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Ensaios baseados em CRISPR para a detecção de infecções por vírus BK e JC após transplante renal

  • role： Contribute equally, First author同等贡献者, 第一作者
  • Affiliation：

    Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China

Yu Liu1,  
  • role： Contribute equally同等贡献者
  • Affiliation：

    Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China

Jing-Song Xu1,  
  • role： Contribute equally同等贡献者
  • Affiliation：

    Department of Pathology, the Fifth People’s Hospital of Shanghai, Fudan University, Shanghai 200240, China

Li Cao2,  
  • Affiliation：

    Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China

Shuang Yang1,  
  • Affiliation：

    Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China

Tian-Ming Li1,  
  • Affiliation：

    Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China

Hai-Qian Huang1,  
  • Affiliation：

    Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China

Jun-Heng Zhang1,  
  • Affiliation：

    Central Laboratory, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China

Xue Zhao3,  
  • role： Corresponding author通信作者
  • Affiliation：

    Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China

  • Email：qq2005011@163.com
  • Introduction：* Qian Liu qq2005011@163.com
Qian Liu1*,  
  • role： Corresponding author通信作者
  • Affiliation：

    Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, China

  • Email：lishun86@126.com
  • Introduction：Shun Li lishun86@126.com
Shun Li4*,  
  • role： Corresponding author通信作者
  • Affiliation：

    Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China

  • Email：rjlimin@shsmu.edu.cn
  • Introduction：Min Li rjlimin@shsmu.edu.cn
Min Li1*,  
  • role： Corresponding author通信作者
  • Affiliation：

    Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China

  • Email：renjijywh@shsmu.edu.cn
  • Introduction：Hua Wang renjijywh@shsmu.edu.cn
Hua Wang1*

Resumo

Os recipientes de transplante de órgãos enfrentam riscos significativos de infecções agudas ou crônicas que ameaçam a sobrevivência do enxerto. Os vírus BK (BKV) e JC (JCV) são dois vírus oportunistas proeminentes e podem causar nefrite associada ao poliomavírus e perda do rim enxertado em pacientes em estado imunossuprimido após o transplante renal. Portanto, a detecção oportuna e o monitoramento contínuo da carga viral são indispensáveis. No entanto, os métodos diagnósticos atuais permanecem limitados, e o desenvolvimento de novas tecnologias moleculares de detecção é extremamente urgente. As sequências e concentrações do RNA CRISPR (crRNA), a concentração de Cas13a e os primers para amplificação por polimerase de recombinase (RPA) foram otimizados para a detecção de BKV e JCV. Em seguida, um novo chip microfluídico de gota dupla foi projetado e fabricado, e integrado ao CRISPR (ddCRISPR) para detectar qualitativamente simultaneamente BKV e JCV. Posteriormente, o ensaio ddCRISPR foi verificado usando amostras clínicas. Então, uma tira de fluxo lateral combinada com CRISPR (LFCRISPR) foi desenvolvida para a detecção de BKV e JCV em ambientes com recursos limitados. Foi estabelecido e otimizado um sistema de reação em um pote único RPA-CRISPR para detecção de BKV e JCV. O ddCRISPR pode detectar simultânea e rapidamente BKV e JCV com alta sensibilidade (10 cópias/ml para BKV e 1 cópia/ml para JCV) e fornecer quantificação absoluta, adequado para detecção da carga viral e propício para tratamento personalizado e preciso de receptores de transplante de órgãos. LFCRISPR simplificou o processo operacional por meio de uma leitura visual simples, facilitando a triagem do vírus após o transplante de órgãos. Essas plataformas incorporam testes moleculares no modelo de tratamento de transplante, reduzindo custos, prolongando o tempo de sobrevivência do enxerto, melhorando os resultados clínicos do manejo pós-operatório no transplante renal e melhorando a qualidade de vida dos pacientes.

Palavras-chave

vírus BK (BKV); vírus JC (JCV); transplante renal; repetições palindrômicas curtas espaçadas regularmente (CRISPR); microfluídica; ensaio de fluxo lateral (LFA)

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