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Quantitative detection kit for orientia tsutsugamushi based on droplet PCR (polymerase chain reaction)

A kit and technology of scrub bugs, applied in the biological field, can solve problems such as false negatives, missed detection, and failure to take into account internal control channels, etc., to achieve the effects of reducing pollution, accurate quantitative results, and avoiding false negatives

Active Publication Date: 2022-04-22
GUANGDONG GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004]The sensitivity of fluorescent quantitative PCR is lower than that of digital PCR, which may cause missed detection and false negatives; moreover, fluorescent quantitative PCR can usually only achieve two fluorescent channels Simultaneous detection, unable to take into account the internal control channel
Or detect two copies, that is, two genes and one internal control gene are used to form the detection system; finally, the quantification of copy number by fluorescent quantitative PCR depends on the standard curve, and the samples with unknown concentration are estimated from the known concentration standard, which does not realize the real absolute quantification

Method used

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  • Quantitative detection kit for orientia tsutsugamushi based on droplet PCR (polymerase chain reaction)
  • Quantitative detection kit for orientia tsutsugamushi based on droplet PCR (polymerase chain reaction)
  • Quantitative detection kit for orientia tsutsugamushi based on droplet PCR (polymerase chain reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: sample detection

[0058] 1. Primer probe sequence

[0059] Based on the sequence of the conserved region of Orient tsutsugamushi, primers and probes were designed using Primer express software. After multiple comparisons and sequence analysis, 2 pairs of primers with excellent specificity were selected. The amplified product was 73-74 bp, which could be amplified efficiently. At the same time, in order to reduce the non-specific binding of the system, the probe sequence CCCCCAAAAA exists in the selected amplification product at the same time (two pairs of primers share one probe), and does not exist in other sequences of oriental tsutsugamushi, which is unique. The specific information is shown in Table 1:

[0060] Table 1 Primer and probe sequences

[0061]

[0062] When designing and selecting primers in the early stage, other primers have problems such as insufficient sensitivity of false positive results, such as the following primer-probe sets: ...

Embodiment 2

[0106] Embodiment 2: specificity test

[0107] Use the primers (F1-R1-F2-R2-P) of the present invention to simultaneously detect a variety of common pathogens, including Streptococcus, Staphylococcus aureus, coagulase-negative Staphylococcus, Enterococcus, Candida albicans, Pseudomonas aeruginosa , Stenophagous maltophilia, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii. All pathogens were pure cultures identified by MALDI-TOF, mixed into negative plasma to form mock samples. The specificity test was carried out according to the test method described in Example 1, and no positive result was detected, indicating that the specificity of the primers was high, and the test results are shown in Table 9.

[0108] Table 9 specificity test results

[0109]

Embodiment 3

[0110] Embodiment 3: sensitivity test

[0111] Negative plasma was added to Orient tsutsugamushi (10-20copies / mL), and digital PCR and real-time quantitative PCR were used to test the samples of each group simultaneously. The digital PCR test was positive, while the real-time quantitative PCR test was negative. The relevant test results are shown in Table 10.

[0112] Table 10 Sensitivity test results

[0113]

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Abstract

The invention relates to the technical field of biology, in particular to a quantitative detection kit for orientia tsutsugamushi based on droplet PCR (Polymerase Chain Reaction). The invention provides primers with nucleotide sequences as shown in SEQ ID NO.1-6, and probes with nucleotide sequences as shown in SEQ ID NO.7 and SEQ ID NO.8. The invention also provides a kit for detecting the content of the gene. The kit is good in sensitivity which is far lower than the detection limit of fluorescent quantitative PCR, and possible false negative is avoided; detection of two detection channels and one internal control channel can be carried out at the same time, and pollution possibly caused by repeated sample adding is reduced; absolute quantification of the number of liquid drops in the chip is achieved through multiple digital PCR, dependence on a standard curve is not needed, and the quantification result is more accurate.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a quantitative detection kit for oriental tsutsugamushi based on droplet PCR. Background technique [0002] Scrub typhus is a natural foci disease caused by oriental tsutsugamushi infection. The intermediate host is chigger mites, which are mainly transmitted to humans through bites. The main symptoms of scrub typhus include fever, rash, myalgia, swollen lymph nodes, nausea, vomiting, eschar (black spots appearing at the site of mite bites), abdominal pain and non-specific flu, etc., and there may be multiple complications: Jaundice, acute renal failure and disseminated intravascular coagulation (DIC), acute respiratory distress syndrome, myocarditis and meningoencephalitis, and even multi-organ failure. Troublingly, symptoms of scrub typhus are similar to diseases such as leptospirosis, dengue, brucellosis and typhoid fever, making differential diagnosis difficult. The presence of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/689C12Q1/6851C12Q1/04
CPCC12Q1/689C12Q1/6851C12Q2600/16C12Q2600/166C12Q2563/159C12Q2563/107C12Q2537/143Y02A50/30
Inventor 顾兵胡雪姣赵云虎刘素玲周晖张莉滟凌勇张鑫强袁凯旋
Owner GUANGDONG GENERAL HOSPITAL
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