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Multiplex fluorescence PCR (Polymerase Chain Reaction) detection method and detection kit for typhoid/paratyphoid saimonella

A technology of Salmonella typhi and detection kit, which is applied in the direction of fluorescence/phosphorescence, biochemical equipment and methods, microbiological determination/inspection, etc., to achieve objective and accurate results and easy operation

Active Publication Date: 2011-06-22
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the fluorescent PCR technology reported so far generally detects one type of Salmonella typhi in a single tube, and there is no multiple fluorescent PCR technology using the improved molecular beacon fluorescent probe combined with the HAND system to simultaneously detect typhoid, type A, type B, and type C in one tube. Literature reports on paratyphoid fever

Method used

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  • Multiplex fluorescence PCR (Polymerase Chain Reaction) detection method and detection kit for typhoid/paratyphoid saimonella
  • Multiplex fluorescence PCR (Polymerase Chain Reaction) detection method and detection kit for typhoid/paratyphoid saimonella
  • Multiplex fluorescence PCR (Polymerase Chain Reaction) detection method and detection kit for typhoid/paratyphoid saimonella

Examples

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Effect test

Embodiment 1

[0042] A multiple fluorescent PCR detection kit for typhoid and paratyphi Salmonella, said kit comprising:

[0043] Tailing primers for specifically amplifying the ViaB gene sequences of Salmonella typhi and Salmonella paratyphi C, the base sequences of which are shown in SEQ ID NO: 1 (ViaB-TF) and SEQ ID NO: 2 (ViaB-TR) ;

[0044]A molecular beacon probe for detecting the ViaB gene of Salmonella typhi and Salmonella paratyphi C, the base sequence of which is shown in SEQ ID NO: 3 (ViaB-P);

[0045] Tailing primers for specifically amplifying the fliC-a gene sequence of Salmonella paratyphi A, the base sequences of which are as SEQ ID NO: 4 (fliC-a-TF) and SEQ ID NO: 5 (fliC-a-TR) shown;

[0046] A molecular beacon probe for detecting the fliC-a gene of Salmonella paratyphi A, the base sequence of which is shown in SEQ ID NO: 6 (fliC-a-P);

[0047] A tailing primer for specifically amplifying the fliC-b gene sequence of Salmonella paratyphi beta, its base sequence is as SEQ...

Embodiment 3

[0091] Embodiment 3 carries out methodological evaluation to the kit described in embodiment 1

[0092] 1. Method

[0093] The 8 positive reference products and 8 negative reference products (see Table 4 for details) purchased from the Central Inspection Institute were serially diluted as required, and then the DNA was prepared according to the bacterial liquid boiling method (take 1ml bacterial liquid, centrifuge at 12000rpm for 5min, discard Supernatant: Add 50 μl of double distilled water to suspend the precipitate, boil at 100°C for 5 minutes, centrifuge at 12000 rpm for 2 minutes to take the supernatant as a template), and carry out experiments on the specificity, sensitivity, precision and detection limit of the fluorescent PCR system. Among them, the precision experiment needs to process 10 samples of the precision reference product in the same batch, and calculate the Ct value CV% in the test.

[0094] For the recipe and PCR program of the PCR system used, see Multipl...

Embodiment 4 Embodiment 1

[0099] Embodiment 4 Embodiment 1 described kit clinical assessment comparative experiment

[0100] Use the typhoid and paratyphi Salmonella multiplex PCR detection kit and gold standard culture method described in this application to carry out blind detection on blood samples. The negative and positive test results of the specimen are based on the culture method, and the detection performance indicators such as sensitivity and specificity of the present invention are measured with this.

[0101]A total of 538 cases of clinical trial samples from 3 provincial laboratories were detected, 212 cases were detected positive and 326 cases were negative by culture method; 218 cases were detected by the kit of the present invention and 320 cases were negative. Compared with the culture method, the sensitivity of the kit of the present invention to detect Salmonella typhi and paratyphi is 100%, and the specificity is 98.2%, as shown in Table 5.

[0102] Table 5 culture method compares ...

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Abstract

The invention discloses a multiplex fluorescence PCR (Polymerase Chain Reaction) detection kit for typhoid / paratyphoid saimonella. The detection method comprises the following step of: specifically amplifying tailed primers of a ViaB gene sequence, a fliC-a gene sequence, a fliC-b gene sequence and a ssaR gene sequence of typhoid saimonella, C type paratyphoid saimonella, Atype paratyphoid saimonella and B type paratyphoid saimonella respectively, wherein the base sequences of the tailed primers are shown as SEQ ID NO:1, 2, 4, 5, 7, 8, 10 and 11; and the molecular beacon probes for detecting the ViaB gene sequence, the fliC-a gene sequence, the fliC-b gene sequence and the ssaR gene sequence of the typhoidsaimonella, the C type paratyphoid saimonella, the A type paratyphoid saimonella and the B type paratyphoid saimonella are shown as SEQ ID NO: 3, 6, 9 and 12. The detection method and the detection kit provided by the invention reach the sensitivity level of a gold standard culture method in the aspect of detection sensitivity index, and have the advantages of quickness, sensitivity, easiness and convenience in operating, objective and correct result and the like compared with the conventional culture method.

Description

technical field [0001] The invention relates to the field of detection of Salmonella typhi, in particular to a multiple fluorescent PCR detection method and detection kit for Salmonella typhi and paratyphi. Background technique [0002] Typhoid fever and paratyphoid fever are the statutory B infectious diseases in my country. Typhoid fever is caused by Salmonella typhi, and paratyphoid is caused by Salmonella paratyphi A, B, and C. Acute intestinal infectious diseases caused by typhoid fever are still one of the major public health problems worldwide, especially in developing countries. There are approximately 17 million cases of typhoid fever and 600,000 deaths worldwide each year. After 1990, the average incidence rate in my country was between 4.08-10.45 / 100,000, and 51,000-120,000 people were reported to be sick and 33-374 people died of illness every year. [0003] At present, the existing diagnostic methods for typhoid and paratyphoid fever are mainly based on bacter...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10G01N21/64
CPCY02A50/30
Inventor 扈庆华石晓路朱玉梅李庆阁汪再兴李迎慧邱亚群林一曼刘涛
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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