Search

CN-121992127-A - Primer probe composition for detecting plasmodium, kit and application

CN121992127ACN 121992127 ACN121992127 ACN 121992127ACN-121992127-A

Abstract

The invention relates to the technical field of molecular biology, in particular to a primer probe composition for detecting plasmodium, a kit and application, wherein the primer probe composition comprises a first primer and a first probe for detecting plasmodium falciparum; the kit comprises a second primer and a second probe for detecting plasmodium falciparum pfhrp2 genes, a third primer and a third probe for detecting plasmodium falciparum pfhrp genes, a fourth primer and a fourth probe for detecting plasmodium vivax, a fifth primer and a fifth probe for detecting plasmodium vivax, and a sixth primer and a sixth probe for detecting plasmodium ovale. According to the invention, through the designed primer probe composition, the gene deletion states of plasmodium falciparum, plasmodium vivax, plasmodium malariae, plasmodium ovale and plasmodium falciparum pfhrp, pfhrp3 can be synchronously detected, the problem that the existing detection method is difficult to consider plasmodium typing and gene deletion identification is solved, and the method has the advantages of high detection efficiency, strong specificity, high sensitivity and the like.

Inventors

  • LI XIAOTONG
  • LU FENG
  • ZHAO CHUNZHONG
  • YE JIANZHONG
  • DIAO MUYAN
  • YE YING
  • HE JIANAN
  • ZHANG RAN
  • ZHU LI
  • LIU JUN

Assignees

  • 深圳国际旅行卫生保健中心(深圳海关口岸门诊部)

Dates

Publication Date
20260508
Application Date
20260324

Claims (10)

  1. 1. A primer probe composition for detecting plasmodium, comprising: The kit comprises a first primer and a first probe, wherein the nucleotide sequence of the first primer is shown as SEQ ID No. 1-2, and the nucleotide sequence of the first probe is shown as SEQ ID No. 3; The second primer and the second probe are used for detecting plasmodium falciparum pfhrp genes, the nucleotide sequence of the second primer is shown as SEQ ID No. 4-5, and the nucleotide sequence of the second probe is shown as SEQ ID No. 6; The third primer and the third probe are used for detecting plasmodium falciparum pfhrp gene, the nucleotide sequence of the third primer is shown as SEQ ID No. 7-8, and the nucleotide sequence of the third probe is shown as SEQ ID No. 9; The kit comprises a fourth primer and a fourth probe, wherein the nucleotide sequence of the fourth primer is shown as SEQ ID No. 10-11, and the nucleotide sequence of the fourth probe is shown as SEQ ID No. 12; The kit comprises a fifth primer and a fifth probe, wherein the nucleotide sequence of the fifth primer is shown as SEQ ID No. 13-14, and the nucleotide sequence of the fifth probe is shown as SEQ ID No. 15; The kit comprises a sixth primer and a sixth probe, wherein the nucleotide sequence of the sixth primer is shown as SEQ ID No. 16-17, and the nucleotide sequence of the sixth probe is shown as SEQ ID No. 18.
  2. 2. The primer probe composition according to claim 1, further comprising a seventh primer and a seventh probe for plasmodium pass through, wherein the nucleotide sequence of the seventh primer is shown as SEQ ID No. 19-20, and the nucleotide sequence of the seventh probe is shown as SEQ ID No. 21.
  3. 3. The primer probe composition of claim 1 or 2, further comprising an internal reference probe set for detecting amplification of an internal reference gene, wherein the internal reference probe set comprises an internal reference primer having a nucleotide sequence shown as SEQ ID nos. 22 to 23 and an internal reference probe having a nucleotide sequence shown as SEQ ID No. 24.
  4. 4. The primer probe composition of claim 1 or 2, wherein the nucleotide sequence of the first probe is 5 'tagged with a fluorescent group and 3' tagged with a quenching group; And/or, the nucleotide sequence of the second probe is marked with a fluorescent group at the 5 'end and a quenching group at the 3' end; and/or, the nucleotide sequence of the third probe is marked with a fluorescent group at the 5 'end and a quenching group at the 3' end; and/or, the nucleotide sequence of the fourth probe is marked with a fluorescent group at the 5 'end and a quenching group at the 3' end; and/or, the nucleotide sequence of the fifth probe is marked with a fluorescent group at the 5 'end and a quenching group at the 3' end; And/or, the nucleotide sequence of the sixth probe is marked with a fluorescent group at the 5 'end and a quenching group at the 3' end.
  5. 5. The primer probe composition according to claim 4, wherein the fluorescent group is at least one selected from FAM, ROX, VIC, CY and HEX; And/or the quenching group is selected from at least one of BHQ1, BHQ2 and BHQ 3.
  6. 6. A kit for plasmodium detection comprising a primer probe set according to any one of claims 1-5.
  7. 7. Use of the primer probe composition of any one of claims 1 to 5 or the kit of claim 6 for detecting plasmodium.
  8. 8. A method for detecting plasmodium, comprising the steps of: using the DNA of the sample to be detected as a template, performing PCR amplification by using the primer probe composition according to any one of claims 1 to 5 or the kit according to claim 6, and judging according to the PCR amplification result and the Ct value.
  9. 9. The method according to claim 8, wherein the reaction system for PCR amplification comprises water, a PCR premix, an enzyme cocktail, and the primer probe composition according to any one of claims 1 to 5.
  10. 10. The method for detecting plasmodium according to claim 8 or 9, wherein the reaction procedure of the PCR amplification includes 50 ℃ reverse transcription for 30min, 95 ℃ pre-denaturation for 3min, 95 ℃ denaturation for 15s, 60 ℃ annealing for 30s, and 40 cycles are performed.

Description

Primer probe composition for detecting plasmodium, kit and application Technical Field The invention relates to the technical field of molecular biology, in particular to a primer probe composition for detecting plasmodium, a kit and application. Background Malaria is a major public health problem to be managed and controlled in the global scope, has a wide epidemic range and high infection risk, seriously threatens human health, and is a key premise for realizing effective treatment and blocking a transmission chain. Malaria is mainly caused by four plasmodium infections of plasmodium falciparum, plasmodium vivax, plasmodium malariae and plasmodium ovale, and the severity and treatment scheme of the diseases caused by different insect strains are different, so that in diagnosis, whether the infection exists or not is definitely required, and the plasmodium species are accurately distinguished. Currently, rapid diagnostic Reagents (RDT) based on histidine protein 2 rich (HRP 2) of plasmodium falciparum are widely used in malaria endemic areas worldwide, especially where resources are scarce, by virtue of their outstanding advantages of easy operation and rapid detection. However, in recent years, a plurality of malaria high-incidence areas successively report plasmodium falciparum strains with natural deletions pfhrp2 and/or pfhrp3 genes, and the deletion strains can not synthesize HRP2/HRP3 antigens, so that false negative results appear in RDT detection depending on the antigens, meanwhile, the accurate identification of four plasmodium falciparum is difficult to synchronously finish by the existing RDT and partial detection technology, and the accuracy of clinical accurate diagnosis and treatment decisions and epidemic dynamic monitoring is seriously interfered. Currently, the confirmation of pfhrp/3 gene deletion mainly depends on the traditional nested PCR combined gel electrophoresis technology, and the technology has the inherent defects of complicated steps, long time consumption, easiness in occurrence of cross contamination and the like, cannot realize accurate quantitative analysis, is more difficult to synchronously finish the distinguishing detection of plasmodium falciparum, plasmodium vivax, plasmodium malariae and plasmodium ovale, and is difficult to meet the actual requirements of large-scale epidemic situation screening and clinical rapid auxiliary diagnosis. Although the fluorescent quantitative PCR technology is gradually applied to plasmodium detection, has the advantages of high sensitivity, strong specificity, low pollution risk and the like, the prior art is limited to general plasmodium screening (such as targeted 18S rRNA genes) or single target quantitative detection, or can only judge whether plasmodium infection exists and cannot distinguish the species of insect strains, or only detect a single plasmodium falciparum strain and ignore other pathogenic plasmodium, and generally lacks an integrated detection scheme capable of synchronously solving three core problems of 'whether plasmodium infection exists', 'which plasmodium is infected' and 'whether the plasmodium falciparum is carrying Pfhrp/3 gene deletion which causes RDT failure' through a single experiment in a closed reaction system. Furthermore, the genome of different strains of plasmodium has a difference, wherein the plasmodium falciparum genome has extremely high AT content, and pfhrp/3 genes are positioned in a subtelomere area with high variation, which brings great challenges to the design of a targeting primer and a probe in the area, and simultaneously, in order to realize synchronous specific detection of four strains of plasmodium falciparum, plasmodium vivax, plasmodium malariae and plasmodium ovale, cross reaction among different strains is avoided, and higher requirements are put forward on the specificity and the stability of the primer and the probe. Disclosure of Invention In view of the above, the invention provides a primer probe composition, a kit and application for detecting plasmodium, which can synchronously detect plasmodium falciparum, plasmodium vivax, plasmodium malariae, plasmodium ovale and plasmodium falciparum pfhrp, pfhrp3 in a gene deletion state by the designed primer probe composition, and overcomes the problem that the existing detection method is difficult to consider plasmodium typing and gene deletion identification, and has the advantages of high detection efficiency, strong specificity, high sensitivity and the like. In a first aspect, the present invention provides a primer probe composition for detecting plasmodium, comprising: The kit comprises a first primer and a first probe, wherein the nucleotide sequence of the first primer is shown as SEQ ID No. 1-2, and the nucleotide sequence of the first probe is shown as SEQ ID No. 3; The second primer and the second probe are used for detecting plasmodium falciparum pfhrp genes, the nucleotide sequence of the second primer is shown