Search

US-20260125771-A1 - MPX-HA Assay For Real-Time Detection Of Monkeypox

US20260125771A1US 20260125771 A1US20260125771 A1US 20260125771A1US-20260125771-A1

Abstract

Disclosed are an assay, method, and kit for detecting a monkeypox virus (MPXV) and the application thereof. The assay may be used alone or in combination with another assay or assays. The method of using and kit includes a composition used for detecting MPXV, wherein the composition is composed of a primer pair and a probe. The primer pair is composed of two single-stranded nucleic acid molecules shown in the first sequence and the second sequence in a sequence list, and the sequence of the probe is the third sequence in the sequence list. The kit is used for detecting MPXV, and has advantages of providing real-time screening of MPXV reducing cross-reaction to other pox diseases, and increasing specificity to MPXV for disease prevention and improvement of public health.

Inventors

  • Yanan Zhao
  • David S. Perlin

Assignees

  • HACKENSACK MERIDIAN HEALTH, INC.

Dates

Publication Date
20260507
Application Date
20231003

Claims (10)

  1. 1 . An assay composition for detecting a monkeypox virus (MPXV), comprising: a primer pair and a probe; wherein the primer pair includes a forward primer and a reverse primer having the sequences in [SEQ.1] and [SEQ.2], respectively; and the probe includes the sequence in [SEQ.3] for increasing specificity and reducing cross-reaction as compared to conventional rt-PCR assays.
  2. 2 . The composition according to claim 1 , wherein the probe is characterized in that: 5′ end of the probe is marked with a fluorescent reporter group FAM, and 3′ end is marked with a fluorescent quenching group Dabcyl.
  3. 3 . The composition according to claim 1 , wherein the primer pair has a concentration in 0.8 μl of 1 μM for the forward primer, and 2 μl of 10 μM for the reverse primer, and the probe has a concentration in 0.4 μl of 5 μM.
  4. 4 . The composition according to claim 1 , wherein the probe is a MPX-specific HA MB probe that ensures during the post PCR melt curve analysis it melts from the HA target amplified from MPXV at a temperature significantly higher than that from other orthopoxvirus species which may be likely amplified by the same PCR.
  5. 5 . The composition according to claim 4 , further including a second assay MPX-F3L having a second primer pair and a second probe; wherein the second primer pair includes a second forward primer and a second reverse primer having the sequences in [SEQ.4] and [SEQ.5], respectively; and the second probe includes the sequence in [SEQ.6].
  6. 6 . A test kit for detecting a monkeypox virus (MPXV), comprising the composition described in claim 1 or the compositions in claim 5 , and following material: a 2× master mix for real-time PCR (qPCR); a PCR enzyme, anti-Taq antibody, a qPCR-optimized buffer; and a heat-resistant RNase enzyme.
  7. 7 . The test kit according to claim 6 , further includes a DNA template; wherein the DNA template is a 5 μl DNA template having a synthetic oligo target or a monkeypox genomic DNA template.
  8. 8 . The test kit according to claim 7 , wherein the test kit utilizes a DNA sample from a patient for the detection of MPXV prior to appearance of lesions or symptomatology of MPXV.
  9. 9 . A method of using an assay in the detection of monkeypox virus (MPXV), comprising the steps of: providing a MPX-HA assay includes setting up in the 20 μl reaction material comprising of 10 μl of Premix Ex Taq (Probe qPCR), 0.8 μl of 1 μM a forward primer [SEQ. 1], 2 μl of 10 μM a reverse primer [SEQ.2], 0.4 μl of 5 μM MPX-HA MB probe [SEQ. 3], and 5 μl of DNA template; running a reaction on the qPCR cycler with thermal profile of 95° C. for 2 min, 50 cycles of 95° C. for 5 sec and 56° C. for 20 sec, followed by 95° C. for 20 sec, then melted from 53° C. to 63° C. with a ramp rate of 0.1° C./s; and obtaining rt-PCR test results at least after 1 hour and 7 min to complete the reaction.
  10. 10 . The method according to claim 9 , further including the steps of: preparing a MPX-F3L second assay that includes setting up in a 20 μl reaction material comprising of 10 μl of Premix Ex Taq (Probe qPCR), 0.4 μl of 10 μM second forward primer [SEQ.4], 0.4 μl of 10 μM second reverse primer [SEQ.5], 0.4 μl of 5 μM MPX-F3L MB second probe [SEQ.6], and 5 μl of DNA template; running a second reaction on a qPCR cycler with a thermal profile of 95° C. for 2 min followed by 40 cycles of 95° C. for 5 sec and 60° C. for 20 sec; and obtaining rt-PCR test results at least after 48 minutes to complete the second reaction.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. provisional application No. 63/413,089, filed Oct. 4, 2022, the disclosures of which are hereby incorporated herein by reference. TECHNICAL FIELD The present disclosure relates to the biological technical field relating to an assay, method, and test kit for detecting monkeypox virus (MPXV) and application thereof. The present disclosure also relates to an assay, or combination of assays, method, and test kit that are specific for MPXV without cross reaction to other pox viruses that allow real-time detection of monkeypox virus. BACKGROUND The emergence of disease-causing viruses is a constant threat to humanity as each year novel viruses are being found (Woolhouse et al., 2012). Unprecedented global changes—population growth, increased trade and travel, and climate change—in the recent decades have made the threat of re-emergence of viruses even more likely (Baker et al., 2022). The recent emergence of monkeypox disease by monkeypox virus (MPXV) has made global headlines. In May 2022, over 92 monkeypox cases were reported from 12 countries (WHO, 2022a). Although there was a major outbreak of monkeypox in the United States in 2003 via prairie dogs infected from an imported Gambian pouched rat (Karem et al., 2005; Reed et al., 2004), the recent unrelated outbreaks in multiple countries have caused serious concerns post-COVID and pressed for the need of disease surveillance and viral detection. Indeed, the recent widespread emergence of monkeypox, a rare and endemic zoonotic disease by monkeypox virus (MPXV), has made global headlines. While transmissibility (R0≈0.58) and fatality rate (0-3%) are low, its prolonged morbidity of two to four weeks, and lengthy incubation period of up to 21 days has caused monkeypox to become a considerable public health concern. Thus, effective containment and disease management require quick and efficient detection of MPXV. Monkeypox virus is a double-stranded DNA virus, Poxviridae, that may be categorized together with variola virus. The symptoms to a person infected with MPXV is similar to smallpox, such as body temperature increase or a fever, headache, lymphadenectasis, cough and extreme whole-body pain. Compared to smallpox, monkeypox has a much lower infectivity or human to human transmission with R0<1 (Learned et al., 2005; Sklenovská and Van Ranst, 2018), perhaps ≈0.576 (McMullen, 2015), and an attack rate of ≈50% (Luciani et al., 2021). While antiviral drug ST-246, a potent Orthopoxvirus egress inhibitor, can protect non-human primates from MPXV (Huggins et al., 2009), smallpox vaccine (for example, Dryvax) seems 85% effective in humans against monkeypox (Edghill-Smith et al., 2005). The MPXV has two distinct clades (Likos et al., 2005). The Congo basin clade causes illness similar to smallpox and has a case fatality rate of up to 10% in unvaccinated populations. The West African clade causes less severe and less interhuman transmissible disease (Chen et al., 2005; Li et al., 2010). The disease severity of monkeypox, compared to smallpox, is considerably less with milder rash. Prior to the 2022 outbreak, monkeypox was mostly endemic in West and Central African countries. However, monkeypox cases are rising at an alarming rate worldwide since May 2022, with over 25,600 confirmed cases in the US and 68,000 globally as of Sep. 7, 2022. See for example: (https://www.cdc.gov/poxvirus/monkeypox/response/2022/world-map.html). Diagnosis for human orthopoxvirus infections is largely limited to polymerase chain reaction (PCR) based molecular testing. As part of public health preparedness for infectious disease threats, the Center for Disease Control (CDC) has made diagnostic testing for orthopoxviruses available at Laboratory Response Network (LRN). More recently, the CDC developed a non-variola Orthopoxvirus (NVO) real-time PCR diagnostic test (with FDA 510(k) clearance) to facilitate monkeypox detection. However, this NVO test does not differentiate monkeypox virus from other NVOs. To date, there is currently no commercially available assay to specifically detect monkeypox virus. Therefore, a detection technique or assay to specifically detect monkeypox virus has great importance. There is a need for an improved assay, kit, and method for real-time detection of the monkeypox virus before any symptomatology arises. There is also a need to have such a detection without cross reaction to other pox virus such as cowpox and the like. SUMMARY Compared to the above prior attempts, the present disclosure fulfills the above criteria and provides additional benefits that state of the art systems cannot provide. The following monkeypox specific real-time PCR assays can be used as a sensitive and accurate tool for monkeypox diagnosis. It is based on detection the detection of MPXV using an assay of MPX-HA with a specialized molecular beacon and, depending on the implementation, this MPX-HA assay may also be