Evaluation and validation of an RT‐PCR assay for specific detection of monkeypox virus (MPXV)

Mondolfi, Alberto Paniz; Guerra, Susana; Muñoz, Marina; Luna, Nicolás; Hernandez, Matthew M.; Patiño, Luz Helena; Reidy, Jason; Banu, Radhika; Shrestha, Paras; Umeaku, Audrey; Chen, Feng; Cao, Liyong; Patel, Armi; Hanna, Ayman; Li, Sunny; Look, Andy; Pagani, Nina; Albrecht, Randy A.; Pearl, Raymond; García‐Sastre, Adolfo; Bogunovic, Dusan; Palacios, Gustavo; Bonnier, Lucia; Cera, Freddy; Lopez, Heidi; Calderon, Yvette; Eiting, Erick; Mullen, Karr; Shin, Sang-Goo; Lugo, Luz Amarilis; Urbina, Antonio; Starks, Carlotta; Koo, Tonny; Uychiat, Patricia; Look, Avery; Bakel, Harm van; Gonzalez‐Reiche, Ana S.; Betancourt, Adolfo Firpo; Reich, David; Cordon‐Cardo, Carlos; Simon, Viviana; Sordillo, Emilia Mia; Ramírez, Juan David

doi:10.1002/jmv.28247

Cited by 32 publications

(22 citation statements)

References 42 publications

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“…Monkeypox is a reemerging zoonotic viral disease caused by the monkeypox virus (MPXV) [ 5 ]. MPXV is a double-stranded DNA virus of the genus Orthopoxvirus of the family Poxviridae known for more than half a century [ 6 ] but geographically endemic to Central and West Africa [ 7 , 8 ]. MPXV has two distinct clades: the Central Basin clade and the West African clade [ 9 ].…”

Section: Introductionmentioning

confidence: 99%

Detection of Monkeypox Virus according to The Collection Site of Samples from Confirmed Cases: A Systematic Review

et al. 2022

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Due to the rapid evolution of the monkeypox virus, the means by which the monkeypox virus is spread is subject to change. Therefore, the present study aims to analyze the detection of the monkeypox virus according to the collection site of samples from confirmed monkeypox cases. A systematic literature review was performed using PubMed, Scopus, Web of Science, and Embase databases until 5 October 2022. A total of 1022 articles were retrieved using the search strategy. After removing duplicates (n = 566) and examining by title, abstract, and full text, 65 studies reporting monkeypox case reports were included with a detailed description of risk factors, sexually transmitted infections (STIs), site of monkeypox virus-positive specimens, location of skin lesions, and diagnostic test. A total of 4537 confirmed monkeypox cases have been reported, of which 98.72% of the cases were male with a mean age of 36 years, 95.72% had a sexual behavior of being men who have sex with men, and 28.1% had human immunodeficiency virus (HIV). The most frequent locations of lesions in patients diagnosed with monkeypox were: 42.85% on the genitalia and 37.1% in the perianal region. All confirmed monkeypox cases were diagnosed by reverse transcriptase polymerase chain reaction (RT-PCR), and the most frequent locations of samples collected for diagnosis that tested positive for monkeypox virus were: 91.85% from skin lesions, 20.81% from the oropharynx, 3.19% from blood, and 2.43% from seminal fluid. The disease course of the cases with monkeypox was asynchronous, with no severe complications, and most patients did not report specific treatment but simply followed a symptomatic treatment.

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Section: Introductionmentioning

confidence: 99%

Detection of Monkeypox Virus according to The Collection Site of Samples from Confirmed Cases: A Systematic Review

et al. 2022

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“…By extracting from up to 250 μL of specimen, we determined the LoD for MPXV-F3L, MPXV-G2R, and OPXV-E9L assays to be as low as 66 copies/mL, or 3.3 copies per PCR reaction. Our analytical sensitivity compares favorably with a recent evaluation of the PKamp Monkeypox Virus RT-PCR assay, which also uses the F3L gene as its target (21). Furthermore, all three assays did not cross react with HSV or VZV, demonstrating that both assays are highly specific to MPXV even in mixed samples with high HSV/VZV viral titer.…”

Section: Discussionmentioning

confidence: 77%

“…Virus RT-PCR assay, which also uses the F3L gene as its target (21). Furthermore, all three assays did not cross react with HSV or VZV, demonstrating that both assays are highly specific to MPXV even in mixed samples with high HSV/VZV viral titer.…”

Section: Discussionmentioning

confidence: 90%

Evaluation and Clinical Validation of Monkeypox Virus Real-Time PCR Assays

Mills¹,

Juergens²,

Gov³

et al. 2022

Preprint

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BACKGROUND: In spring of 2022, an outbreak of monkeypox spread worldwide. Here, we describe performance characteristics of monkeypox virus (MPXV)-specific and pan-orthopoxvirus qPCR assays for clinical use. METHODS: We validated probe-based qPCR assays targeting MPXV-specific loci F3L and G2R (genes MPXVgp052/OPG065 and MPXVgp002 and gp190/OPG002, respectively) and a pan-orthopoxvirus assay targeting the E9L locus (MPXVgp057/OPG071). Clinical samples and synthetic controls were extracted using the Roche MP96 or Promega Maxwell 48 instrument. qPCR was performed on the AB7500 thermocycler. Synthetic control DNA and high concentration clinical samples were quantified by droplet PCR. Cross-reactivity was evaluated for camelpox and cowpox genomic DNA, vaccinia culture supernatant, and HSV- and VZV-positive clinical specimens. We also determined analytical performance of the F3L assay using dry swabs, Aptima vaginal and rectal swabs, nasopharyngeal, rectal, and oral swabs, cerebrospinal fluid, plasma, serum, whole blood, breastmilk, urine, saliva, and semen. RESULTS: The MPXV-F3L assay is reproducible at a limit of detection (LoD) of 65.6 copies/mL of viral DNA in VTM/UTM, or 3.3 copies/PCR reaction. No cross-reactivity with herpesviruses or other poxviruses was observed. MPXV-F3L detects MPXV DNA in alternative specimen types, with an LoD ranging between 260-1000 copies/mL, or 5.7-10 copies/PCR reaction. In clinical swab VTM specimens, MPXV-F3L and MPXV-G2R assays outperformed OPXV-E9L by an average of 2.4 and 2.8 Cts, respectively. MPXV-G2R outperformed MPXV-F3L by 0.4 Cts, consistent with presence of two copies of G2R present in labile inverted terminal repeats (ITRs) of MPXV genome. CONCLUSIONS: MPXV is readily detected by qPCR using three clinically validated assays.

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“…The assay yielded a limit of detection (LOD) of 13.5 copies/µl and 15 copies/µl by using a microtiter plate reader and one-pot system, respectively. Also, RT-PCR assay was developed by Paniz-Mondolfi et al [88] , which targets F3L- gene of MPXV with a LOD of 7.2 genome copies/reaction. In a recent preprint, Singh et al [89] described a method to distinguish MPXV from other related orthopoxviruses by targeting a single nucleotide polymorphism (SNP) on the polA gene.…”

Section: Diagnostic Assaysmentioning

confidence: 99%

Monkeypox (mpox) virus: Classification, origin, transmission, genome organization, antiviral drugs, and molecular diagnosis

Karagöz¹,

Tombuloğlu²,

Alsaeed³

et al. 2023

Journal of Infection and Public Health

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Evaluation and validation of an RT‐PCR assay for specific detection of monkeypox virus (MPXV)

Cited by 32 publications

References 42 publications

Detection of Monkeypox Virus according to The Collection Site of Samples from Confirmed Cases: A Systematic Review

Detection of Monkeypox Virus according to The Collection Site of Samples from Confirmed Cases: A Systematic Review

Evaluation and Clinical Validation of Monkeypox Virus Real-Time PCR Assays

Monkeypox (mpox) virus: Classification, origin, transmission, genome organization, antiviral drugs, and molecular diagnosis

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