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Microdroplet digital PCR (polymerase chain reaction) kit used for quantitatively detecting human cytomegalovirus, and detection method

A technology for human cytomegalovirus and cytomegalovirus, which is applied in the field of viral nucleic acid detection, can solve the problems of low virus titer, false positives, and affect the accuracy of HCMV, and achieves good specificity, high sensitivity, and reduced PCR amplification efficiency. low effect

Pending Publication Date: 2020-01-14
苏州云泰生物医药科技有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this detection method needs to establish a standard curve, and the virus titer is usually at a low level, which affects the accuracy of HCMV detection, and can only achieve qualitative and relative quantitative detection of HCMV
At the same time, the RT-PCR method has certain limitations in sensitivity and specificity. It is sensitive to certain chemical substances in the extraction process of viral nucleic acid, which in turn inhibits the PCR reaction, resulting in "false positive" results.

Method used

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  • Microdroplet digital PCR (polymerase chain reaction) kit used for quantitatively detecting human cytomegalovirus, and detection method
  • Microdroplet digital PCR (polymerase chain reaction) kit used for quantitatively detecting human cytomegalovirus, and detection method
  • Microdroplet digital PCR (polymerase chain reaction) kit used for quantitatively detecting human cytomegalovirus, and detection method

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Experimental program
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Effect test

Embodiment 1

[0069] Design and screening of embodiment 1 primers and probes

[0070] 1. Design of primers and probes: Obtain the human cytomegalovirus gene sequence through the online tools of the National Center for Biotechnology Information (NCBI), and compare and analyze the obtained sequences at www.ebi.ac.uk / online. Specific primers and probes suitable for ddPCR were designed using PrimeExpress software.

[0071] Alternative primers are shown in Table 3:

[0072] table 3

[0073]

[0074]

[0075] 2. Screening of primers

[0076] (1) The primers were randomly matched into 4 pairs, F1R1, F1R2, F2R1, F2R2;

[0077] (2) Fluorescence quantitative PCR by dye method, PCR system formula: 10 μL of 2×one-step SYBR Green Supermix, 1 μL of forward primer and reverse primer, diH 2 O 3 μL, positive template 5 μL, total volume 20 μL. Finally, a pair of primers F1R1 with strong specificity and suitable for HCMV droplet digital PCR was obtained.

[0078] 3. Screening of probes

[0079] (...

Embodiment 2

[0081] Embodiment 2 kit composition

[0082] The specific composition of the kit is shown in Table 4:

[0083] Table 4

[0084]

Embodiment 3

[0085] Embodiment 3 detection method

[0086] 1. Extraction of DNA: Use the kit of Kangwei Century Company to extract DNA, the specific operation is as follows:

[0087] 1.1 Take 1ml sample, add 100μL proteinase K, and mix well;

[0088] 1.2 Add 800μL Buffer CL and shake vigorously for 30s;

[0089] 1.3 Incubate at 60°C for 30 minutes, and keep mixing during the period;

[0090] 1.4 Add 1800μL Buffer CB (add isopropanol) and mix well;

[0091] 1.5 After 5 minutes in ice bath, transfer the solution to the extension tube, turn on the negative pressure device, wait for the solution in the tube to be sucked away, and then turn off the negative pressure;

[0092] 1.6 Add 500 μL Buffer GW1 (add absolute ethanol) to the adsorption column, turn on the negative pressure device, wait for the solution in the tube to be sucked away, and turn off the negative pressure;

[0093] 1.7 Add 750 μL Buffer GW2 (add absolute ethanol) to the adsorption column, turn on the negative pressure devi...

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Abstract

The invention belongs to the field of viral nucleic acid detection, and particularly relates to a microdroplet digital PCR (polymerase chain reaction) kit used for quantitatively detecting a human cytomegalovirus, and a detection method. The microdroplet digital PCR kit comprises a microdroplet digital PCR detection agent, wherein the microdroplet digital PCR detection agent contains a primer anda probe used for detecting human cytomegalovirus nucleic acids; the primer and the probe used for detecting human cytomegalovirus nucleic acids comprise a forward primer indicated by SEQ ID NO:1, a reverse primer indicated by SEQ ID NO:2 and a probe disclosed by SEQ ID NO:3; and two ends of the probe are labeled with fluorophores. The kit is quick, accurate, good in specificity and high in sensitivity, and can directly carry out absolute quantification without depending on a standard product.

Description

technical field [0001] The invention belongs to the field of virus nucleic acid detection, and in particular relates to a droplet digital PCR kit and a detection method for quantitative detection of human cytomegalovirus. Background technique [0002] Human cytomegalovirus (HCMV) belongs to the genus Cytomegalovirus of the family Herpesviridae, with a diameter of 150-200nm, a double-stranded, linear DNA structure at the core, and an icosahedral symmetric capsid with a diameter of 100nm. The capsid consists of 162 Capsomers consist of capsomers, 150 of which are hexons and 12 of which are pentons. There is a cortex wrapping outside the nucleocapsid, and the outermost layer of the virion is a lipid bilayer envelope containing a variety of virus-encoded glycoproteins. There are 5 kinds of polypeptides in the virus capsid, and 2 kinds are the main structural proteins, namely the main protein and the minor protein of the capsid. The main protein of the capsid has a molecular wei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/705C12Q1/6851C12Q2531/113C12Q2563/159
Inventor 严晓锋刘凤麟熊慧
Owner 苏州云泰生物医药科技有限公司
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