotide sequences shown in SEQ ID NO:28-29, and the probes for detecting Haemophilus influenzae contain the nucleotide sequence shown in SEQ ID NO:30.

[0046] According to a preferred embodiment of the present invention, the primers for detecting Mycobacterium tuberculosis contain the nucleotide sequences shown in SEQ ID NO:31-32, and the probes for detecting Mycobacterium tuberculosis contain the nucleotide sequence shown in SEQ ID NO:33.

[0047] According to a preferred embodiment of the present invention, the primers for detecting Listeria monocytogenes contain the nucleotide sequences shown in SEQ ID NO:34-35, and the probes for detecting Listeria monocytogenes contain the nucleotide sequence shown in SEQ ID NO:36.

[0048] According to a preferred embodiment of the present invention, the primers for detecting Klebsiella pneumoniae contain the nucleotide sequences shown in SEQ ID NO:37-38, and the probes for detecting Klebsiella pneumoniae contain the nucleotide sequence shown in SEQ ID NO:39.

[0049] According to a preferred embodiment of the present invention, the primers for detecting Cryptococcus neoformans contain the nucleotide sequences shown in SEQ ID NO:40-41, and the probes for detecting Cryptococcus neoformans contain the nucleotide sequence shown in SEQ ID NO:42.

[0050] According to a preferred embodiment of the present invention, the primer for detecting Group B Streptococcus comprises the nucleotide sequence shown in SEQ ID NO:43-44, and the probe for detecting Group B Streptococcus comprises the nucleotide sequence shown in SEQ ID NO:45.

[0051] The primers and probes for each pathogen contained in the composition provided by the present invention can be primers and probes as shown in the aforementioned sequences, or primers and probes can be obtained by (independently) adding several nucleotides (e.g., 1, 2, 3, 5, 10, or more nucleotides) upstream (5' end) and / or downstream (3' end) to these primers and probes, as long as they can achieve (simultaneous) efficient detection of the aforementioned pathogen target sequences.

[0052] According to a preferred embodiment of the present invention, the probe may also be modified with a labeling group (in order to distinguish different target sequences in the sample, or to perform quantitative analysis of target sequences in the sample).

[0053] Any labeling group commonly used in the field for nucleic acid detection, especially labeling groups that can be modified in nucleic acid probes, can be used in this invention, such as fluorescent labeling groups, nanoluminescent labeling groups, enzyme labels, etc.

[0054] Preferably, the labeling group is a fluorescent labeling group.

[0055] Since the composition provided by this invention can be used simultaneously for the detection of multiple different target sequences, in order to more clearly distinguish the detection results of different target analytes, it is preferable to modify the probes for different target sequences with different fluorescent labeling groups. Preferably, the different fluorescent labeling groups can be detected simultaneously through different channels.

[0056] According to some preferred embodiments of the present invention, the fluorescent labeling group may be selected from at least one of ATTO 425, HEX, FAM, ROX, CY5, CY7, Quasar705, VIC, and Alexa Fluor 405.

[0057] This invention does not impose any particular restrictions on the modification site of the fluorescent labeling group, and can be adjusted according to actual needs. Typically, it can be modified at the 5' end of the probe.

[0058] Preferably, the probe (3' end) is further modified with a fluorescence quenching group. Preferably, the fluorescence quenching group is selected from at least one of BHQ0, BHQ1, BHQ2, BHQ3, SQ1, and SQ2. Those skilled in the art are familiar with the combination of fluorescent labeling groups and fluorescence quenching groups, and will not elaborate further here.

[0059] According to some preferred embodiments of the present invention, the composition may further include primers for internal standards and optional probes.

[0060] Preferably, the internal standard includes at least one human housekeeping gene. This invention does not impose any particular limitation on the specific human housekeeping gene used; it can be selected according to actual needs. For example, it can select… GAPDH Genes or fragments thereof are used as internal standards.

[0061] According to a preferred embodiment of the present invention, the target sequence of the internal standard has at least 80% identity with the nucleotide sequence shown in SEQ ID NO:60.

[0062] When using the composition of the present invention to detect multiple (potentially present) pathogens in a sample, the primers and probes for the aforementioned 14 pathogens and the internal standard can be added together to the reaction tube (i.e., a "one-tube reaction"), or multiple tubes can be used for detection depending on the actual situation. For example, when using an eight-channel PCR instrument, detection can be performed in two tubes, with each tube containing the internal standard and primers and probes for 7 different pathogens. The present invention does not impose any particular limitation on the specific method of grouping primers and probes when performing detection in multiple tubes; the primers and probes for each pathogen in the composition of the present invention can be combined arbitrarily.

[0063] According to some preferred embodiments of the present invention, the composition comprises: (A) Primers and optional probes for amplifying the target sequence of at least one of Coxsackievirus A10, Neisseria meningitidis, Japanese encephalitis virus, herpes simplex virus type 1, herpes simplex virus type 2, human herpesvirus type 6, and human diicovirus; and / or (B) Primers and optional probes for amplifying the target sequence of at least one of Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Listeria monocytogenes, Klebsiella pneumoniae, Cryptococcus neoformans and Group B Streptococcus.

[0064] In the composition provided by this invention, each primer / probe can be packaged independently or in mixed packaging. Since non-specific binding does not occur between the primers / probes in the composition provided by this invention, preferably, to save packaging materials and reduce production and usage costs, the composition is presented in mixed packaging. For example, primers / probes can be divided into group (A) and group (B) according to the detection target and then mixed and packaged separately.

[0065] A second aspect of the present invention provides a kit comprising the composition described in the first aspect.

[0066] According to a preferred embodiment of the present invention, the kit further comprises at least one of a buffer, an enzyme, a magnesium ion source, an RNase inhibitor (RNasin), and deoxyribonucleoside triphosphates (dNTPs). "Magnesium ion source" refers to a reagent that provides magnesium ions, typically a water-soluble inorganic magnesium salt, such as MgCl2.

[0067] Preferably, the buffer solution comprises tris-hydroxymethylaminomethane hydrochloride buffer (Tris-HCl buffer).

[0068] Preferably, the enzyme includes at least one of UDG enzyme (also known as UNG enzyme), DNA polymerase (e.g., H-Taq enzyme), and reverse transcriptase (also known as RT enzyme).

[0069] The deoxyribonucleoside triphosphates included in the kit of this invention are raw materials used for complementary pairing with template sequences during in vitro nucleic acid amplification. The specific types of deoxyribonucleoside triphosphates included can be selected according to actual needs. Preferably, the deoxyribonucleoside triphosphates may include dATP (containing adenine), dGTP (containing guanine), dTTP (containing thymine), dCTP (containing cytosine), and dUTP (containing uracil). This invention does not have any particular restrictions on the specific source of the deoxyribonucleoside triphosphates in the kit; they can be self-prepared compounds or commercially available compounds. For example, commercially available dNTPs reagents (containing dATP, dGTP, dTTP, dCTP) or dNTP(U)s reagents (containing dATP, dGTP, dTTP, dCTP, dUTP) can be used directly.

[0070] More preferably, the kit may also include a positive control reagent and / or a negative control reagent.

[0071] This invention does not impose any particular restrictions on the specific selection of reagents used as positive and negative controls in the kit, and they can be selected according to conventional techniques in the art. For example, reagents that do not contain the target sequence, such as physiological saline or buffer, can be used as negative controls, as can internal standard gene plasmids, etc., as negative controls. Alternatively, mixed plasmids containing the corresponding target sequence, DNA or RNA fragments containing the target sequence, or pseudoviruses can be used as positive controls.

[0072] A third aspect of the present invention provides a method for detecting meningitis pathogens in a sample, the method comprising performing in vitro nucleic acid amplification on the sample to be tested using the composition described in the first aspect or the kit described in the second aspect.

[0073] Preferably, the meningitis pathogen is selected from at least one of Coxsackievirus A10, Neisseria meningitidis, Japanese encephalitis virus, herpes simplex virus type 1, herpes simplex virus type 2, human herpesvirus type 6, human diico virus, Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Listeria monocytogenes, Klebsiella pneumoniae, Cryptococcus neoformans, and Group B Streptococcus.

[0074] In this invention, nucleic acid amplification and detection of the sample to be tested can be performed using methods conventional in the art. For example, in some embodiments, the method provided by this invention may include the following steps: (1) Nucleic acid extraction and purification from samples; (2) The nucleic acid extracted in step (1) is amplified in vitro, that is, the purified sample nucleic acid is used as a template for in vitro amplification of nucleic acid using the composition described in the first aspect of the present invention or the kit described in the second aspect of the present invention.

[0075] Preferably, the method further includes analyzing the amplification results. In a preferred embodiment, since the probe of the present invention is modified with fluorescent labeling groups or other markers, pathogens can be qualitatively or quantitatively detected and identified by detecting the markers through fluorescence detection or other methods. For example, multiplex PCR can be used to amplify and detect different target sequences through different detection channels using the fluorescent labeling groups modified on the probe, ultimately determining the type of pathogen in the sample.

[0076] In some preferred embodiments, the above method can be a method for joint detection of meningitis pathogens in a sample. "Joint detection" refers to detecting multiple pathogens that may be present in the sample to determine whether the sample contains these pathogens, or to further determine the specific content of these pathogens in the sample. It only means that the method can identify / detect multiple pathogens, and does not mean that the sample must contain multiple pathogens simultaneously. In practical applications, the sample to be tested may or may not contain the meningitis pathogens. If the sample contains meningitis pathogens, it may contain only one of the aforementioned pathogens, or it may contain multiple pathogens simultaneously.

[0077] It should be noted that the methods provided in this invention can be diagnostic or non-diagnostic. For example, diagnostic aspects may include using the compositions, kits, or methods of this invention to detect patient-derived samples to identify the pathogens infecting meningitis patients; and non-diagnostic aspects may include using the compositions, kits, or methods of this invention to detect samples from various sources (such as human, animal, environmental, or biological materials cultured in vitro in experiments, such as cells, tissues, microbial colonies, viruses, etc.) to complete related research, drug development or screening, detection, disease prevention and control, and other related work.

[0078] Furthermore, the present invention also provides the use of the aforementioned composition and / or kit in the preparation of products for the diagnosis / identification of meningitis pathogens. The products can be any product in the art that can be used for the diagnosis / identification of pathogens of infectious diseases, such as kits or detection platforms containing detection reagents and accompanying detection instruments (and optional analysis software, etc.).

[0079] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0080] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available products purchased from regular chemical or biological reagent / material suppliers, and all reagents are of analytical grade.

[0081] Example 1 This embodiment illustrates the preparation of the reagent kit provided by the present invention.

[0082] 1. Prepare primers and probes Primers and probes were designed for the target sequences of the following 14 meningitis pathogens and internal standards. Primers and probes were synthesized according to the sequences in Table 1.

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] Table 1

[0099]

[0100] 2. Prepare the unit reaction reagent kit for PCR reaction. Prepare the reagents according to the required amounts of unit PCR reaction solution (i.e., PCR reaction solution used to detect one sample in one PCR reaction) and unit enzyme solution (i.e., enzyme solution used to detect one sample in one PCR reaction) as shown in Tables 2-1 to 2-3. In Tables 2-1 and 2-2, the PCR buffer is PCR buffer (S09) purchased from Hunan Kangde Biotechnology Co., Ltd.; the amounts of upstream primer, downstream primer, and probe are for one sequence.

[0101] Table 2-1

[0102] Table 2-2

[0103] Table 2-3

[0104] 3. Prepare control reagents Negative control: sterile saline.

[0105] Positive control: A mixture of target sequence plasmids for each pathogen. Target sequence plasmids for each pathogen were prepared by inserting the target sequence of each pathogen into the PUC57 plasmid. Specific target sequences are shown in SEQ ID NO:46-60. The concentration of each target sequence in the positive control was 1×10⁻⁶. 6 Copy number / mL.

[0106] 4. Prepare the reagent kit According to the target detection capacity of a single kit, combine and package the reagents prepared in steps 1-3. Specifically, take one portion of enzyme solution (2 μL) and mix it with PCR reaction solution A (38 μL), and add the corresponding pathogen primers and probes as shown in Table 3 to prepare PCR reaction reagent kit A; take one portion of enzyme solution (2 μL) and mix it with PCR reaction solution B (38 μL), and add the corresponding pathogen primers and probes as shown in Table 3 to prepare PCR reaction reagent kit B.

[0107] Table 3

[0108] Example 2 This embodiment illustrates the effectiveness of the kit provided by the present invention in the combined detection of simulated mixed samples.

[0109] (a) PCR detection 1. Prepare the sample to be tested. This embodiment uses a simulated sample prepared from the positive control in Example 1 for testing.

[0110] Simulated sample preparation: Target sequence plasmids of 14 pathogens and internal standards were mixed and diluted with human serum to a concentration of 1 × 10⁻⁶ for each target sequence. 5 Copy number / mL.

[0111] Sample preparation: Take 200 μL of positive control, negative control and simulated sample into 1.5 mL centrifuge tubes respectively, and use the nucleic acid extraction and purification reagent of Sansure Biotech Co., Ltd. (product number S10015-48) to perform nucleic acid extraction and purification according to its instructions.

[0112] 2. Sample addition and PCR amplification Take two portions each of the purified simulated sample, purified positive control, and purified negative control, 10 μL each, and add them to different PCR reaction tubes. Add 40 μL of unit PCR reaction reagent kit A to one tube and 40 μL of unit PCR reaction reagent kit B to the other tube (refer to Tables 2-1, 2-2, 2-3, and 3 for the specific components of unit PCR reaction reagent kits A and B). After mixing, place the tubes in the Hongshi Real-Time PCR Analyzer and perform the PCR reaction according to the reaction conditions in Table 4.

[0113] Table 4

[0114] The reaction results were analyzed according to the standards in Table 5.

[0115] Table 5

[0116] See the test results for the simulated sample. Figure 1A (Detected using PCR reaction solution A) and Figure 1B (Detection was performed using PCR reaction solution B). Furthermore, the results for the negative control showed no significant amplification curves for any of the detected targets. Based on the above judgment criteria, it was determined that all detected targets in the simulated sample used in this embodiment were positive, indicating that the composition and kit of the present invention can perform combined detection of the aforementioned 14 different meningitis pathogens.

[0117] (ii) Sensitivity test Sensitivity (LOD) testing was performed on each target, using the following method: Using the plasmid from Example 1 as a positive control, gradient dilution solutions of each target sequence were prepared. Then, the method described in Experiment (I) was used to detect each gradient dilution solution of the target sequence to determine the detection limit for each pathogen target sequence. Table 6 shows the detection rate of each target sequence at different concentrations (detection rate = number of detected target sequences / total number of detected target sequences). The detection rate of the target sequences for each pathogen reached 100% at a minimum concentration of 200 copies / mL. Therefore, the detection limit of the composition and kit of this invention is determined to be 200 copies / mL.

[0118] Table 6

[0119] (iii) Specificity test Other similar pathogens (yellow fever virus, mumps virus, Zika virus, chikungunya virus, adenovirus, human parvovirus, West Nile virus, tick-borne encephalitis virus, and rubella virus) were detected using the method described in Experiment (I). The results are as follows: Figure 2A(Detected using PCR reaction solution A) and Figure 2B As shown in the figure (using PCR reaction solution B for detection), these pathogens all showed obvious negative detection results, indicating that the composition and kit provided by the present invention have good specificity.

[0120] (iv) Anti-interference test Using the method described in Experiment (I), PCR reaction solutions A and B from the kit prepared in Example 1 were used to detect the simulated samples. The difference was that different interfering substances were added (the final concentration of each interfering substance was 50 μg / mL). The detection results (Ct values ​​for each channel) are shown in Tables 7 and 8.

[0121] Table 7

[0122] Table 8

[0123] As can be seen from the data in Tables 7-8, the kit of the present invention exhibits good tolerance to PCR interference when used for sample detection.

[0124] (v) Stability test Using the method described in Experiment (I), freshly prepared kits were used to detect simulated samples (the concentration of each pathogen target sequence plasmid was 200 copies / mL), and the detection was repeated 10 times.

[0125] Place the freshly prepared kit at 37°C (also known as "37°C acceleration") for 24 hours, and then test the simulated samples (the concentration of each pathogen target sequence plasmid is 200 copies / mL). Repeat the test 10 times.

[0126] The freshly prepared kits were stored under actual storage conditions (-20±5℃) for 11 months, and then the simulated samples (the concentration of each pathogen target sequence plasmid was 200 copies / mL) were tested, with the test repeated 10 times.

[0127] The freshly prepared kits were stored under actual storage conditions (-20±5℃) for 11 months. During this period, the kits were thawed and tested on simulated samples (the concentration of each pathogen target sequence plasmid was 200 copies / mL) every 80 days, with the tests repeated 10 times. After each test, the kits were returned to the actual storage conditions and stored again. Each time the kits were thawed and tested on simulated samples, it was counted as one freeze-thaw test, for a total of 4 freeze-thaw tests.

[0128] The test results of each of the above treatments were recorded and compared. According to the results, whether it is the repeated test results of the same sample in each batch or the test results of the sample after different treatments, the precision (reflected by the coefficient of variation CV value) is ≤5% and is highly consistent, indicating that the kit of the present invention has good detection stability.

[0129] Comparative Example 1 (a) Target sequence substitution and primer / probe substitution Comparative kit I was prepared using the method described in Example 1, except that the target sequence of Cryptococcus neoformans in tube B was replaced with the target sequence of SEQ ID NO:61. The primer and probe sequences for this pathogen are shown in Table 9.

[0130]

[0131] Table 9

[0132] When a single-detection kit for Cryptococcus neoformans was prepared by adding only primers and probes containing the sequences shown in SEQ ID NO 62-64 to the kit, the amplification efficiency for detecting Cryptococcus neoformans was good, and the detection results were as follows: Figure 3A As shown. The primer probe was mixed with the primer probes of the remaining targets in tube B to prepare a combined detection kit, for the concentration of 1×10⁻⁶ in Example 2. 5 Simulated samples of copies / mL were tested, and the results are as follows: Figure 3B As shown in Table 9, the results indicate that while the amplification efficiency of Cryptococcus neoformans was good when using the primers and probes in the assay reagent, it had a significant impact on the amplification efficiency of Mycobacterium tuberculosis primers and probes, resulting in a significant decrease in the relative fluorescence intensity of the Mycobacterium tuberculosis target and a noticeable delay in the CT value.

[0133] (ii) Primer / probe replacement The kit was prepared using the method described in Example 1, except that the primers and probes for detecting Japanese encephalitis virus in tube A were replaced with those in Table 10, resulting in a control kit.

[0134] Table 10

[0135] When a single-detection kit for Japanese encephalitis virus is prepared by adding only primers and probes with the sequences shown in SEQ ID NO 65-66 to the kit, the amplification efficiency is good, and the amplification results are as follows: Figure 4A As shown. The primer probe was mixed with the primer probes of the remaining targets in tube A to prepare a combined detection kit, for the concentration of 1×10⁻⁶ in Example 2. 5Simulated samples of copies / mL were tested, and the results are as follows: Figure 4B As shown in the figure. The results indicate that the relative fluorescence increment of Japanese encephalitis virus was significantly reduced, and the Japanese encephalitis primer probe had a significant impact on the amplification efficiency of the human herpesvirus type 6 primer probe in the same tube. The relative fluorescence increment of the two targets was significantly reduced, and the CT value was delayed.

[0136] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A composition for detecting a meningitis pathogen, characterized by, The composition comprises primers and optionally probes for amplifying a target sequence of the pathogen, wherein the meningitis pathogen comprises at least one of Coxsackievirus A10, Neisseria meningitidis, Japanese encephalitis virus, Herpes simplex virus type 1, Herpes simplex virus type 2, Human herpesvirus type 6, Human bocavirus, Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Listeria monocytogenes, Klebsiella pneumoniae, Cryptococcus neoformans and Group B Streptococcus; the target sequence of Coxsackievirus A10 has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 46; the target sequence of Neisseria meningitidis has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 47; the target sequence of Japanese encephalitis virus has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 48; the target sequence of Herpes simplex virus type 1 has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 49; the target sequence of Herpes simplex virus type 2 has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 50; the target sequence of Human herpesvirus type 6 has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 51; the target sequence of Human bocavirus has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 52; the target sequence of Streptococcus pneumoniae has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 53; the target sequence of Haemophilus influenzae has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 54; the target sequence of Mycobacterium tuberculosis has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 55; the target sequence of Listeria monocytogenes has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 56; the target sequence of Klebsiella pneumoniae has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 57; the target sequence of Cryptococcus neoformans has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 58; the target sequence of Group B Streptococcus has at least 80% identity to the nucleotide sequence set forth in SEQ ID NO:

59.

2. The composition of claim 1, wherein, the primers for detecting Coxsackievirus A10 comprise the nucleotide sequences set forth in SEQ ID NOs: 1-2, and the probe for detecting Coxsackievirus A10 comprises the nucleotide sequence set forth in SEQ ID NO: 3; and / or, the primers for detecting Neisseria meningitidis comprise the nucleotide sequences set forth in SEQ ID NOs: 4-5, and the probe for detecting Neisseria meningitidis comprises the nucleotide sequence set forth in SEQ ID NO: 6; and / or, the primers for detecting Japanese encephalitis virus comprise the nucleotide sequences set forth in SEQ ID NOs: 7-8, and the probe for detecting Japanese encephalitis virus comprises the nucleotide sequence set forth in SEQ ID NO: 9; and / or, the primers for detecting Herpes simplex virus 1 comprise nucleotide sequences as set forth in SEQ ID NOs: 10-11, the probe for detecting Herpes simplex virus 1 comprises a nucleotide sequence as set forth in SEQ ID NO: 12; and / or, the primers for detecting Herpes simplex virus 2 comprise nucleotide sequences as set forth in SEQ ID NOs: 13-14, the probe for detecting Herpes simplex virus 2 comprises a nucleotide sequence as set forth in SEQ ID NO: 15; and / or, the primers for detecting Human herpesvirus 6 comprise nucleotide sequences as set forth in SEQ ID NOs: 16-17, the probe for detecting Human herpesvirus 6 comprises a nucleotide sequence as set forth in SEQ ID NO: 18; and / or, the primers for detecting Human bocavirus comprise nucleotide sequences as set forth in SEQ ID NOs: 19-20, the probe for detecting Human bocavirus comprises a nucleotide sequence as set forth in SEQ ID NO: 21; and / or, the primers for detecting Streptococcus pneumoniae comprise nucleotide sequences as set forth in SEQ ID NOs: 25-26, the probe for detecting Streptococcus pneumoniae comprises a nucleotide sequence as set forth in SEQ ID NO: 27; and / or, the primers for detecting Haemophilus influenzae comprise nucleotide sequences as set forth in SEQ ID NOs: 28-29, the probe for detecting Haemophilus influenzae comprises a nucleotide sequence as set forth in SEQ ID NO: 30; and / or, the primers for detecting Mycobacterium tuberculosis comprise nucleotide sequences as set forth in SEQ ID NOs: 31-32, the probe for detecting Mycobacterium tuberculosis comprises a nucleotide sequence as set forth in SEQ ID NO: 33; and / or, the primers for detecting Listeria monocytogenes comprise nucleotide sequences as set forth in SEQ ID NOs: 34-35, the probe for detecting Listeria monocytogenes comprises a nucleotide sequence as set forth in SEQ ID NO: 36; and / or, the primers for detecting Klebsiella pneumoniae comprise nucleotide sequences as set forth in SEQ ID NOs: 37-38, the probe for detecting Klebsiella pneumoniae comprises a nucleotide sequence as set forth in SEQ ID NO: 39; and / or, the primers for detecting Cryptococcus neoformans comprise nucleotide sequences as set forth in SEQ ID NOs: 40-41, the probe for detecting Cryptococcus neoformans comprises a nucleotide sequence as set forth in SEQ ID NO: 42; and / or, the primers for detecting Group B Streptococcus comprise nucleotide sequences as set forth in SEQ ID NOs: 43-44, the probe for detecting Group B Streptococcus comprises a nucleotide sequence as set forth in SEQ ID NO:

45.

3. The composition according to claim 1 or 2, wherein, The composition further comprises primers and optionally probes for detecting a target sequence of an internal standard; Preferably, the target sequence of the internal standard has at least 80% identity to a nucleotide sequence as set forth in SEQ ID NO: 60; Preferably, the target sequence of the internal standard has at least 80% identity to a nucleotide sequence as set forth in SEQ ID NO: 60; More preferably, the primer for detecting the internal standard comprises a nucleotide sequence as set forth in SEQ ID NO: 22-23, and the probe for detecting the internal standard comprises a nucleotide sequence as set forth in SEQ ID NO:

24.

4. The composition according to any one of claims 1-3, wherein, The composition comprises: (A) primers and optionally probes for amplifying target sequences of at least one of Coxsackievirus A10, Neisseria meningitidis, Japanese encephalitis virus, Herpes simplex virus 1, Herpes simplex virus 2, Human herpesvirus 6, and Human bocavirus; and / or (B) primers and optionally probes for amplifying target sequences of at least one of Streptococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Listeria monocytogenes, Klebsiella pneumoniae, Cryptococcus neoformans, and Group B Streptococcus.

5. A kit for detecting a meningitis pathogen, characterized by, The kit comprises the composition of any one of claims 1-4.

6. The kit of claim 5, wherein, The kit further comprises at least one of a buffer, an enzyme, a magnesium ion source, an RNAse inhibitor, and deoxyribonucleotide triphosphates.

7. The kit of claim 6, wherein, The buffer comprises Tris-HCl buffer; and / or, the enzyme comprises at least one of UNG enzyme, DNA polymerase, and reverse transcriptase; Preferably, the kit further comprises a positive control reagent and / or a negative control reagent.

8. The kit of any one of claims 5-7, wherein, The kit further comprises reagents for extracting and / or purifying nucleic acids from a sample.

9. A method of detecting a meningitis pathogen in a sample other than for diagnosis, characterized in that, The method comprises in vitro amplification of nucleic acids from a sample to be tested using the composition of any one of claims 1-4 or the kit of any one of claims 5-8.

10. The method of claim 9, wherein, The method comprises: (1) sample nucleic acid extraction and purification; (2) in vitro amplification of the extracted nucleic acids from step (1). (1) sample nucleic acid extraction and purification; (2) in vitro amplification of the extracted nucleic acids from step (1).

Images