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[EXPERIENCE OF STUDY AND POSSIBLE WAYS OF ELIMINATION OF FALSE POSITIVE AND FALSE NEGATIVE RESULTS DURING EXECUTION OF POLYMERASE CHAIN REACTION ON AN EXAMPLE OF JUNIN VIRUS RNA DETECTION].

Abstract

AIM

Experience of study and possible ways of elimination of false positive and false negative results during execution of polymerase chain reaction on an example of Junin virus RNA detection. MATERIALSS AND METHODS: Junin virus--causative agent of Argentine hemorrhagic fever (AHF) strain XJpR37/5787 was obtained from the State collection of pathogenicity group I causative agents of the 48th Central Research Institute. Reagent kit for detection of Junin virus RNA by RT-PCR was developed in the Institute and consists of 4 sets: for isolation of RNA, execution of reverse-transcription reaction, execution of PCR and electrophoretic detection of PCR products. RT-PCR was carried out by a standard technique. Continuous cell cultures of African green monkey Vero B, GMK-AH-1(D) were obtained from the museum of cell culture department of the Centre.

RESULTS

An experimental study of the effect of various factors of impact on the sample under investigation ("thawing-freezing", presence of formaldehyde, heparin) on the obtaining of false negative results during Junin virus RNA detection by using RT-PCR was studied. Addition of 0.01% heparin to the samples was shown to completely inhibit PCR. Addition of 0.05% formaldehyde significantly reduces sensitivity of the method. A possibility of reduction of analysis timeframe from 15 to 5 days was shown during detection of the causative agent in samples with low concentration of the latter by growing the samples and subsequent analysis of the material obtained by using RT-PCR.

CONCLUSION

During detection of causative agent by using RT-PCR false negative results could appear in the presence of formaldehyde and heparin in the sample. A possibility of elimination of false negative PCR results due to concentration of the causative agent in the sample under investigation at a level below sensitivity threshold was shown on the example of Junin virus RNA detection by using growing of the pathogen in appropriate accumulation system with subsequent analysis of the material obtained using PCR.

Authors

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Pub Type(s)

Journal Article

Language

rus

PubMed ID

26950993

Citation

Sizikova, T E., et al. "[EXPERIENCE of STUDY and POSSIBLE WAYS of ELIMINATION of FALSE POSITIVE and FALSE NEGATIVE RESULTS DURING EXECUTION of POLYMERASE CHAIN REACTION ON an EXAMPLE of JUNIN VIRUS RNA DETECTION]." Zhurnal Mikrobiologii, Epidemiologii I Immunobiologii, 2015, pp. 78-82.
Sizikova TE, Lebedev VN, Pantyukhov VB, et al. [EXPERIENCE OF STUDY AND POSSIBLE WAYS OF ELIMINATION OF FALSE POSITIVE AND FALSE NEGATIVE RESULTS DURING EXECUTION OF POLYMERASE CHAIN REACTION ON AN EXAMPLE OF JUNIN VIRUS RNA DETECTION]. Zh Mikrobiol Epidemiol Immunobiol. 2015.
Sizikova, T. E., Lebedev, V. N., Pantyukhov, V. B., Borisevich, S. V., & Merkulov, V. A. (2015). [EXPERIENCE OF STUDY AND POSSIBLE WAYS OF ELIMINATION OF FALSE POSITIVE AND FALSE NEGATIVE RESULTS DURING EXECUTION OF POLYMERASE CHAIN REACTION ON AN EXAMPLE OF JUNIN VIRUS RNA DETECTION]. Zhurnal Mikrobiologii, Epidemiologii I Immunobiologii, (6), 78-82.
Sizikova TE, et al. [EXPERIENCE of STUDY and POSSIBLE WAYS of ELIMINATION of FALSE POSITIVE and FALSE NEGATIVE RESULTS DURING EXECUTION of POLYMERASE CHAIN REACTION ON an EXAMPLE of JUNIN VIRUS RNA DETECTION]. Zh Mikrobiol Epidemiol Immunobiol. 2015 Nov-Dec;(6)78-82. PubMed PMID: 26950993.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [EXPERIENCE OF STUDY AND POSSIBLE WAYS OF ELIMINATION OF FALSE POSITIVE AND FALSE NEGATIVE RESULTS DURING EXECUTION OF POLYMERASE CHAIN REACTION ON AN EXAMPLE OF JUNIN VIRUS RNA DETECTION]. AU - Sizikova,T E, AU - Lebedev,V N, AU - Pantyukhov,V B, AU - Borisevich,S V, AU - Merkulov,V A, PY - 2016/3/9/entrez PY - 2016/3/10/pubmed PY - 2016/3/25/medline SP - 78 EP - 82 JF - Zhurnal mikrobiologii, epidemiologii i immunobiologii JO - Zh Mikrobiol Epidemiol Immunobiol IS - 6 N2 - AIM: Experience of study and possible ways of elimination of false positive and false negative results during execution of polymerase chain reaction on an example of Junin virus RNA detection. MATERIALSS AND METHODS: Junin virus--causative agent of Argentine hemorrhagic fever (AHF) strain XJpR37/5787 was obtained from the State collection of pathogenicity group I causative agents of the 48th Central Research Institute. Reagent kit for detection of Junin virus RNA by RT-PCR was developed in the Institute and consists of 4 sets: for isolation of RNA, execution of reverse-transcription reaction, execution of PCR and electrophoretic detection of PCR products. RT-PCR was carried out by a standard technique. Continuous cell cultures of African green monkey Vero B, GMK-AH-1(D) were obtained from the museum of cell culture department of the Centre. RESULTS: An experimental study of the effect of various factors of impact on the sample under investigation ("thawing-freezing", presence of formaldehyde, heparin) on the obtaining of false negative results during Junin virus RNA detection by using RT-PCR was studied. Addition of 0.01% heparin to the samples was shown to completely inhibit PCR. Addition of 0.05% formaldehyde significantly reduces sensitivity of the method. A possibility of reduction of analysis timeframe from 15 to 5 days was shown during detection of the causative agent in samples with low concentration of the latter by growing the samples and subsequent analysis of the material obtained by using RT-PCR. CONCLUSION: During detection of causative agent by using RT-PCR false negative results could appear in the presence of formaldehyde and heparin in the sample. A possibility of elimination of false negative PCR results due to concentration of the causative agent in the sample under investigation at a level below sensitivity threshold was shown on the example of Junin virus RNA detection by using growing of the pathogen in appropriate accumulation system with subsequent analysis of the material obtained using PCR. SN - 0372-9311 UR - https://www.unboundmedicine.com/medline/citation/26950993/[EXPERIENCE_OF_STUDY_AND_POSSIBLE_WAYS_OF_ELIMINATION_OF_FALSE_POSITIVE_AND_FALSE_NEGATIVE_RESULTS_DURING_EXECUTION_OF_POLYMERASE_CHAIN_REACTION_ON_AN_EXAMPLE_OF_JUNIN_VIRUS_RNA_DETECTION]_ L2 - https://medlineplus.gov/bloodthinners.html DB - PRIME DP - Unbound Medicine ER -