Come evolveranno e come sono cambiate le metodiche di laboratorio negli ultimi anni? Quali sono le più comuni e che specificità e sensibilità riescono a raggiungere nella diagnosi di situazioni patologiche? Ecco le risposte!

La PCR è una reazione di polimerizzazione in vitro di un segmento specifico di DNA per mezzo di un enzima chiamato DNA polimerasi. La reazione permette di ottenere quindi ampliconi di un determinato tratto di DNA utile a fini diagnostici e di ricerca. E’ un tipo di analisi che può avere numerose applicazioni come ad esempio, in un contesto di analisi di secondo livello, quantificare la viremia del virus HIV di un soggetto affetto che viene sottoposto ad un trattamento antivirale.

Nel 1983 venne isolato il retrovirus HIV (Human Immunodeficiency Virus). Oggi sono noti due virus responsabili della sindrome da immunodeficienza acquisita (AIDS) umana: HIV-1 e HIV-2.
La tipologia HIV-1 è diffusa in tutto il mondo ed è responsabile della maggior parte dei casi di AIDS, mentre la tipologia HIV-2 è presente soprattutto in Africa Occidentale, nei Caraibi e in America Meridionale con la caratteristica di essere meno virulenta del primo tipo provocando una patologia a decorso relativamente più attenuato.

L’infezione da HIV rappresenta la tappa iniziale che porta ad un quadro patologico e clinico decisamente drammatico che si conclude, nel corso di diversi anni, con la morte del paziente.
La trasmissione avviene tramite liquidi biologici da un soggetto infetto ad uno sano, attraverso ad esempio rapporti sessuali non protetti o l’uso di siringhe tra tossicodipendenti. L’infezione può inoltre essere trasmessa tra madre e feto per via transplacentare, durante il parto o l’allattamento.

Virus HIV
Virus HIV

L’HIV è un retrovirus il cui genoma è definito diploide in quanto costituito da due filamenti di RNA parzialmente embricate che fungono da stampo per la sintesi di DNA bicatenario.
Il virus aderisce alla cellula tramite recettori specifici, successivamente penetra nella cellula. La fase di uncoating successiva consiste nell’esposizione vera e propria del genoma all’interno della cellule nel sito target di replicazione. La moltiplicazione dei retrovirus è caratteristica dato che all’interno del virione è contenuto l’enzima trascrittasi inversa che permette appunto la trascrizione inversa di ciascuna molecola di RNA in una molecola di DNA bicatenario che grazie all’enzima integrasi si integra nel DNA cellulare e viene successivamente trascritto e tradotto in proteina virale.

Per poter procedere alla valutazione della viremia totale di un certo soggetto è necessario però, prima di eseguire una PCR, estrarre l’acido nucleico caratteristico del virus. Nel caso in cui si voglia ricercare il virus HIV per valutarne la viremia sarà indispensabile estrarre l’RNA virale dal campione biologico del paziente in questione. In questo caso l’acido nucleico virale verrà poi retrotrasritto in cDNA, subendo un ulteriore passaggio, in quanto la reazione di polimerizzazione avviene solo a partire da una molecola di DNA.

L’estrazione dell’RNA può essere fatta sia manualmente, sia in maniera automatizzata sfruttando differenti principi di estrazione e separazione dell’acido nucleico. Esiste una tecnica che si basa sull’utilizzo di particelle magnetiche che vengono dispensate, insieme ad un buffer di lisi, nelle provette di reazione nelle quali viene aggiunto il plasma del paziente. Questa fase di incubazione, che si serve anche del riscaldamento delle provette, permette il rilascio dell’acido nucleico che andrà a legarsi alla fase solida, in questo caso rappresentata dalle biglie magnetiche presenti in soluzione. Successivamente le provette di reazione vengono sottoposte ad un campo magnetico che cattura in una zona della provetta le particelle a cui è adeso l’RNA. Al termine di questa fase si elimina il materiale non legato presente nella provetta di reazione e si effettuano una serie di lavaggi, interrompendo o meno il campo magnetico, così da eliminare ogni sorta di aspecificità.

Infine viene dispensato un buffer di eluizione molto affine all’acido nucleico rispetto alle biglie magnetiche che permette la risospensione dell’RNA in soluzione. L’acido nucleico così estratto viene successivamente trasferito in una piastra a 96 pozzetti per la fase successiva.

La retrotrascrizione dell’RNA avviene grazie all’utilizzo di una particolare mix di retrotrascrizione composta da RNAasi STOP che inibisce l’effetto di degradazione dell’acido nucleico da parte di enzimi, DNA polimerasi RNA dipendente ossia un enzima che permette di sintetizzare DNA a partire da una molecola stampo di RNA, relativi primers di innesco per la sintesi del DNA, deossinucleotidi e acqua ai quali viene aggiunto l’acido nucleico estratto precedentemente.

Una volta ottenuto il cDNA si procede con la reazione di polimerizzazione che avviene grazie l’utilizzo di un termociclatore, uno strumento che permette di sottoporre l’acido nucleico miscelato ad una master mix di amplificazione ad una serie ripetuta di cicli nei quali avvengono le seguenti fasi:
1 – denaturazione a 95 ° C, fase in cui avviene la separazione della catena di DNA;
2 – annealing a 60° C, fase in cui si ha l’appaiamento dei primers con il filamento di DNA;
3 – estensione a 72 ° C, fase di formazione della nuova catena.
Questa reazione permette quindi, grazie ad una serie di cicli, di ottenere un accumulo esponenziale del frammento target che ha una lunghezza pari alla somma della lunghezza dei primers e della distanza tra esse.
E’ importante ottimizzare le condizioni di reazione agendo su una serie di parametri relativi in buona parte alle componenti della master mix di amplificazione e alla loro concentrazione. Innanzi tutto l’acido nucleico estratto deve essere il più possibile puro e quindi privo di interferenze e secondariamente deve essere in una quantità sufficiente da poter essere amplificato. I primers devono essere specifici, avere una lunghezza ottimale ed una concentrazione utile. Inoltre deve esserci una concentrazione ottimale sia del tampone contenente magnesio, sia dei deossinucleotidi che andranno poi a costituire il filamento di DNA grazie alla DNA polimerasi che deve essere presente in concentrazione ottimale ed essere del tipo adatto. Infine è opportuno avere uno strumento preciso che funzioni secondo le modalità impostate e quindi utilizzando una temperatura, tempi e cicli opportuni.

Piastra da PCR a 96 pozzetti.
Piastra da PCR a 96 pozzetti.

La PCR si serve di svariati metodi di rilevazione uno dei quali sfrutta le sonde TAQ-MAN inserite nei pozzetti di reazione insieme al campione e alla master mix. Sono costituite da un’estremità detta Quencher (inibitore di fluorescenza) e da un’estremità detta Reporter (fluorocromo). Queste si legano istantaneamente ad una determinata sequenza di DNA. Nel momento in cui avviene la sintesi del filamento, la DNA polimerasi elimina la sonda che si separa in Q ed R emettendo fluorescenza.
Si parla in questo caso di PCR- Real Time in quanto queste sonde permettono allo strumento di rivelare alla fine di ogni ciclo, la quantità di DNA replicato ottenendo quindi un valore numerico preciso che sarà proporzionale alla viremia ricercata.

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