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Melting curve analysis based oral pathogen multiple PCR (polymerase chain reaction) detection method

A melting curve analysis and detection method technology, applied in microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of low detection efficiency, long detection period, easy to cause pollution, etc. specific effect

Active Publication Date: 2016-01-20
XIAMEN JIKE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems of complex operation, long detection cycle, limited specificity and sensitivity, high rate of false positives, need to open the tube, easy to cause pollution, low detection efficiency, and high cost in the prior art, the present invention adopts the following technical solutions:

Method used

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  • Melting curve analysis based oral pathogen multiple PCR (polymerase chain reaction) detection method
  • Melting curve analysis based oral pathogen multiple PCR (polymerase chain reaction) detection method
  • Melting curve analysis based oral pathogen multiple PCR (polymerase chain reaction) detection method

Examples

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Embodiment 1

[0055] Example 1 Investigate artificial synthesis of different complementary target sequences Investigate the improved Taqman probe melting curve method to detect five oral pathogens: Porphyromonas gingivalis, Helicobacter pylori, Fusobacterium nucleatum, Streptococcus mutans, aureus staphylococcal capacity;

[0056] In this example, an improved Taqman probe for the above five kinds of oral pathogens was designed, the target sequences of the above five kinds of oral pathogens were designed, and the improved Taqman probe melting curve method was used to detect the five kinds of oral pathogens. The sequences of the modified Taqman probes and targets used were:

[0057] The probe for Porphyromonas gingivalis is 5'-HEX-catcaccacataccacagatg-BHQ2-3'

[0058] The probe for Helicobacter pylori is 5’-HEX-ctgcaacgtgacccattagcag-BHQ2-3’

[0059] The probe for Fusobacterium nucleatum is 5'-HEX-aaaatagcatactttacatattcatttt-BHQ2-3'

[0060] The probe for Streptococcus mutans is 5'-FAM-t...

Embodiment 2

[0070] Embodiment 2 investigates the artificially synthesized improved Taqman probe melting curve method after multiplex PCR to detect five kinds of oral pathogenic bacteria (Porphyromonas gingivalis, Helicobacter pylori, Fusobacterium nucleatum, Streptococcus mutans, Staphylococcus aureus at the same time )Ability.

[0071] In this embodiment, the improved Taqman probes and amplification primers for the above five oral pathogens are designed, and the whole genome DNA of the above five oral pathogens is used to investigate the improved Taqman probe melting curve method for simultaneous detection of five oral pathogens. ability of germs. The modified Taqman probes used were:

[0072] The probe for Porphyromonas gingivalis is 5'-HEX-catcaccacataccacagatg-BHQ2-3'

[0073] The probe for Helicobacter pylori is 5’-HEX-ctgcaacgtgacccattagcag-BHQ2-3’

[0074] The probe for Fusobacterium nucleatum is 5'-HEX-aaaatagcatactttacatattcatttt-BHQ2-3'

[0075] The probe for Streptococcus m...

Embodiment 3

[0094] Example 3 investigates the sensitivity of the artificially synthesized and improved Taqman probe melting curve method to detect different concentrations of Fusobacterium nucleatum plasmid standard substances after multiplex PCR.

[0095] In this example, the improved Taqman probes and amplification primers for the above five oral pathogenic bacteria were designed, and the plasmid standards for the complementary target sequence of Fusobacterium nucleatum were artificially synthesized, and the melting curve of the improved Taqman probe was investigated to detect the complementation of Fusobacterium nucleatum. Detection sensitivity of the target sequence.

[0096] The modified Taqman probes used were:

[0097] The probe for Porphyromonas gingivalis is 5'-HEX-catcaccacataccacagatg-BHQ2-3'

[0098] The probe for Helicobacter pylori is 5’-HEX-ctgcaacgtgacccattagcag-BHQ2-3’

[0099] The probe for Fusobacterium nucleatum is 5'-HEX-aaaatagcatactttacatattcatttt-BHQ2-3'

[0100...

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Abstract

The invention discloses a melting curve analysis based oral pathogen multiple PCR (polymerase chain reaction) detection method. The method is characterized by achieving multiple detection of five common oral pathogens by simultaneously amplifying specific target sequences of porphyromonas gingivalis, helicobacter pylori, fusobacterium nucleatum, streptococcus mutans and staphylococcus aureus in the oral pathogens by adopting multiple PCR and then carrying out improved Taqman probe melting curve analysis and identification. By adopting multiple PCR, the method is simple in detection operation, is short in detection cycle, can avoid false positive, also dispenses with pipe opening operation, can be reused, has the effect of saving resources and the cost and has high detection sensitivity and specificity.

Description

technical field [0001] The invention belongs to the field of molecular biology, and particularly relates to a multiplex PCR rapid detection method for five common oral pathogenic bacteria, Porphyromonas gingivalis, Helicobacter pylori, Fusobacterium nucleatum, Streptococcus mutans and Staphylococcus aureus. Background technique [0002] The human oral microbiota is an extremely complex ecosystem, including viruses, fungi, protozoa and bacteria. Among them, the number of bacteria is the largest. Statistics show that there are about 100 million bacteria per milliliter of saliva, and the bacterial species in the entire oral cavity exceeds There are 600 species. Porphyromonas gingivalis is currently considered to be one of the main pathogenic bacteria of chronic periodontitis. The local inflammatory response caused by adhesion and invasion of the arterial wall after entering the blood is considered to accelerate cardiovascular disease (cardiovascular disease, CVD) is one of the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/04C12R1/145C12R1/46C12R1/445C12R1/01
CPCC12Q1/686C12Q2527/107C12Q2537/143C12Q2561/101C12Q2561/113
Inventor 刘旭宏
Owner XIAMEN JIKE BIOTECH
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