Use of a panel of mnp markers for identification of plasmodium species, primer sets, kits and uses thereof

Abstract

The application discloses a kind of identification of plasmodium MNP marker combination, application, primer group, kit and its application, the application includes: the MNP marker combination is used for the genomic region that is screened on falciparum genome and is distinguished from other species and has multiple nucleotide polymorphisms within the species for the purpose of non-disease diagnosis, the primer group includes: 1st primer pair to 3rd primer pair, the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the forward primer of the 3rd primer pair and the reverse primer of the 3rd primer pair, sequentially as shown in SEQ ID NO:1-6 in sequence table.The application of the identification of plasmodium MNP marker combination, primer group, kit and its application, based on the scheme of multi-target detection, can realize efficient, accurate and sensitive identification, variation monitoring and cross-host tracing of falciparum.

Description

Technical Field

[0001] This disclosure relates to the field of biotechnology, and in particular to the application of an MNP marker combination for identifying Plasmodium, primer sets, kits, and their applications. Background Technology

[0002] Plasmodium is the parasitic protozoan that causes malaria, primarily transmitted through the bite of Anopheles mosquitoes, also known as Anopheles malaria mosquitoes, which cause significant harm. Currently, artemisinin-based drugs are the main treatment. Among the Plasmodium species that infect humans, Plasmodium falciparum is the most common and pathogenic, and also the species with the greatest concern regarding resistance to artemisinin-based drugs, posing a significant challenge to prevention and control. Therefore, developing a highly sensitive detection technology for Plasmodium falciparum applicable to both human and mosquito-borne samples, while also considering genetic variation detection and cross-species tracing, has significant clinical and public health value.

[0003] Traditional detection methods primarily rely on microscopic examination, rapid diagnostic test strips, and PCR (Polymerase Chain Reaction)-based nucleic acid detection technology. Observation of stained blood smears from patients is the traditional gold standard, directly identifying the morphology and species of Plasmodium falciparum. However, it is dependent on professional operation and prone to missed detections in early stages or with low infection levels. Rapid diagnostic test strips are prone to false positives / false negatives. PCR detection technology targets only one marker of the pathogen in a single reaction, resulting in low efficiency and potential failure due to primer variations, leading to false negatives. Therefore, a more efficient and accurate method for detecting Plasmodium falciparum is urgently needed. Summary of the Invention

[0004] To address the problems of existing technologies, this disclosure provides an application of MNP marker combinations for identifying Plasmodium falciparum, a primer set, a reagent kit, and their applications. The technical solution is as follows: On one hand, this disclosure provides an application of an MNP marker combination for identifying Plasmodium falciparum, the application including: using the MNP marker combination for non-disease diagnostic purposes to screen for genomic regions on the Plasmodium falciparum genome that are distinct from other species and have multiple nucleotide polymorphisms within said species, the MNP marker combination including at least one of marker site MNP-1, marker site MNP-2, and marker site MNP-3, the positions of marker sites MNP-1 to MNP-3 on the reference sequence are shown in the table below:

[0005] Specifically, the application includes: using the MNP marker combination for non-disease diagnostic purposes such as identification, mutation monitoring, source tracing, and database construction of the Plasmodium falciparum.

[0006] On the other hand, embodiments of this disclosure provide a primer set for identifying Plasmodium, the primer set comprising: a first primer pair to a third primer pair, each primer pair comprising a forward primer and a reverse primer, the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the third primer pair and the reverse primer of the third primer pair being as shown in SEQ ID NO: 1 to SEQ ID NO: 6 in the sequence listing.

[0007] In another aspect, embodiments of this disclosure provide a kit for identifying MNP marker combinations for Plasmodium, the kit comprising the aforementioned primer set.

[0008] Furthermore, the kit also includes a multiplex PCR premix.

[0009] In another aspect, embodiments of this disclosure provide an application of the primer set or the kit as described, the application including using the primer set or the kit for the identification of the Plasmodium falciparum.

[0010] Furthermore, the application includes using the MNP marker combination, the primer set, or the kit for monitoring mutations in Plasmodium falciparum.

[0011] Furthermore, the application includes using the MNP marker combination, the primer set, or the kit for tracing the source of Plasmodium falciparum.

[0012] Furthermore, the application includes using the MNP marker combination, the primer set, or the kit for the construction of a database of Plasmodium falciparum.

[0013] The beneficial effects of the technical solutions provided in this disclosure are: This disclosure provides an application of MNP marker combinations for identifying Plasmodium falciparum, a primer set, a kit, and their applications. Through analysis of the Plasmodium falciparum genome sequence, three MNP marker combinations were screened, and three primer sets were designed based on the sequence information of these three MNP marker combinations. Both the MNP marker combinations and the primer sets exhibit high specificity in identifying Plasmodium falciparum. Multiplex PCR amplification using the designed primer sets, followed by sequencing of the amplified products using a next-generation sequencing platform, allows for the simultaneous detection of all three MNP marker combinations for Plasmodium falciparum. Compared to fluorescence PCR methods that use only one primer pair to detect one site and detect only the fluorescence signal, the primer set provided in this disclosure offers high efficiency, accuracy, and sensitivity in identifying Plasmodium falciparum, providing technical support for identification, variation monitoring, cross-species tracing, and database construction for Plasmodium falciparum. The kit provided in this disclosure can detect all MNP marker sites in a single reaction. In the reproducibility test, all three MNP marker loci were stably detected, and the logarithm of the major genotype differences in MNP markers between different libraries and between different library preparation batches for each sample tested by this kit was 0, resulting in a high reproducibility rate. r =100%, accuracy a The 100% accuracy rate demonstrates the high accuracy and stability of this kit in detecting Plasmodium falciparum. During identification, samples are tested using high-throughput sequencing. By adding a unique tag to each sample, hundreds or even thousands of samples can be tested simultaneously, further improving detection efficiency. Plasmodium falciparum is identified based on the detected MNP-tagged base sequences, eliminating the need for parallel testing of standard samples. Obtaining the detected MNP-tagged base sequences allows for not only Plasmodium falciparum identification in a single reaction but also simultaneous detection of genetic variations between samples, source tracing, and database construction. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a schematic diagram illustrating the polymorphism principle of MNP marker sites provided in Embodiment 2 of this disclosure; Figure 2 This is a flowchart of the detection process for MNP marker combinations provided in Embodiment 2 of this disclosure. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below with reference to the accompanying drawings. Example

[0017] This disclosure provides an application of an MNP marker combination for identifying Plasmodium falciparum. The application includes: using the MNP marker combination for non-disease diagnostic purposes to screen for genomic regions on the Plasmodium falciparum genome that are distinct from other species and have multiple nucleotide polymorphisms within a species. The MNP marker combination includes at least one of marker site MNP-1, marker site MNP-2, and marker site MNP-3. The positions of marker sites MNP-1 to MNP-3 on the reference sequence are shown in Table 1.

[0018] Table 1 shows the MNP marker combinations.

[0019] Example 2 This disclosure provides a primer set for identifying MNP marker combinations for Plasmodium falciparum. The primer set includes primer pairs 1 to 3, each primer pair including a forward primer and a reverse primer. The forward primer of primer pair 1, the reverse primer of primer pair 1, the forward primer of primer pair 3, and the reverse primer of primer pair 3 are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 6 in the sequence listing. The specific sequences are shown in Table 2.

[0020] Table 2 lists the primer pairs corresponding to MNP marker combinations.

[0021] The primer pairs in the primer set provided in this embodiment do not conflict with each other, and can be efficiently amplified by multiplex PCR.

[0022] Detection efficiency evaluation of primer sets: Positive samples of Plasmodium falciparum were tested using the primer set provided in this embodiment, with a total of 3 replicates. The sequencing data were analyzed, and 3 pairs of highly compatible and product-specific primer sets were selected for identification of the specificity of the 3 MNP markers of Plasmodium falciparum.

[0023] The three MNP marker combinations for screening Plasmodium falciparum disclosed in this embodiment are as follows: Figure 1 As shown.

[0024] Positive samples of Plasmodium falciparum with known copy numbers, identified by digital PCR and collected by the Hubei Provincial Center for Disease Control and Prevention, were used to prepare positive samples with copy numbers of 1 copy / reaction, 10 copies / reaction, and 100 copies / reaction. An equal volume of sterile water was also included as a negative control. Three replicate libraries were analyzed for each sample over four consecutive days, resulting in 12 sequencing data sets per sample. Based on the data analysis results for each sample shown in Table 3, the reproducibility and accuracy of the detection method were evaluated, and thresholds for quality control system contamination and detection of the target Plasmodium falciparum were established. The detection procedure for MNP marker combinations is as follows: Figure 2 As shown.

[0025] Table 3 shows the sensitivity and stability of primer sets in identifying Plasmodium falciparum.

[0026] As shown in Table 3, all three MNP marker sites of Plasmodium falciparum were detected in the positive samples with 10 copies / reaction and 100 copies / reaction, and the sequence was specifically aligned with the reference sequence of Plasmodium falciparum. This indicates that the primer set provided in this embodiment has good technical stability, high specificity and sensitivity as low as 10 copies / reaction.

[0027] Assessment of the reproducibility and accuracy of detection of Plasmodium falciparum: The reproducibility and accuracy of primer set identification of Plasmodium falciparum were evaluated based on whether the genotypes of the common detection sites could be reproduced in two replicates. Specifically, the genotypes of each MNP marker site generated from 12 sets of data from 100 copies / reaction positive samples were compared pairwise, and the results are shown in Table 4.

[0028] Table 4 shows the reproducibility and accuracy of the primer sets.

[0029] Table 4 shows that the number of MNP markers with different major genotypes is 0; based on the principle that genotypes that can be reproduced between two replicate experiments are considered accurate, the accuracy rate a = 1 - (1 - r ) / 2 = 0.5 + 0.5 r , r Reproducible rate, which is the ratio of the number of reproducible loci with the major genotype to the number of shared loci. In this example, the logarithm of the difference in the major genotype of MNP markers between different libraries and between different library preparation batches for each sample in the reproducibility test is 0, i.e., reproducibility r = 100% and accuracy a = 100%.

[0030] In positive samples with 10 copies / reaction and 100 copies / reaction, the primer set of this embodiment can reliably detect the three MNP marker sites of Plasmodium falciparum, while the negative control can occasionally detect one MNP marker site. Therefore, the criterion for determining the positivity of Plasmodium falciparum provided in this embodiment is: when no less than two MNP marker combinations of Plasmodium falciparum are detected in the test sample and the abundance of the detected sequences (i.e., the ratio of the number of sequences in the test sample that are matched to the MNP marker combination of Plasmodium falciparum to the total number of sequences in the test sample) is at least 10 times that in the blank control, it is determined that the nucleic acid of Plasmodium falciparum is detected in the test sample. Example

[0031] This disclosure provides a kit for identifying MNP marker combinations for Plasmodium, the kit comprising the primer set provided in Example 2.

[0032] Furthermore, the kit may also include a multiplex PCR premix. Example

[0033] This disclosure provides an application of an MNP marker combination, primer set, or kit, including the use of the MNP marker combination, primer set, or kit for the identification of Plasmodium falciparum.

[0034] Six nucleic acid samples provided by the Hubei Provincial Center for Disease Control and Prevention were identified using MNP marker combinations, primer sets, or kits. Four of the six samples were human-derived, and two were mosquito-derived, named S1 to S6 respectively (where S1 was a negative sample of Plasmodium falciparum, and S2 to S6 were positive samples of Plasmodium falciparum). The identification results are shown in Table 5.

[0035] Table 5 shows the analysis of Plasmodium falciparum detection in 6 nucleic acid samples.

[0036] Table 5 shows that the primer set and kit can accurately detect the three MNP marker sites of Plasmodium falciparum in each sample with a single reaction. The simultaneous detection of the three MNP marker sites ensures a true positive result for Plasmodium falciparum in the sample. Furthermore, combined with... Figure 1 It is known that fluorescent PCR-based methods detect one marker per reaction and determine the result based on a single marker, which easily leads to false positives and false negatives. To achieve identification based on three markers, fluorescent PCR requires three detections. This demonstrates that the primer set and kit provided in this embodiment have high accuracy and efficiency in identifying Plasmodium falciparum. Example

[0037] MNP marker combinations, primer sets, or kits can be used for variation monitoring and cross-host tracing of Plasmodium falciparum.

[0038] As shown in Table 5 of Example 3, the primer set and kit provided in this example can detect all three MNP marker sites of Plasmodium falciparum, with an average sequencing coverage of 4000-fold for each MNP marker site. Pairwise comparisons were performed on the genotypes of the three markers in the five positive samples (S2-S6) in Table 5, and the results are shown in Table 6. The genotypes of the MNP markers detected in the three human samples (S2-S4) and the two mosquito samples (S5 and S6) were completely identical.

[0039] Table 6 shows the detection analysis of seven positive samples of Plasmodium falciparum.

[0040] As shown in Table 6, the primer set and kit provided in this embodiment can directly identify sequences in real samples without culture, and can be used to monitor the variation of Plasmodium. In addition, as shown in Table 6, the primer set and kit provided in this embodiment can simultaneously monitor human and mosquito-borne samples. Based on sequence comparison, it is determined that the Plasmodium falciparum in the human-borne samples is very likely to be infected by mosquitoes of types S5 and S6. Therefore, the primer set and kit provided in this embodiment can be used for infection tracing, and can clarify whether mosquitoes in a specific area carry Plasmodium, that is, whether they are "malaria vectors", providing a basis for the deployment of prevention and control resources. Example

[0041] This embodiment provides a method for constructing a database of Plasmodium falciparum using MNP marker sites, primer sets, or kits. Specifically, the database can be an MNP fingerprint database.

[0042] As shown in Table 5 of Example 2, after testing the samples using this primer set, the major genotype of the MNP marker locus of Plasmodium falciparum in each sample is obtained. The major genotypes of the strains obtained from all samples are compared, and the major genotypes with differences are entered into the database file to form the Plasmodium falciparum MNP fingerprint database. Each time the major genotype of the MNP marker locus of a sample or strain is compared with the constructed MNP fingerprint database, the MNP fingerprint profiles of strains with different major genotypes can be entered into the constructed MNP fingerprint database. Therefore, theoretically, the constructed MNP fingerprint database can be continuously updated and enriched. Because the constructed database is based on the gene sequence of the detected strains, it is compatible with all high-throughput sequencing data and has the characteristics of being fully co-constructable, shareable, and updatable.

[0043] The above description is merely an optional embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A primer set for identifying Plasmodium, characterized in that, The primer set includes: primer pairs 1 to 3, each primer pair including a forward primer and a reverse primer, the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the third primer pair and the reverse primer of the third primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 6 in the sequence listing.

2. A kit for identifying MNP marker combinations for Plasmodium, characterized in that, The kit includes the primer set as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes a multiplex PCR premix.

4. The application of the primer set as described in claim 1 or the kit as described in claim 2, characterized in that, The applications include using the primer set or the kit for non-disease diagnostic purposes to identify Plasmodium falciparum.

5. The application according to claim 4, characterized in that, The applications include using the primer set or the kit for monitoring mutations in Plasmodium falciparum.

6. The application according to claim 4, characterized in that, The applications include using the primer set or the kit for tracing the origin of Plasmodium falciparum.

7. The application according to claim 4, characterized in that, The applications include using the primer set or the kit for constructing a database of Plasmodium falciparum.

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