Performance of a Multiplex Qualitative PCR LCx Assay for Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Group M Subtypes, Group O, and HIV-2

Abravaya, Klara; Esping, Claudia; Hoenle, Robert; Gorzowski, Jacek; Perry, Robert T.; Kroeger, Paul E.; Robinson, John; Flanders, R

doi:10.1128/jcm.38.2.716-723.2000

Cited by 23 publications

(5 citation statements)

References 44 publications

(55 reference statements)

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“…However, this limit was significantly higher, 50.5 TCID 50 for VSV‐IND and 284 TCID 50 for VSV‐NJ, when tri‐reagent method was used for the extraction of total RNA from the samples. These detection limits are superior or comparable to previously reported detection limits of similar assays 10–15. Negative controls confirmed that only RNA was transcribed and amplified, no virus had contaminated the control cultures, and no contaminating RNA or DNA was present in any of the reagents used in this study.…”

Section: Discussionsupporting

confidence: 85%

Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey

Arshed¹,

Magnuson

Triantis

et al. 2011

Clinical Laboratory Analysis

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Two methods for the extraction of RNA of vesicular stomatitis virus (VSV) Indiana1 and New Jersey and their simultaneous amplification by one-step polymerase chain reaction using reverse transcriptase were evaluated. A guanidine-thiocyanate-based RNA extraction (Qiagen RNeasy Mini Kit, Qiagen, Valencia, CA ) followed by column-based purification coupled with one-step RT-PCR proved to be a simple, safe, practicable, and reliable tool for rapid, highly sensitive, and specific differential diagnosis of both types of VSV in cell lysate and spiked tissue samples as compared with the tri-phasic extraction method (Tri-reagent method). When RNA was extracted either from VSV cell culture stock or from VSV spiked bovine lymph nodes by using Qiagen RNeasy Mini Kit, the detection limit in the multiplex RT-PCR was as low as 0.505 to 2.84 TCID(50) for VSV-IND and VSV-NJ, respectively. The multiplex RT-PCR consistently detected VSV-IND and NJ RNA in as little as 0.1-1.0 fg of total RNA from spiked BHK-21 cell suspension when Qiagen RNeasy mini kit was used. The multiplex RT-PCR assay was capable of detecting both types of VSV in a one-step reaction tube. The minimum sensitivity of this assay in various experiments was 0.1683 TCID(50) (IND), 0.0946 TCID(50) (NJ), and 0.057 fg (IND and NJ) per 2 µl PCR sample, which is significantly more sensitive than reported previously (0.28-2.8 TCID50/1 µl). So the present study improved the sensitivity of previously reported multiplex RT-PCR for the detection and differentiation of VSV-IND and VSV-NJ in a single assay.

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Section: Discussionsupporting

confidence: 85%

Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey

Arshed¹,

Magnuson

Triantis

et al. 2011

Clinical Laboratory Analysis

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“…This detection limit is superior or comparable to reported detection limits of similar assays. 1,3,5,10,24 The significant increase in the level of virus assayed by tissue culture after day 1 postinfection indicates that the virus replicated in the mosquitoes, as noted by other researchers. 13 The ability to detect VS RNA by this multiplex RT-PCR in as little as 20 fg or in as much as 430.2 ng of insect total RNA demonstrates the wide range of acceptable template concentrations that may be used.…”

Section: Discussionsupporting

confidence: 72%

A Single-Tube Multiplex Reverse Transcription—Polymerase Chain Reaction for Detection and Differentiation of Vesicular Stomatitis Indiana 1 and New Jersey Viruses in Insects

et al. 2003

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Abstract.A multiplex single-tube reverse transcription-polymerase chain reaction (RT-PCR) has been developed for the detection and differentiation of vesicular stomatitis viruses (VSV), Indiana 1 and New Jersey, from insect samples. Using this assay, detection of either or both viruses in as little as 20 fg of total RNA from tissue culture was achieved, along with detection of vesicular stomatitis (VS) RNA from macerates containing 2 infected mosquitoes in pools of 10-30 noninfected mosquitoes. Vesicular stomatitis virus was detected by RT-PCR in all culture-positive samples, and detection as low as 4 plaque forming units per milliliter was achieved. Comparison between RT-PCR and tissue culture revealed that RT-PCR was able to detect VSV in a volume of insect macerate averaging almost 100 times less than that required for detection by tissue culture. The reported RT-PCR is a potential valuable tool for rapid and sensitive detection and differentiation of VS in insects because intense work associated with viral isolation, the cytotoxicity of insect extracts, and separate virus identification steps can be avoided. Potential application to detection and differentiation of VSV serotypes from vertebrate hosts is addressed.Vesicular stomatitis virus (VSV) causes vesicular lesions on the epithelia of the tongue and mouth, as well as coronary bands of hooves of cattle, pigs, and horses. 9 Humans, rodents, and numerous other mammals and fowl can also be infected. 12,16 Vesicular stomatitis virus has been isolated on many occasions in several species of insects, including mosquitoes, during viral epidemics and in forested endemic foci. 2,21-23 At least 2 groups of insects, sand flies (Lutzomyia spp) and black flies (Diptera: Simuliidae), have been shown to carry the virus in nature and to replicate and transmit VSV to susceptible hosts after laboratory infection. 2,13,20 In addition, it has been shown that black flies can act as vectors to disseminate VSV by cofeeding on a nonviremic host. 14 However, the exact role played by insects in VSV's natural cycle has not been definitely proven. Vesicular stomatitis (VS) is of considerable economic importance as quarantines must be enforced until diagnosis is complete because its clinical symptoms are indistinguishable from those of foot and mouth disease (FMD).

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“…Several other molecular methods have been developed which are more accurate (but more complex) than that described in the present study. One uses inosine‐containing primers for the detection of HIV‐1 (13), while others detect both HIV‐1 and HIV‐2 by multiplex PCR (27–29). Whether the simple molecular approach described here is indeed highly accurate, both in blood and saliva, or the obtained results are caused by studying an infected group with rather homogeneous characteristics (nationality, residence, asymptomatic status, periodontal disease) has to be tested in a greater series of individuals in a multiethnic environment.…”

Section: Discussionmentioning

confidence: 99%

Immunological and molecular detection of human immunodeficiency virus in saliva, and comparison with blood testing

Yapijakis

Panis

Koufaliotis³

et al. 2006

European J Oral Sciences

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In order to test the detection feasibility of human immunodeficiency virus (HIV) in saliva, a three-method blind screening analysis was conducted. Sixty-eight individuals were studied, comprising 34 HIV carriers and 34 noncarriers (controls) of matched gender and age. An oral examination preceded saliva and blood sampling of studied individuals. All samples were tested blind for HIV by using two immunological methods [Oraquick-compatible enzyme-linked immunosorbent assay (ELISA) and a fluorescent immunoenzymatic method (ELFA)], confirmed by western blotting, and a simple molecular method (polymerase chain reaction amplification of a relatively constant viral DNA region), confirmed by DNA hydridization. Compared with the controls, about twice as many HIV carriers had oral health problems, including periodontal disease. ELFA resulted in 33/34 positives and 34/34 negatives in saliva, while it detected 34/34 positives and 34/34 negatives in blood. ELISA performed even better, with correct assignment of all positives and negatives in both saliva and blood. The PCR method, at three annealing temperatures, surprisingly detected all positive samples, while it gave no false-positive result. In conclusion, the detection of anti-HIV in saliva may achieve accuracy of 97.1-100%, comparable with that in blood. Furthermore, this study suggests that a highly accurate molecular method of HIV detection may be feasible, although the studied carriers had rather homogeneous characteristics.

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Performance of a Multiplex Qualitative PCR LCx Assay for Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Group M Subtypes, Group O, and HIV-2

Cited by 23 publications

References 44 publications

Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey

Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey

A Single-Tube Multiplex Reverse Transcription—Polymerase Chain Reaction for Detection and Differentiation of Vesicular Stomatitis Indiana 1 and New Jersey Viruses in Insects

Immunological and molecular detection of human immunodeficiency virus in saliva, and comparison with blood testing

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